Endothelin analogs and uses thereof

Disclosed are a peptide represented by formula (I) or a pharmaceutically acceptable salt thereof: ##STR1## wherein M represents a mercaptoacyl group; P, Q, R, S, T, U, V, W, X, Y and Z each represent amino acid residues, wherein an amino acid side chain of Y is either a substituted saturated aliphatic hydrocarbon group having 1 to 15 carbon atoms or an unsubstituted saturated aliphatic hydrocarbon group having 4 to 15 carbon atoms other than (1S)-1-methylpropyl; (2) a method for producing the above-mentioned peptide or the salt thereof, which comprises subjecting a peptide represented by formula (II) or a salt thereof to an oxidation reaction: EQU M-P-Cys-Q-R-S-T-Asp-U-Glu-Cys-Val-Tyr-V-Cys-His-W-X-Y-Ile-Z-OH(II) wherein M, P, Q, R, S, T, U, V, W, X, Y and Z are as diefined above; and (3) use of the above-mentioned peptide or the pharmaceutically acceptable salt thereof as an anti-endothelin agent.

BACKGROUND OF THE INVENTION 
The present invention relates to novel peptides having antagonistic 
activity to endothelin receptors. These novel peptides are useful as 
prophylactic and therapeutic drugs for hypertension, cardiac or cerebral 
circulatory diseases, renal diseases and asthma, and to the production 
thereof. The present invention further relates to the use thereof. 
Endothelin (ET) is a vasoconstrictive peptide composed of 21 amino acid 
residues. Endothelin was isolated from the culture supernatant of the 
endothelial cells of porcine aortas. Its structure was determined by M. 
Yanagisawa et al. in 1988 [M. Yanagisawa et al., Nature 332, 411-415 
(1988)]. More recently, the research on genes coding for endothelin 
revealed the presence of peptides similar to endothelin in structure. 
These peptides are named endothelin-1 (ET-1), endothelin-2 (ET-2) and 
endothelin-3 (ET-3), respectively, and their structures are as follows: 
##STR2## 
(All of the amino acids constituting ET-1, ET-2 and ET-3 take the L-form.) 
[Inoue et al., Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867 (1989)] 
The above-mentioned peptides of the endothelin family exist in vivo and 
have vasopressor activity. For this reason, these peptides are anticipated 
to be intrinsic factors responsible for the control of circulatory 
systems, and deduced to be related to hypertension, cardiac or cerebral 
circulatory diseases (for example, cardiac infarction) and renal diseases 
(for example, acute renal insufficiency). In addition, these peptides also 
have bronchial smooth muscle constrictor activity, and therefore deduced 
to be related to asthma. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide novel 
peptides having antagonistic activity to the endothelin receptors. 
The present inventors further studied, the activity of endothelin having 
strong vascular smooth muscle constrictor activity, and the antagonistic 
effect of the synthesized novel peptides by amino acid substitution mainly 
at the 19-position of endothelin. 
Namely, the present invention provides 
(1) a peptide represented by formula (I) (SEQ ID NO: 1) or a 
pharmaceutically acceptable salt thereof: 
##STR3## 
wherein M represents a mercaptoacyl group; P, Q, R, S, T, U, V, W, X, Y 
and Z each represent amino acid residues, wherein an amino acid side chain 
of Y is either a substituted saturated aliphatic hydrocarbon group having 
1 to 15 carbon atoms or an unsubstituted saturated aliphatic hydrocarbon 
group having 4 to 15 carbon atoms other than (1S)-1-methylpropyl; 
(2) a method of producing the above-mentioned peptide represented by 
formula (I) or the salt thereof, which comprises subjecting a peptide 
represented by formula (II) (SEQ ID NO: 2) or a salt thereof to an 
oxidation reaction: 
EQU M-P-Cys-Q-R-S-T-Asp-U-Glu-Cys-Val-Tyr-V-Cys-His-W-X-Y-Ile-Z-OH(II) 
wherein M represents a mercaptoacyl group; P, Q, R, S, T, U, V, W, X, Y and 
Z each represent amino acid residues, wherein an amino acid side chain of 
Y is either a substituted saturated aliphatic hydrocarbon group having 1 
to 15 carbon atoms or an unsubstituted saturated aliphatic hydrocarbon 
group having 4 to 15 carbon atoms other than (1S)-1-methyl propyl; 
(3) a pharmaceutical composition comprising the above-mentioned peptide 
represented by formula (I) or a pharmaceutically acceptable salt thereof; 
and 
(4) use of the above mentioned peptide represented by formula (I) or a 
pharmaceutically acceptable salt thereof as an anti-endothelin agent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In the peptides of the present invention represented by formula (I), the 
saturated aliphatic hydrocarbon groups with 1 to 15 carbon atoms as the 
amino acid side chains of Y include alkyl, cycloalkyl or cycloalkyl-alkyl 
groups in which the alkyl groups may be straight or branched. As alkyl 
groups, preferred are C.sub.1-10 alkyl groups, which include, for example, 
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, 
n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and 
n-decyl. As cycloalkyl groups, preferred are C.sub.3-10 cycloalkyl groups, 
which include, for example, cyclopropyl, cyclobutyl, cyclopentyl, 
cyclohexyl, cycloheptyl, cyclooctyl and norbornyl. As cycloalkyl-alkyl 
groups, preferred are C.sub.3-10 cycloalkyl-C.sub.1-6 alkyl groups, which 
include, for example, cyclopropylmethyl, cyclobutylmethyl, 
cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, 
1-cyclopentylethyl, 1-cyclohexylethyl, 2-cyclopentylethyl, 
2-cyclohexylethyl, 3-cyclohexylpropyl, 4-cyclohexylbutyl and 
5-cyclohexylpropyl. 
The above-mentioned saturated hydrocarbon groups may also be substituted, 
and the substituent groups include sulfur-containing groups (such as 
thione, mercapto, methylthio, ethylthio, n-propylthio, isopropylthio, 
n-butylthio, isobutylthio, t-butylthio, phenylthio, cyclopentylthio, 
cyclohexylthio and thienyl), oxygen-containing substituents (such as 
ketone, hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 
isobutoxy, t-butoxy, n-pentyloxy, cyclohexyloxy, phenoxy, benzyloxy and 
furyl), nitrogen-containing groups (such as amino, N-methylamino, 
N-ethylamino, N-n-propylamino, N-isopropylamino, N-n-butylamino, 
N-isobutylamino, N-t-butylamino, N-n-pentylamino, N-n-hexylamino, 
N-cyclohexylamino, N,N-dimethylamino, N,N-diethylamino, 
N,N-di-n-propylamino, N,N-di-isopropylamino, N,N-di-n-butylamino, 
N,N-diisobutylamino, N,N-di-t-butylamino, N,N-di-n-pentylamino, 
N,N-di-n-hexylamino, N,N-dicyclohexylamino, nitro, guanidino, pyrrolidino, 
piperidino, indolyl and imidazolyl), aromatic hydrocarbon group (such as 
phenyl, 1-naphtyl, 2-naphtyl) and halogen groups (such as chloro, bromo 
and fluoro). 
The unsubstituted saturated aliphatic hydrocarbon groups with 4 to 15 
carbon atoms other than (1S)-1-methylpropyl as the amino acid side chains 
of Y, are alkyl, cycloalklyl or cycloalkyl-alkyl groups in which alkyl may 
be straight or branched. As alkyl groups, preferred are C.sub.4-10 alkyl 
groups, which include, for example, n-butyl, isobutyl, t-butyl, n-pentyl, 
isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. As 
cycloalkyl groups, preferred are C.sub.4-10 cycloalkyl groups, which 
include, for example, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 
cyclooctyl and norbornyl. As cycloalkyl-alkyl groups, preferred are 
C.sub.3-10 cycloalkyl-C.sub.1-6 alkyl groups, which include, for example, 
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 
cycloheptylmethyl, 1-cyclopentylethyl, 1-cyclohexylethyl, 
2-cyclopentylethyl, 2-cyclohexylethyl, 2,2-dicyclopentylethyl, 
2,2-dicyclohexylethyl, 3-cyclohexylpropyl, 4-cyclohexylbutyl and 
5-cyclohexylpropyl. 
As an amino acid side chain of Y, a hydrocarbon group branched at its 
.beta.-positioned carbon atom is more preferred, for example, isobutyl, 
neopentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 
cyclohexylmethyl, cycloheptylmethyl, 2-cyclohexylpropyl, 
2,2-dicyclohexylethyl, 2-mercaptopropyl, 2-thienylmethyl, 2-hydroxypropyl, 
2-furylmethyl, 3-indolylmethyl, 4-imidazolylmethyl and benzyl. 
In this specification, amino acids and peptides are indicated by the 
abbreviations commonly used in the art or adopted by the IU-IUB 
Commission on Biochemical Nomenclature. For example, the following 
abbreviations are used: 
Gly: Glycine 
Ala: Alanine 
Val: Valine 
Leu: Leucine 
Ile: Isoleucine 
Ser: Serine 
Thr: Threonine 
Cys: Cysteine 
Met: Methionine 
Glu: Glutamic acid 
Asp: Aspartic acid 
Lys: Lysine 
Arg: Arginine 
His: Histidine 
Phe: Phenylalanine 
Tyr: Tyrosine 
Trp: Tryptophan 
Pro: Proline 
Asn: Asparagine 
Gln: Glutamine 
Tyr(Et): O-Ethyltyrosine 
Nal(1): 1-Naphthylalanine 
Nal(2): 2-Naphthylalanine 
Cha: Cyclohexylalanine 
Thi: .beta.-2-Thienylalanine 
Phe(4F): 4-Fluorophenylalanine 
Phg: Phenylglycine 
Cyt: Cystine 
Abu: 2-Aminobutyric acid 
Nva: Norvaline 
Nle: Norleucine 
tLeu: tert-Leucine 
.gamma.Leu: .gamma.-Methylleucine 
Mpr: 3-Mercaptopropionic acid 
Protective groups and reagents commonly used in this specification are 
indicated by the following abbreviations: 
Boc: t-Butoxycarbonyl 
Bzl: Benzyl 
BrZ: 2-Bromobenzyloxycarbonyl 
ClZ: 2-Chlorobenzyloxycarbonyl 
Tos: p-Toluenesulfonyl 
Dnp: 2,4-Dinitrophenyl 
OcHex: Cyclohexyl ester 
For: Formyl 
MeBzl: 4-Methylbenzyl 
Acm: Acetamidomethyl 
TFA: Trifluoroacetic acid 
HF: Anhydrous hydrogen fluoride 
HOBt: 1-Hydroxybenzotriazole 
DMF: N,N-Dimethylformamide 
In the present invention, the mercaptoacyl groups represented by M which 
may be substituted are carboxylic acid-derived acyl groups having mercapto 
groups, which include aliphatic, alicyclic and aromatic carboxylic acids. 
Preferred examples of the aliphatic mercaptoacyl groups include mercapto 
C.sub.2 -C.sub.10 alkanoyl groups such as 3-mercaptopropionyl and 
4-mercaptobutyryl. Preferred examples of the alicyclic mercaptoacyl groups 
include mercapto C.sub.3 -C.sub.8 cycloalkylcarbonyl groups such as 
3-mercaptocyclopentylcarbonyl. Preferred examples of the aromatic 
mercaptoacyl groups include C.sub.6 -C.sub.14 arylcarbonyl groups such as 
3-mercapto-3-phenylpropionyl. The above-mentioned aliphatic and alicyclic 
groups are preferably used. As noted above, the mercaptoacyl groups may be 
substituted. Substituent groups on the mercaptoacyl groups include amino 
and hydroxyl groups. A mercaptoacyl group substituted by an amino group at 
the .alpha.-position of the acyl group is more preferred. Preferred 
examples thereof include Cys, homocysteine and 3-mercapto-D-valine 
(penicillamine). The most preferred examples of the unsubstituted 
mercaptoacyl groups and the substituted mercaptoacyl groups are 
3-mercaptopropionyl and Cys, respectively. 
In the present invention, the amino acid residue represented by P, Q, R, S, 
T, U, V, W, X, Y or Z may be either a natural amino acid residue or a 
synthetic amino acid residue, and may be any of the L-, D- and DL-forms. 
Accordingly, P, Q, R, S, T, U, V, W, X, Y and Z can also be expressed as 
##STR4## 
respectively. The compound of formula (I) (SEQ ID NO: 1) can therefore be 
represented by formula (I') (SEQ ID NO: 1): 
##STR5## 
wherein P', Q', R', S', T', U', V', W', X' and Z' each represent hydrogen 
atoms or hydrocarbon groups with 1 to 15 carbon atoms which may be 
substituted, and Y' represents either a substituted saturated aliphatic 
hydrocarbon group having 1 to 15 carbon atoms or an unsubstituted 
saturated aliphatic hydrocarbon group having 4 to 15 carbon atoms other 
than (1S)-1-methylpropyl. The hydrocarbon groups having 1 to 15 carbon 
atoms include aliphatic hydrocarbon groups, aromatic hydrocarbon groups 
and aliphatic-aromatic hydrocarbon groups. 
The aliphatic hydrocarbon groups represented by P' to X' and Z' described 
above may be straight, branched or cyclic groups which may be saturated. 
Examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, 
isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, cyclopentyl, 
n-hexyl, cyclohexyl, n-heptyl, cycloheptyl, n-octyl, n-nonyl, n-decyl, 
cyclopentylmethyl and cyclohexyl-methyl. The substituted aliphatic 
hydrocarbon groups include methylthiomethyl, ethylthiomethyl, 
n-propylthiomethyl, isopropylthiomethyl, n-butylthiomethyl, 
t-butylthiomethyl, 2-methylthioethyl, 2-ethylthioethyl, 
2-t-butylthioethyl, mercaptomethyl, 1-mercaptoethyl, 2-mercaptoethyl, 
phenylthiomethyl, 1-phenylthioethyl, 2-phenylthioethyl, benzylthiomethyl, 
4-methoxyphenylthiomethyl, 4-methoxybenzylthiomethyl, 
4-methylbenzylthiomethyl, 4-nitrobenzylthiomethyl, 
4-pyridylbenzylthiomethyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 
methoxy-methyl, ethoxymethyl, n-propoxymethyl, isopropoxymethyl, 
n-butoxymethyl, t-butoxymethyl, n-pentyloxymethyl, cyclo-pentyloxymethyl, 
n-hexyloxymethyl, cyclohexyloxyInethyl, 1-methoxyethyl, 1-ethoxyethyl, 
1-propoxyethyl, 1-isopropoxyethyl, 1-n-butoxyethyl, 1-isobutoxyethyl, 
1-t-butoxyethyl, phenoxymethyl, 1-phenoxyethyl, 2-phenoxyethyl, 
benzyloxymethyl, 2-benzyloxyethyl, carboxymethyl, 1-carboxyethyl, 
2-carboxyethyl, methoxycarbonylmethyl, ethoxycarbonylmethyl, 
n-propoxycarbonylmethyl, isopropoxycarbonylmethyl, n-butoxycarbonylmethyl, 
isobutoxycarbonylmethyl, t-butoxycarbonylmethyl, 
n-pentyloxycarbonylmethyl, cyclopentyloxycarbonylmethyl, 
n-hexyloxycarbonylmethyl, cyclohexyloxycarbonylmethyl, 
cycloheptyloxycarbonylmethyl, cyclooctyloxycarbonylmethyl, carboxyethyl, 
methoxycarbonylethyl, ethoxycarbonylethyl, n-propoxycarbonylethyl, 
isopropoxycarbonylethyl, n-butoxycarbonylethyl, isobutoxycarbonylethyl, 
t-butoxycarbonylethyl, n-pentyloxycarbonylethyl, 
cyclopentyloxycarbonylethyl, n-hexyloxycarbonylethyl, 
cyclohexyloxycarbonylethyl, cycloheptyloxycarbonylethyl, 
cyclooctyloxycarbonylethyl, 2-aminoethyl, 2-(N-methylamino)ethyl, 
2-(N,N-dimethylamino)ethyl, 3-aminopropyl, 3-(N,N-diethylamino)propyl, 
2-guanidinoetyl, 3-guanidinopropyl, aminocarbonylmethyl, 
N-methylaminocarbonylmethyl, N-ethylaminocarbonylmethyl, 
N-n-propyl-aminocarbonylmethyl, N-isopropylaminocarbonylmethyl, 
N-n-butylaminocarbonylmethyl, N-isobutylaminocarbonylmethyl, 
N-t-butylaminocarbonylmethyl, N-n-pentylamino-carbonylmethyl, 
N-isopentylaminocarbonylmethyl, N-neopentylaminocarbonylmethyl, 
N-n-hexylaminocarbonylmethyl, N-cyclohexylaminocarbonylmethyl, 
N,N-dimethylaminocarbonylmethyl, N,N-diethylaminocarbonylmethyl, 
N,N-di-n-propylaminocarbonylmethyl, N,N-diisopropylaminocarbonylmethyl, 
N,N-di-n-butylaminocarbonylmethyl, N,N-diisobutylaminocarbonylmethyl, 
N,N-di-t-butylaminocarbonylmethyl, N,N-di-n-pentylaminocarbonylmethyl, 
N,N-diisopentylaminocarbonylmethyl, N,N-dineopentylaminocarbonylmethyl, 
N,N-di-n-hexylaminocarbonylmethyl, N,N-dicyclohexylaminocarbonylmethyl, 
pyrrolidinocarbonylmethyl, piperidinocarbonylmethyl, aminocarbonylethyl, 
N-methylaminocarbonylethyl, N-ethylaminocarbonylethyl, 
N-n-propylaminocarbonylethyl, N-isopropylaminocarbonylethyl, 
N-n-butylaminocarbonylethyl, N-isobutylaminocarbonylethyl, 
N-t-butylaminocarbonylethyl, N-n-pentylaminocarbonylethyl, 
N-cyclopentylaminocarbonylethyl, N-n-hexylaminocarbonylethyl, 
N-cyclohexylaminocarbonylethyl, N,N-dimethylaminocarbonylethyl, 
N,N-diethylaminocarbonylethyl, N,N-di-n-propylaminocarbonylethyl, 
N,N-diisopropylaminocarbonylethyl, N,N-di-n-butylaminocarbonylethyl, 
N,N-diisobutylaminocarbonylethyl, N,N-di-t-butylaminocarbonylethyl, 
N,N-di-n-pentylaminocarbonylethyl, N,N-dicyclopentylaminocarbonylethyl, 
N,N-di-n-hexylaminocarbonylethyl, N,N-dicyclohexylaminocarbonylethyl, 
3-indolylmethyl, 4-imidazolylmethyl, 2-thienylmethyl, 2-furylmethyl, 
pyrrolidinocarbonylethyl and piperidinocarbonylethyl. 
Examples of the aromatic hydrocarbon groups and aliphatic-aromatic 
hydrocarbon groups represented by P' to X' and Z' include phenyl, 
1-naphthyl, 2-naphthyl, phenylmethyl, 1-phenylethyl, 2-phenylethyl, 
1-naphthylmethyl, 2-naphthylmethyl and 9-anthranylmethyl. Examples of the 
substituted aromatic hydrocarbon groups and aliphatic-aromatic hydrocarbon 
groups include 4-hydroxyphenyl, 4-hydroxyphenylmethyl, 
4-methoxyphenylmethyl, 4-ethoxyphenylmethyl, 4-n-propoxyphenylmethyl, 
4-isopropoxyphenylmethyl, 4-n-butoxyphenylmethyl, 4-isobutoxyphenylmethyl, 
4-t-butoxyphenylmethyl, 4-n-pentyloxyphenylmethyl, 
4-cyclopentyloxyphenylmethyl, 4-n-hexyloxyphenylmethyl, 
4-cyclohexyloxyphenylmethyl, 4-aminophenylmethyl, 
4-dimethylaminophenylmethyl, 4-diethylaminophenylmethyl, 
4-di-n-propylaminophenylmethyl, 4-diisopropylaminophenylmethyl, 
4-di-n-butylaminophenylmethyl, 4-pyrrolidinophenylmethyl, 
4-piperidinophenylmethyl, 4-nitrophenylmethyl, 4-fluorophenylmethyl, 
3-fluorophenylmethyl, 2-fluorophenylmethyl, 4-chlorophenylmethyl, 
3-chlorophenylmethyl and 2-chlorophenylmethyl. 
Y' corresponds to the amino acid side chain of Y as described above. 
Therefore, Y' has the same meaning as the amino acid side chain of Y, and 
represents a substituted saturated aliphatic hydrocarbon group having 1 to 
15 carbon atoms or an unsubstituted saturated aliphatic hydrocarbon group 
having 4 to 15 carbon atoms other than (1S)-1-methylpropyl. 
Preferred examples of the amino acid residues represented by P to Z (having 
P' to Z', respectively) are more specifically described below. 
P is an amino acid residue having an alkyl group which may be substituted 
as the amino acid side chain (P'). And hydroxy group is preferred as this 
substituent group. Specific examples of the substituent groups include Ala 
as well as Ser and Thr. 
Q includes, for example, Ser, Thr, Phe and Ala. Ser and Ala are preferred 
among others. 
R is an amino acid residue having an alkyl group which may be substituted 
as the amino acid side chain (R'). A OH is preferred as this substituent 
group. Specific examples of the substituent groups include Ala as well as 
Ser and Thr. 
S is an amino acid residue having a lipophilic portion as the amino acid 
side chain (S'). Specific examples thereof include Leu, Ala, Tyr, Trp and 
Met, and Leu is preferred. 
T includes Met, Leu, Lys, Ala, Nle and Glu, and Met, Ala and Nle are 
preferred. 
U includes Lys, Ala and Glu. 
As V, aromatic amino acids are preferred, with the monocyclic ones 
preferred over the bicyclic ones. Preferred examples thereof include Trp 
as well as Phe and Tyr. 
W includes Gln as well as Leu. 
As Z, aromatic amino acids are preferred, with bicyclic ones being more 
preferred. Preferred examples thereof include Trp, and Trp having a 
substituent group [for example, N-(indole)-formyltryptophan], 
.alpha.-naphthylalanine and .beta.-naphthylalanine. Substituted compounds 
such as N-(indole)-formyl compounds are often used in place of tryptophan 
easily decomposed by oxidation. 
As X, amino acid residues other than Asp are preferred, and particularly, 
amino acid residues having hydroxyl groups are preferred due to their 
strong binding affinity for endothelin receptors. Preferred examples 
thereof include Ser and Thr. In addition, amino acid residues such as Asn 
and Gly are also preferably used. 
Preferred examples of Y include amino acid residues having the amino acid 
side chain (Y') branched at the 2-position, for example, Leu, Cha, Phe, 
.gamma.Leu and Asn. 
Although embodiments of the present invention have emphasized substitution 
of (Y) at the 19-position, further substitution at the 18-position is also 
within the scope of the invention. Preferred combinations of the 
18-position and the 19-position include Thr-Leu, Thr-.gamma.Leu, Thr-Cha, 
Thr-Phe, Thr-Asn, Ser-Leu, Asn-Leu and Gly-Leu. The combinations of 
Thr-Leu, Thr-.gamma.Leu and Thr-Cha are especially preferred. 
The pharmaceutically acceptable salts of the peptides represented by 
formula (I) or (I') include sodium salts, potassium salts and calcium 
salts as well as addition salts of inorganic acids such as hydrochlorides, 
sulfates and phosphates, and salts of organic acids such as acetates, 
propionates, citrates, tartarates, malates and oxalates. 
The peptides of the present invention represented by formula (I) or (I') 
can be produced by methods for peptide synthesis known in the art, which 
may be either solid phase synthesis methods or liquid phase synthesis 
methods. Examples of such methods for peptide synthesis include methods 
described in M. Bodansky and M. A. Ondetti, Peptide Synthesis, 
Interscience, New York (1966); F. M. Finn and K. Hofmann, The Proteins, 
Vol. 2, edited by H. Nenrath and R. L. Hill, Academic Press, New York, 
(1976); N. Izumiya et al., Peptide Gosei no Kiso to Jikken (Fundamentals 
and Experiments of Peptide Synthesis), Maruzen (1985); H. Yazima, S. 
Sakakibara et al., Seikagaku Jikken Koza (Course of Biochemical 
Experiments), 1, edited by Biochemical Society of Japan, Tokyo Kagaku 
Dojin (1977); H. Kimura et al., Zoku Seikagaku Jikken Koza (Course of 
Biochemical Experiments, second series), 2, edited by Biochemical Society 
of Japan, Tokyo Kagaku Dojin (1987); and J. M. Stewart and J. D. Young, 
Solid Phase Peptide Synthesis, Pierce Chemical Company, Illinois (1984), 
which describe azide methods, chloride methods, acid anhydride methods, 
mixed acid anhydride methods, DCC methods, active ester methods, methods 
using Woodward reagent K, carbodiimidazole methods, oxidation-reduction 
methods, DCC/HONB methods and methods using BOP reagents. 
The peptides of the present invention represented by formula (I) or (I') 
can be produced by condensing a raw material having a reactive carboxyl 
group corresponding to one of two kinds of fragments which are separated 
at any position of its peptide bond with a raw material having a reactive 
amino group corresponding to the other fragment, and then, eliminating a 
protective group by methods known in the art, if the resulting condensed 
product has any protective group, followed by further oxidation reaction. 
In particular, in the solid phase synthesis methods, an amino acid whose 
functional groups which should not affect the reaction are protected, is 
combined with an insoluble resin such as a Pam resin through the carboxyl 
group of the amino acid. After elimination of the N.sup..alpha. 
-protective group, an amino acid, whose functional groups which should not 
affect the reaction are protected, is condensed therewith. This procedure 
is repeated until a desired protected peptide is obtained. Then, the 
protective group is eliminated and the desired peptide is released from 
the resin by methods known in the art such as hydrogen fluoride treatment, 
trifluoromethanesulfonic acid treatment and trifluoroacetic acid 
treatment, followed by further oxidation reaction, whereby the compound of 
the present invention is produced. 
When the M.sup.1 -Cys.sup.15 and Cys.sup.3 -Cys.sup.11 bonds are formed by 
the oxidation reaction, a compound represented by formula (II) or (II') is 
oxidized by methods known in the art. 
##STR6## 
In this case, (1) the two di-sulfide bonds may be concurrently formed by 
the oxidation reaction, (2) the oxidation reaction may be conducted with 
the protective groups of M.sup.1 and Cys.sup.15 to form the Cys.sup.3 
-Cys.sup.11 bond, and then the protective groups may be eliminated, 
followed by further oxidation to form the M.sup.1 -Cys.sup.15 bond, or (3) 
the oxidation reaction may be conducted with the protective groups of 
Cys.sup.3 and Cys.sup.11 to form the M.sup.1 -Cys.sup.15 bond, and then 
the protective groups may be eliminated, followed by further oxidation to 
form the Cys.sup.3 -Cys.sup.11 bond. 
Protection of the functional groups which should not affect the reaction of 
the raw materials, the protective groups, elimination of the protective 
groups, and activation of the functional groups related to the reaction 
can also be suitably selected from groups or methods known in the art. 
Examples of the protective groups for the amino group of the raw materials 
include carbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl, 
isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 
2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, 
phthalyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl and 
9-fluorenylmethyloxycarbonyl. The protective groups for the carboxyl group 
include, for example, alkyl esters (such as esters of methyl, ethyl, 
propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl 
and 2-adamantyl), benzyl esters, 4-nitrobenzyl esters, 4-methoxybenzyl 
esters, 4-chlorobenzyl esters, benzhydryl esters, phenacyl esters, 
carbobenzoxyhydrazide, t-butyloxycarbonylhydrazide and tritylhydrazide. 
Examples of the protective groups for the thiol group of cysteine include 
4-methoxybenzyl, 4-methylbenzyl, benzyl, t-butyl, adamantyl, trityl, 
acetamidomethyl, carbomethoxysulfenyl, 3-nitro-2-pyridinesulfenyl and 
trimethylacetamidomethyl. 
The hydroxyl group of serine can be protected, for example, by 
esterification or etherification. Examples of groups suitable for this 
esterification include lower alkanoyl groups such as acetyl, aroyl groups 
such as benzoyl, and carbonic acid-derived groups such as 
benzyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable for 
the etherification include benzyl, tetrahydropyranyl and t-butyl. However, 
the hydroxyl group of serine is not always required to be protected. 
Examples of the protective groups for the phenolic hydroxyl group of 
tyrosine include benzyl, 2,6-dichlorobenzyl, 2-nitrobenzyl, 
2-bromobenzyloxycarbonyl and t-butyl. However, the phenolic hydroxyl group 
of tyrosine is not always required to be protected. 
Methionine may be used in the form of sulfoxides. 
The protective groups for the imidazole ring of histidine include 
p-toluenesulfonyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, 
2,4-dinitrophenyl, benzyloxymethyl, t-butoxymethyl, t-butoxycarbonyl, 
trityl and 9-fluorenylmethyloxycarbonyl. However, the imidazole ring is 
not always required to be protected. 
The protective groups for the indole ring of tryptophan include formyl, 
2,4,6-trimethylbenzensulfonyl, 2,4,6-trimethoxybenzenesulfonyl, 
4-methoxy-2,3,6-trimethylbenzenesulfonyl, 
.beta.,.beta.,.beta.-trichloroethyloxycarbonyl and 
diphenylphosphinothioyl. However, the indole ring is not always required 
to be protected. 
Examples of the reactive carboxyl groups of the raw materials include the 
corresponding acid anhydrides, azide and active esters (esters of alcohols 
such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, 
cyanomethyl alcohol, p-nitrophenol, 
N-hydroxy-5-norbornene-2,3-dicarboxyimide, N-hydroxysuccinimide, 
N-hydroxyphthalimide and N-hydroxybenzotriazole. Examples of the activated 
amino acid groups of the raw materials include the corresponding 
phosphoric acid amides. 
Condensation reaction can be conducted in the presence of a solvent(s). The 
solvent(s) can be appropriately selected from the solvents commonly used 
in peptide condensation reactions. Examples of the solvents include 
anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, 
chloroform, dioxane, dichloromethane, tetrahydrofuran, acetonitrile, ethyl 
acetate, N-methylpyrrolidone and appropriate mixtures thereof. 
The reaction temperature is appropriately selected from the temperature 
range commonly used in peptide bond-forming reactions, usually from the 
range of about -20.degree. to about 30.degree. C. 
After protection is accomplished, the protected peptide or the protected 
peptide resin thus obtained is subjected to protective group-eliminating 
reaction. Although this reaction varies depending on the kind of 
protective group, it is in any event industrially advantageous to 
eliminate all protective groups in one step without affecting the peptide 
bonds. As to the cysteine-containing peptides, it is more advantageous 
from the viewpoint of ease of purification to eliminate the protective 
groups in two steps, namely, to eliminate the protective groups other than 
the protective groups for the thiol group first, followed by elimination 
of the protective groups for the thiol group. The protective groups for 
the thiol group used in such cases include acetamidomethyl and 
trimethyl-acetamidomethyl. 
As described above, in the oxidation reaction of the final stage, the 
peptide represented by formula (II) or (II') from which all of the 
protective groups are eliminated may be oxidized in one step to produce 
the peptide represented by formula (I) or (I'). Alternatively, the peptide 
represented by formula (II) or (II') where only two mercapto group are 
protected, is subjected to the first oxidation, and then the protective 
groups may be eliminated, followed by the second oxidation to produce the 
peptide represented by formula (I) or (I'). In the latter case, the 
oxidative deprotecting reaction is also usable, in which the elimination 
of the protective groups and the oxidation of the resulting thiol groups 
may be conducted in a single reaction. Further, since Trp is easy to be 
oxidized as described above, the above-mentioned oxidation reaction may 
also be conducted before the protective groups for Trp in the molecule are 
eliminated. Thereafter, the protective groups for Trp are eliminated. 
Methods for eliminating the protective groups include, for example, 
reduction with sodium in liquid ammonia, in addition to acid treatment 
with anhydrous hydrogen fluoride, methanesulfonic acid, 
trifluoromethanesulfonic acid, trifluoroacetic acid or mixtures thereof. 
The protective group-eliminating reaction by the above-mentioned acid 
treatment is generally conducted at a proper temperature between about 
-20.degree. and about 40.degree. C. In the acid treatment, it is effective 
to add a cation trapping agent such as anisole, phenol, thioanisole, 
m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol or 
1,2-ethanedithiol. For the protective groups for the thiol group which are 
stable to the acid treatment, acetamidomethyl and 
3-nitro-2-pyridinesulfenyl groups are available, and the former can be 
eliminated with iodine or mercury acetate, and the latter can be 
eliminated with mercaptoethanol. The 2,4-dinitrophenyl group used as the 
protective group for the imidazole ring of histidine is eliminated by 
thiophenol treatment. The formyl group used as the protective group for 
the indole ring of tryptophan may be eliminated by either (i) alkali 
treatment using dilute sodium hydroxide, dilute ammonia or the like, or 
(ii) the above-mentioned elimination by the acid treatment in the presence 
of 1,2-ethanedithiol, 1,4-butanedithiol or the like. 
When the peptide obtained by eliminating the protective groups of the 
protected peptide in this manner is the thiol peptide represented by 
formula (II) or (II'), the thiol peptide is subjected to oxidation. 
Preferred oxidation methods include the methods of oxidizing the thiol 
peptide in a solvent such as water with air, potassium ferricyanide, 
iodine, diiodoethane or the like. It is desirable that the above-mentioned 
oxidation reaction be generally conducted at a high dilution, at a proper 
temperature of about 0.degree. to about 40.degree. C., at a pH of about 6 
to about 8.5. 
After completion of the reaction, the peptide represented by formula (I) or 
(I') thus obtained is collected by conventional separation methods of 
peptide such as extraction, distribution, reprecipitation, 
recrystallization, column chromatography and high performance liquid 
chromatography. 
The peptide of the present invention represented by formula (I) or (I') may 
also be obtained by methods known in the art as salts such as the sodium 
salt, the potassium salt, the calcium salt and the magnesium salt, or as 
acid addition salts, particularly pharmaceutically acceptable acid 
addition salts. Examples thereof include salts of inorganic acids (such as 
hydrochloric acid, sulfuric acid and phosphoric acid) or organic acids 
(such as acetic acid, propionic acid, citric acid, tartaric acid, malic 
acid, oxalic acid and methanesulfonic acid). 
The peptides of the present invention or the pharmaceutically acceptable 
salts thereof bind to endothelin receptors of warm-blooded animals as 
shown in the experimental examples described below, but do not exhibit 
endothelin-like constrictor activity. Therefore, they function as 
endothelin antagonists. To bring about an anti-endothelin activity in 
warm-blooded animals, an effective amount of the peptides or the 
pharmaceutically acceptable salts thereof is administered to the 
warm-blooded animals. 
The novel peptides of the present invention in which any amino acids are 
substituted at the 1-, 2-, 4-, 5-, 6-, 7-, 9-, 14-, 17- 18- 19- and 21 
positions of endothelin, particularly at the 19-position, have the 
remarkable effect of suppressing the vasopressor activity of endothelin as 
the endothelin antagonists. Thus, the novel peptides of the present 
invention or the salts thereof are the endothelin antagonists having 
vasodilator activity, so that they can be used as agents for improving 
circulatory functions or therapeutic agents for cardiac infarction, acute 
renal insufficiency, asthma and the like. 
The novel peptides of the present invention, when used as the 
above-mentioned therapeutic drugs, can be safely administered orally or 
parenterally in the form of powders, granules, tablets, capsules, 
injections, suppositories, ointments or sustained release preparations, 
alone or in combination with pharmaceutically acceptable carriers, 
excipients or diluents. The peptides of the present invention are 
typically administered parenterally, for example, by intravenous or 
subcutaneous injection, intraventricular or intraspinal administration, 
nasotracheal administration or intrarectal administration. In some cases, 
however, they are administered orally. 
The peptides of the present invention are stable substances, and therefore, 
can be stored as physiological saline solutions. It is also possible to 
lyophilize the peptides, store them in ampules with mannitol or sorbitol, 
and dissolve them in a suitable carrier. The peptides of the present 
invention can be given in their free forms, or in the form of alkali 
addition salts or acid addition salts thereof. Both of the free peptides 
represented by formula (I) and the alkali addition salts or the acid 
addition salts thereof are generally given in a proper dose within the 
range of 1 ng to 10 mg of free peptid per kg of weight. 
More specifically, the dosage varies depending on the type of disease to be 
treated, the symptom of the disease, the object to which the drugs are 
given and the route of administration. For example, when given by 
injection to adult patients of hypertension, it is advantageous that the 
active ingredients (the compounds represented by formula (I)) are normally 
given in one dose of about 1 ng to 10 mg/kg of weight about once to 3 
times a day. Drip infusion is also effective. In this case, the total 
dosage is the same as with injection. 
The peptides of the present invention or the pharmaceutically acceptable 
salts thereof are used as a therapeutic agent such as an anti-endothelin 
agent. In preparing the therapeutic agent, they are carefully purified so 
as to contain no bacteria and no pyrogens. 
The present invention will be described in more detail with the following 
Examples, in which all amino acid residues other than glycine take the 
L-form unless otherwise specified. Table 1 shows the amino acid sequences 
of endothelin-1, endothelin-2, endothelin-3, mouse endothelin (MET) and 
novel peptides obtained in Examples of the present invention, compared to 
one another. 
TABLE 1 
__________________________________________________________________________ 
Known 
ET-1 
##STR7## 
ET-2 
##STR8## 
ET-3 
##STR9## 
MET 
##STR10## 
Example No. 
##STR11## 
2 
##STR12## 
3 
##STR13## 
4 
##STR14## 
5 
##STR15## 
6 
##STR16## 
7 
##STR17## 
8 
##STR18## 
9 
##STR19## 
10 
##STR20## 
11 
##STR21## 
12 
##STR22## 
13 
##STR23## 
14 
##STR24## 
15 
##STR25## 
16 
##STR26## 
17 
##STR27## 
18 
##STR28## 
__________________________________________________________________________ 
In all of the above peptides, Cys.sup.1 -Cys.sup.15 (or Mpr.sup.1 
-Cys.sup.15) and Cys.sup.3 -Cys.sup.11 form S--S bonds. 
EXAMPLE 1 
Production of [Thr.sup.18, Leu.sup.19 ]-ET-1 
Using a Boc-Trp(For)-OCH.sub.2 -Pam resin (0.5 mmole) as a starting 
material, and Boc-amino acid derivative cartridges (2.0 mmoles) (Applied 
Biosystems), the Boc groups were eliminated with trifluoroacetic acid, and 
then, a peptide chain was successively extended from the C-terminal by the 
HOBt active ester method. Boc-Asp(OcHex) and Boc-Glu(OcHex) were used 
after the powders manufactured by Peptide Institute Inc. were enclosed in 
cartridges. In this manner, the protected peptide resin represented by the 
following formula was obtained: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
This peptide resin was suspended in 10 ml of DMF, and 1.0 ml of thiophenol 
was added thereto. The Dnp group, a protective group for the imidazole 
ring of His, was eliminated by stirring the mixture at room temperature 
for 2 hours, and the Boc group was further eliminated by treating with 50% 
TFA/dichloromethane containing 0.1% indole at room temperature for 20 
minutes. Then, 500 mg of the peptide resin thus obtained was treated with 
5 ml of anhydrous hydrogen fluoride in the presence of 500 mg of p-cresol 
and 0.75 ml of 1,4-butanedithiol at 0.degree. C. for 1 hour to remove all 
of the protective groups and to release the peptide from the resin. 
Hydrogen fluoride was removed under reduced pressure, and ethyl ether was 
added to the residue to deposit a precipitate. The precipitate was 
separated by filtration, and 30 ml of TFA was added thereto to dissolve 
the peptide. The resin was removed by filtration, and the filtrate was 
concentrated. Ethyl ether was added to the residue to deposit a 
precipitate. The precipitate was separated by filtration, and dried under 
reduced pressure. The resulting product was dissolved in 500 ml of 0.1M 
ammonium acetate/water-nBuOH-EtOH (2:1:1 v/v) (pH 8.0-8.5), and oxidized 
with air by stirring the solution at room temperature for 15 hours. Then, 
acetic acid was added thereto to adjust the solution to pH 5.0, and the 
solvent was removed by distillation under reduced pressure, followed by 
lyophilization of the residue. The lyophilized product was dissolved in 20 
ml of 60% acetic acid. The resulting solution was subjected to a Sephadex 
G-50 column (5 cm.times.108 cm) and eluted with 60% acetic acid. The 
desired fractions were collected and lyophilized. Finally, the fractions 
were purified by high performance liquid chromatography using a 
YMC-D-ODS-5 column (2 cm.times.25 cm, YMC CO. Ltd.) to obtain the desired 
product. 
Anal. for amino acids (hydrolysis at 110.degree. C. for 24 hours; numerals 
in parentheses indicate theoretical values): Asp 1.00(1); Thr 0.93(1); Ser 
2.56(3); Glu 1.06(1); Cyt 1.85(2); Val 0.99(1); Met 0.99(1); Ile 0.94(1); 
Leu 3.08(3); Tyr 0.97(1); Phe 1.02(1); Lys 1.00(1); His 1.19(1) 
LSIMS (M+H.sup.+)=2477 (theoretical value=2477) 
EXAMPLE 2 
Production of [Thr.sup.18, Cha.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-Cha-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.94(1); Ser 2.55(3); Glu 1.05(1); 
Cyt 1.69(2); Val 0.97(1); Met 1.01(1); Ile 0.94(1); Leu 2.04(2); Tyr 
0.92(1); Phe 1.01(1); Lys 1.00(1); His 1.19(1) 
LSIMS (M+H.sup.+)=2517 (theoretical value=2517) 
EXAMPLE 3 
Production of [Thr.sup.18, Phe.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-Phe-Ile-Trp(For)-O-CH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.88(1); Ser 2.45(3); Glu 1.03(1); 
Cyt 1.48(2); Val 0.88(1); Met 1.00(1); Ile 0.85(1); Leu 1.93(2); Tyr 
0.87(1); Phe 1.81(2); Lys 1.01(1); His 0.88(1) 
LSIMS (M+H.sup.+)=2511 (theoretical value=2511) 
EXAMPLE 4 
Production of [Thr.sup.18, .gamma.Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-.gamma.Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.94(1); Ser 2.51(3); Glu 1.05(1); 
Cyt 1.69(2); Val 0.98(1); Met 1.00(1); Ile 0.92(1); Leu 2.07(2); Tyr 
1.04(1); Phe 0.99(1); Lys 1.01(1); His 1.00(1) 
LSIMS (M+H.sup.+)=2491 (theoretical value=2491) 
.gamma.Leu=.gamma.-Methyl-L-leucine 
EXAMPLE 5 
Production of [Thr.sup.18, Asn.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-Asn-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 2.00(2); Thr 0.96(1); Ser 2.50(3); Glu 1.07(1); 
Cyt 0.75(2); Val 0.91(1); Met 1.03(1); Ile 0.91(1); Leu 2.11(2); Tyr 
0.92(1); Phe 1.04(1); Lys 1.02(1); His 0.99(1) 
LSIMS (M+H.sup.+)=2478 (theoretical value=2478) 
EXAMPLE 6 
Production of [Ser.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Ser(Bz 
l)-Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Ser 3.39(4); Glu 1.06(1); Cyt 1.58(2); 
Val 0.90(1); Met 0.98(1); Ile 0.87(1); Leu 3.05(3); Tyr 0.87(1); Phe 
0.98(1); Lys 0.99(1); His 0.93(1) 
LSIMS (M+H.sup.+)=2463 (theoretical value=2463) 
EXAMPLE 7 
Production of [Asn.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Asn-Le 
u-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 2.00(2); Set 2.40(3); Glu 1.04(1); Cyt 0.76(2); 
Val 0.85(1); Met 1.02(1); Ile 0.85(1); Leu 3.06(3); Tyr 0.85(1); Phe 
1.00(1); Lys 1.00(1); His 0.93(1) 
LSIMS (M+H.sup.+)=2490 (theoretical value=2490) 
EXAMPLE 8 
Production of [Gly.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Gly-Le 
u-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Ser 2.51(3); Glu 1.05(1); Gly 0.99(1); 
Cyt 1.48(2); Val 0.89(1); Met 0.98(1); Ile 0.84(1); Leu 3.01(3); Tyr 
0.86(1); Phe 0.97(1); Lys 0.99(1); His 0.93(1) 
LSIMS (M+H.sup.+)=2433 (theoretical value=2433) 
EXAMPLE 9 
Production of [Thr.sup.18, Leu.sup.19 ]-ET-3 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Thr(Bzl)-Cys(MeBzl)-Phe-Thr(Bzl)-Tyr(BrZ)-Lys(ClZ)-Asp(OcHex 
)-Lys(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Tyr(BrZ)-Cys(MeBzl)-His(Dnp)- 
Leu-Thr(Bzl)-Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 2.71(3); Glu 1.10(1); Cyt 0.77(2); 
Val 0.94(1); Ile 0.91(1); Leu 2.01(2); Tyr 2.84(3); Phe 0.99(1); Lys 
1.96(2); His 0.95(1) 
LSIMS (M+H.sup.+)=2628 (theoretical value=2628) 
EXAMPLE 10 
Production of [Ala.sup.9, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Ala 
-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bzl)-Le 
u-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.91(1); Ser 2.46(3); Glu 1.03(1); 
Ala 0.99(1); Cyt 0.75(2); Val 0.88(1); Met 1.00(1); Ile 0.88(1); Leu 
2.98(3); Tyr 0.86(1); Phe 0.96(1); His 0.93(1) 
LSIMS (M+H.sup.+)=2420 (theoretical value=2420) 
EXAMPLE 11 
Production of [Mpr.sup.1, Thr.sup.18, Leu.sup..multidot. ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Mpr(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys(ClZ 
)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bzl)-L 
eu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.94(1); Ser 2.53(3); Glu 1.07(1); 
Cyt 0.66(1); Val 0.94(1); Met 0.98(1); Ile 0.92(1); Leu 3.03(3); Tyr 
0.88(1); Phe 0.97(1); Lys 0.98(1); His 0.94(1) 
LSIMS (M+H.sup.+)=2462 (theoretical value=2462) 
EXAMPLE 12 
Production of [Ala.sup.2, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ala-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys(ClZ) 
-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bzl)-Le 
u-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.95(1); Ser 1.74(2); Glu 1.06(1); 
Ala 0.96(1); Cyt 0.74(2); Val 0.95(1); Met 0.99(1); Ile 0.94(1); Leu 
3.08(3); Tyr 0.89(1); Phe 0.98(1); Lys 0.99(1); His 0.96(1) 
LSIMS (M+H.sup.+)=2461 (theoretical value=2461) 
EXAMPLE 13 
Production of [Ala.sup.4, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ala-Ser(Bzl)-Leu-Met-Asp(OcHex)-Lys(ClZ) 
-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bzl)-Le 
u-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.95(1); Ser 1.67(2); Glu 1.06(1); 
Ala 0.96(1); Cyt 0.82(2); Val 0.94(1); Met 0.99(1); Ile 0.92(1); Leu 
3.06(3); Tyr 0.88(1); Phe 0.98(1); Lys 0.99(1); His 0.95(1) 
LSIMS (M+H.sup.+)=2461 (theoretical value=2461) 
EXAMPLE 14 
Production of [Ala.sup.5, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-CYs(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ala-Leu-Met-Asp(OcHex)-Lys(ClZ) 
-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bzl)-Le 
u-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.94(1); Ser 1.68(2); Glu 1.05(1); 
Ala 0.98(1); Cyt 0.92(2); Val 0.92(1); Met 0.92(1); Ile 0.90(1); Leu 
2.96(3); Tyr 0.91(1); Phe 0.98(1); Lys 0.98(1); His 0.95(1) 
LSIMS (M+H.sup.+)=2461 (theoretical value=2461) 
EXAMPLE 15 
Production of [Ala.sup.6, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Ala-Met-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val- 
Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bzl)-Leu-Ile-Trp(For)-OCH.sub.2 
-Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.89(1); Ser 2.45(3); Glu 1.04(1); 
Ala 0.98(1); Cyt 0.88(2); Val 0.86(1); Met 0.96(1); Ile 0.85(1); Leu 
1.85(2); Tyr 0.86(1); Phe 0.93(1); Lys 0.98(1); His 0.90(1) 
LSIMS (M+H.sup.+)=2435 (theoretical value=2435) 
EXAMPLE 16 
Production of [Ala.sup.7, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Ala-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.94(1); Ser 2.55(3); Glu 1.06(1); 
Ala 0.97(1); Cyt 0.87(2); Val 0.93(1); Ile 0.92(1); Leu 3.03(3); Tyr 
0.90(1); Phe 0.98(1); Lys 0.98(1); His 0.95(1) 
LSIMS (M+H.sup.+)=2417 (theoretical value=2417) 
EXAMPLE 17 
Production of [Nle.sup.7, Thr.sup.18, Leu.sup.19 ]-ET-1 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Leu-Nle-Asp(OcHex)-Lys 
(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-Thr(Bz 
l)-Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
Anal. for amino acids: Asp 1.00(1); Thr 0.95(1); Ser 2.50(3); Glu 1.07(1); 
Cyt 0.78(2); Val 0.95(1); Ile 0.93(1); Leu 3.07(3); Tyr+Nle 1.97(2); Phe 
0.99(1); Lys 0.99(1); His 0.96(1) 
LSIMS (M+H.sup.+)=2459 (theoretical value=2459) 
EXAMPLE 18 
Production of [Thr.sup.18, Leu.sup.19 ]-ET-2 
The following protected peptide resin was obtained by procedures similar to 
those of Example 1: 
Boc-Cys(MeBzl)-Ser(Bzl)-Cys(MeBzl)-Ser(Bzl)-Ser(Bzl)-Trp(For)-Leu-Asp(OcHex 
)-Lys(ClZ)-Glu(OcHex)-Cys(MeBzl)-Val-Tyr(BrZ)-Phe-Cys(MeBzl)-His(Dnp)-Leu-T 
hr(Bzl)-Leu-Ile-Trp(For)-OCH.sub.2 -Pam resin 
The resulting peptide resin was further deprotected, oxidized and purified 
as with Example 1 to obtain the desired product. 
LSIMS (M+H.sup.+)=2532 (theoretical value=2532) 
Experimental Example 
(1) Assay of Constrictor Suppressing Activity on Porcine Coronary Smooth 
Muscles 
Spiral strips 2 mm.times.15 mm prepared from the coronary right ramus 
circumflexus from which the adventitial connective tissues and the 
endothelial cells were removed were set to 4 ml organ baths. The tension 
of each strip was detected by a force displacement transducer UL-10GR 
(Minebea), and recorded by a polygraph (NEC Sanei). The organ baths were 
maintained at 37.degree. C., and filled with a Krebs-Henseleit solution 
(composition: 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl.sub.2, 1.2 mM KH.sub.2 
PO.sub.4, 25.0 mM NaHCO.sub.3, 1.2 mM MgSO.sub.4, 10.0 mM glucose) gassed 
with 95% O.sub.2 and 5% CO.sub.2. 
A tension of 1.25 to 1.5 g was applied to each of the strips, followed by 
equilibration for 1.5 hours. 60 mM KCl was repeatedly applied thereto at 
intervals of 30 minutes until the constriction response became constant. 
After additional equilibration for 1.5 hours, a sample for assay was given 
thereto. The constriction of the strips was normalized by the constriction 
response of the individual strips to 60 mM KCl and statistically 
processed. 
The suppressing activity was determined as a pA.sub.2 value by giving 
endothelin-1 cumulatively about 15 minutes after the compound having a 
predetermined concentration was given, and comparing the constriction 
thereof with that of a control sample in which only endothelin-1 was 
given. Results thereof are shown in Table 3. 
The novel peptides of the present invention represented by formula (I) and 
the salts thereof showed the activity of suppressing the constriction due 
to endothelin in porcine coronary smooth muscles. Such a case has not been 
reported yet. Hence, the peptides of the present invention represented by 
formula (I) or the salts thereof can be used for the treatment of 
hypertension, cardiac infarction, acute renal insufficiency or asthma of 
mammals such as mice, rats, rabbits, dogs, cats, pigs and humans. 
(2) As to the antagonistic property of the peptides of the present 
invention to endothelin, the affinity for an endothelin receptor and the 
constrictor activity on porcine coronary smooth muscles (according to the 
method described in (1) described above) were assayed. Results thereof are 
shown in Table 2. The affinity for the receptor was assayed by the 
following method. 
Assay of Affinity for Receptor 
A membrane fraction prepared from the porcine heart was diluted to 0.15 
mg/ml by using a buffer solution for assay, and 100 .mu.l of the resulting 
suspension of the membrane fraction was poured into each assay tube to use 
for assay. To this suspension of the membrane fraction was added 2 .mu.l 
of 5 nM .sup.125 I labeled endothelin-1 solution. Further, 3 .mu.l of a 
test peptide solution was added thereto, followed by incubation at a 
temperature of 25.degree. C. for 1 hour. Then, the resulting suspension 
was diluted with 900 .mu.l of the buffer solution for assay cooled with 
ice, and thereafter separated into a supernatant and a precipitate by 
centrifugation at 12,000.times.g for 10 minutes. Cell membranes and an 
endothelin receptor embedded therein were contained in the precipitate, 
and radioactive iodine-labeled endothelin combined with the receptor was 
also recovered in the precipitate. Accordingly, the amount of radioactive 
iodine-labeled endothelin combined with the endothelin receptor was 
determined by measuring the amount of radioactive iodine contained in the 
precipitate with a gamma-ray counter. As shown in Table 2, the peptides of 
the present invention are high in the affinity for the endothelin receptor 
and not high in the maximum constriction. The results reveals that the 
peptides of the the present invention have strong antagonistic activity. 
TABLE 2 
______________________________________ 
Receptor 
binding Constrictor 
Maximum 
activity.sup.1) 
activity.sup.2) 
constriction 
(specific (specific 
(% 60 mM 
Example 
Compound activity) activity) 
KCl) 
No. ET-1 100.sup.3) 
100.sup.4) 
120 
______________________________________ 
1 [Thr.sup.18, Leu.sup.19 ]- 
40 &lt;0.1 4 
ET-1 
2 [Thr.sup.18, Cha.sup.19 ]- 
23 &lt;0.1 2 
ET-1 
3 [Thr.sup.18, Phe.sup.19 ]- 
9.0 &lt;0.1 4 
ET-1 
4 [Thr.sup.18, .gamma.Leu.sup.19 ]- 
23 &lt;0.1 0 
ET-1 
5 [Thr.sup.18, Asn.sup.19 ]- 
1.6 &lt;0.1 9 
ET-1 
6 [Ser.sup.18, Leu.sup.19 ]- 
15 &lt;0.1 1 
ET-1 
7 [Asn.sup.18, Leu.sup.19 ]- 
12 &lt;0.1 4 
ET-1 
8 [Gly.sup.18, Leu.sup.19 ]- 
9.5 &lt;0.1 14 
ET-1 
9 [Thr.sup.18, Leu.sup.19 ]- 
4.0 &lt;0.1 12 
ET-3 
10 [Ala.sup.9, Thr.sup.18, 
16 &lt;0.1 4 
Leu.sup. 19 ]-ET-1 
11 [Mpr.sup.1, Thr.sup.18, 
16 &lt;0.1 1 
Leu.sup.19 ]-ET-1 
12 [Ala.sup.2, Thr.sup.18, 
32 &lt;0.1 5 
Leu.sup.19 ]-ET-1 
13 [Ala.sup.4, Thr.sup.18, 
57 &lt;0.1 6 
Leu.sup.19 ]-ET-1 
14 [Ala.sup.5, Thr.sup.18, 
8.6 &lt;0.1 6 
Leu.sup.19 ]-ET-1 
15 [Ala.sup.6, Thr.sup.18, 
11 &lt;0.1 7 
Leu.sup.19 ]-ET-1 
16 [Ala.sup.7, Thr.sup.18, 
62 &lt;0.1 9 
Leu.sup.19 ]-ET-1 
17 [Nle.sup.7, Thr.sup.18, 
27 &lt;0.1 9 
Leu.sup.19 ]-ET-1 
______________________________________ 
.sup.1) Porcine myocardial membrane fraction 
.sup.2) Porcine coronary artery 
.sup.3) IC.sub.50 = 2.0 .times. 10.sup.-9 M, IC.sub.50 represents the 
concentration of a sample required to prevent 50% of the binding of 
I.sup.125 -ET1 to the porcine myocardial membrane fraction. 
.sup.4) EC.sub.50 (% KCl) = 1.6 .times. 10.sup.-9 M, EC.sub.50 (% KCl) 
represents the concentration of a sample which induces 50% of the 
constriction of the porcine coronary artery due to 60 mM KCl. 
(3) The antagonistic activity on the constriction of porcine coronary 
smooth muscles are shown in Table 3 below. 
TABLE 3 
______________________________________ 
Antagonistic activity on the construction of porcine 
coronary smooth muscles 
Example Relative 
No. Compound pA.sub.2 potency 
______________________________________ 
1 [Thr.sup.18, Leu.sup.19 ]-ET-1 
7.7 100 
2 [Thr.sup.18, Cha.sup.19 ]-ET-1 
7.7 100 
3 [Thr.sup.18, Phe.sup.19 ]-ET-1 
7.2 32 
4 [Thr.sup.18, .gamma.Leu.sup.19 ]-ET-1 
7.4 50 
6 [Ser.sup.18, Leu.sup.19 ]-ET-1 
7.5 63 
8 [Gly.sup.18, Leu.sup.19 ]-ET-1 
6.7 10 
10 [Ala.sup.9, Thr.sup.18, Leu.sup.19 ]-ET-1 
5.9 1.6 
11 [Mpr.sup.1, Thr.sup.18, Leu.sup.19 ]-ET-1 
6.5 6 
12 [Ala.sup.2, Thr.sup.18, Leu.sup.19 ]-ET-1 
6.7 10 
13 [Ala.sup.4, Thr.sup.18, Leu.sup.19 ]-ET-1 
6.9 16 
14 [Ala.sup.5, Thr.sup.18, Leu.sup.19 ]-ET-1 
5.5 0.6 
15 [Ala.sup.6, Thr.sup.18, Leu.sup.19 ]-ET-1 
6.5 6 
16 [Ala.sup.7, Thr.sup.18, Leu.sup.19 ]-ET-1 
7.2 32 
______________________________________ 
pA.sub.2 is a negative logarithm value of a molar concentration of a 
competitive antagonist necessary for shifting in parallel a dose response 
curve for an active drug (for example, ET-1) alone to the high dose side 
by a factor of 2. The higher value shows the stronger antagonistic 
activity. 
As described above, the peptides of the present invention represented by 
formula (1) and the salts thereof have the antagonistic property to 
endothelin, so that they can be used as agents for improving circulatory 
functions, vasodilators or therapeutic agents for asthma. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 25 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
XaaXaaCysXaaXaaXaaXaaAspXaaGluCysValTyrXaaCysHis 
151015 
XaaXaaXaaIleXaa 
20 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
XaaXaaCysXaaXaaXaaXaaAspXaa GluCysValTyrXaaCysHis 
151015 
XaaXaaXaaIleXaa 
20 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CysXaaCysXaaXaaXaaXaaAspLysGluCysValTy rXaaCysHis 
151015 
LeuAspIleIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuAspIleIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii ) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CysSerCysSerSerTrpLeuAspLysGluCysValTyrPheCysHis 
1 51015 
LeuAspIleIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix ) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CysThrCysPheThrTyrLysAspLysGluCysValTyrTyrCysHis 
15 1015 
LeuAspIleIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
( A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CysSerCysAsnSerTrpLeuAspLysGluCysValTyrPheCysHis 
1510 15 
LeuAspIleIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
1510 15 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
( ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
L euThrXaaIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuThrPheIle Trp 
20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(ix) FEATURE: 
(A) NAME/KEY: Modified site 
(B) LOCATION: 19 
(C) OTHER INFORMATION: Leu 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
15 1015 
LeuThrXaaIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
1510 15 
LeuThrAsnIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuSerLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuAsnLeu IleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CysSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuGlyLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CysThrCysPheThrTyrLysAspLysGluCysValTyrTyrCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CysSerCysSerSerLeuMetAspAlaGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(ix) FEATURE: 
(A) NAME/KEY: Modified site 
(B) LOCATION: 1 
(C) OTHER INFORMATION: Mpr 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
XaaSerCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
Leu ThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
( A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
CysAlaCysSerSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
CysSerCysAlaSerLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(x i) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
CysSerCysSerAlaLeuMetAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CysSerCysSerSerAlaMetAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
CysSerCysSer SerLeuAlaAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A ) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(C) OTHER INFORMATION: Nle 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CysSerCysSerSerLeuXaaAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1,15 
(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 3,11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CysSerCysSerSerTrpLeuAspLysGluCysValTyrPheCysHis 
151015 
LeuThrLeuIleTrp 
20