JPH05194592A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To obtain the subject derivative, having remarkable antagonistic action on endothelin receptors and useful as a therapeutic agent, etc., for hypertension and cardio-cerebral circulatory diseases. CONSTITUTION:The objective derivative expressed by formula I [A is D- acidic-alpha-amino acid residue; B is D-neutral-alpha-amino acid residue; C is L-alpha-amino acid residue; D is D-alpha-amino acid residue having aromatic ring group; R1 is H or (substituted)amino; R2 is carboxyl which may be amidated or esterified; m and n are 1 or 2] and its salt, e.g. a peptide derivative expressed by formula II. This derivative expressed by formula I is obtained by oxidizing, e.g. a peptide expressed by formula III with air, potassium ferricyanide, etc., in a solvent such as water at 0-40 deg.C and pH6-8.5.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide derivative having an endothelin receptor antagonistic activity which is useful as a medicine, such as a therapeutic agent for hypertension, a therapeutic agent for cardio-cerebral circulation disease, a therapeutic agent for renal disease, a therapeutic agent for asthma and the like. Regarding its use.

[0002]

BACKGROUND OF THE INVENTION Endothelin (ET) is a vasoconstrictive peptide consisting of 21 amino acids isolated from the culture supernatant of porcine aortic endothelial cells by Yanagisawa et al. In 1988 [Yanagisawa et al., Nature ( Nature), 332, 411-412
page]. Subsequently, studies on the gene encoding endothelin revealed that there are peptides with a structure similar to endothelin, and endothelin-1 (ET
-1), endothelin-2 (ET-2), endothelin-3 (ET-
It is named 3). The structures of these endothelins are as follows (the constituent amino acids in ET-1, 2, and 3 are all L-forms) [Inoue et al., Proc.
The National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 86, 2863-2867
page].

[0003]

[Chemical 2]

Since the above endothelins are present in the body and have a vasoconstrictor action, they are expected to be endogenous factors involved in the regulation of the circulatory system, and hypertension, heart / cerebral circulation diseases (for example, myocardium). Infarction), renal disease (eg acute renal failure)
Has been estimated to be. It also has a bronchial smooth muscle contraction effect, and its relationship with asthma has been estimated.

[0005]

If an antagonist for the above-mentioned endothelin receptor is obtained, it will not only help elucidate the mechanism of action of the endothelin, but also be an effective therapeutic drug for the above-mentioned diseases. Is considered to be large. Until now, a patent application has been made for cyclic pentapeptides having an endothelin receptor antagonistic action obtained from fermentation products (Japanese Patent Application No. 2-413828 and Japanese Patent Application No. 3-126160).
However, it is an object of the present invention to provide a novel peptide having an effect similar to or higher than that.

[0006]

[Means for Solving the Problems] The inventors of the present invention have made earnest studies using the action of suppressing the strong vascular smooth muscle contractile activity of endothelins as an index, and have developed a novel peptide derivative having a remarkable endothelin receptor antagonistic action. The present invention has been completed and further studies have been made to complete the present invention.

That is, the present invention is as follows: Expression [I]

[0008]

[Chemical 3]

[Wherein A is a D-acidic-α-amino acid residue,
B is a D-neutral-α-amino acid residue, C is an L-α-amino acid residue, D is an D-α-amino acid residue having an aromatic ring group, R ₁
Is a hydrogen atom or an optionally substituted amino group, R ₂
Is a carboxyl group which may be amidated or esterified, m and n each represent 1 or 2, and a peptide derivative or a salt thereof, and The present invention relates to an endothelin receptor antagonist comprising a peptide derivative represented by the formula [I] or a pharmacologically acceptable salt thereof.

The above formula [I] indicates that a ring is formed as a whole molecule by the presence of five amide bonds and one SS bond.

In the above formula [I], the amino acid which is the base of the D-acidic-α-amino acid residue represented by A is, for example, an acidic group such as a carboxyl group, a sulfonyl group or a tetrazolyl group in the side chain. Amino acids having Specific examples thereof include D-glutamic acid, D-aspartic acid, D-cysteic acid, D-homocysteic acid, D-
β- (5-tetrazolyl) alanine, D-2-amino-4- (5-
Examples thereof include tetrazolyl) butyric acid, and D-glutamic acid, D-aspartic acid and D-cysteic acid are particularly preferable.

In the above formula [I], the base amino acid for the D-neutral-α-amino acid residue represented by B is, for example, D-alanine, D-valine, D-norvaline or D-leucine. , D-isoleucine, D-alloisoleucine, D-norleucine, D-tert-leucine, D-gamma-methylleucine, D-phenylglycine, D-phenylalanine, etc.
Examples of D-α-amino acids are D-leucine, D-alloisoleucine, D-tert-leucine, D-gamma-methylleucine and D-phenylglycine.

In the above formula [I], L-α represented by C
-Examples of the amino acid that is the base of the amino acid residue include, for example, glycine, L-alanine, L-valine, L-norvaline,
L-leucine, L-isoleucine, L-tertiary leucine, L-norleucine, L-methionine, L-2-aminobutyric acid, L-serine, L-threonine, L-phenylalanine, L-
Aspartic acid, L-glutamic acid, L-asparagine, L-
Commonly known L-α-amino acids such as glutamine, L-lysine, L-tryptophan, L-arginine, L-tyrosine and L-proline are mentioned, and particularly L-leucine, L-norleucine and L-tryptophan. Is preferred.

In the above formula [I], the amino acid which is the base of the D-α-amino acid residue having an aromatic ring group represented by D is, for example, a D-α-amino acid having an aromatic ring group in its side chain. Can be given. Specific examples thereof include D-tryptophan, D-5-methyltryptophan, D-phenylalanine, D-tyrosine, D-1-naphthylalanine and D-2-naphthylalanine, and especially D-tryptophan and D- 5-methyltryptophan is preferred.

In the above formula [I], R ₁ represents a hydrogen atom or an optionally substituted amino group. Examples of the substituent of the amino group include an alkyl group and an acyl group. Here, the alkyl group is, for example, a lower alkyl group which may be substituted with an aromatic ring group. [The aromatic ring group is, for example, phenyl, naphthyl or the like, and may be further substituted with a halogen atom, a lower alkyl group or a lower alkoxy group. Lower alkyl groups are, for example, C _1-3 alkyl groups, such as methyl, ethyl, propyl, etc .; halogen atoms are, for example, fluoro, chloro, bromine, etc .; lower alkoxy groups are, for example, C _1-3 alkoxy groups, methoxy, ethoxy, And a lower alkanoyl group which may be substituted with an aromatic ring group as the acyl group [the aromatic ring group is, for example, phenyl, naphthyl, etc.,
Further, it may be substituted with a halogen atom, a lower alkyl group or a lower alkoxy group; the lower alkyl group is, for example, a C _1-3 alkyl group, such as methyl, ethyl or propyl;
A halogen atom is, for example, fluoro, chloro, bromine, etc .; a lower alkoxy group is, for example, a C _1-3 alkoxy group, methoxy, ethoxy, propoxy, etc .; a lower alkanoyl group is, for example, a C _1-4 alkanoyl group, acetyl, propionyl, butyryl. Etc.] or an optionally substituted aroyl group [the aroyl group is, for example, benzoyl, naphthoyl, etc., and may be further substituted with a halogen atom, a lower alkyl group or a lower alkoxy group; the lower alkyl group is, for example, C _1-3. An alkyl group such as methyl, ethyl, propyl, etc .; a halogen atom is, for example, fluoro,
Chlorine, bromine, etc .; a lower alkoxy group is, for example, C
_1-3 alkoxy groups, such as methoxy, ethoxy, propoxy, etc.], as well as groups formed by removing the hydroxyl groups of α-carboxyl groups of α-amino acids [α-amino acids are glycine, alanine, valine, norvaline, leucine, isoleucine,
Tertiary leucine, norleucine, methionine, 2
-Aminobutyric acid, serine, threonine, phenylalanine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, tryptophan, arginine, tyrosine, proline, and other commonly known α-amino acids, which may be D, L, or DL. ] Is given. Specific examples of the substituted amino group include N-methylamino, N-ethylamino, N, N-dimethylamino, N-benzylamino, N-parachlorobenzylamino, N-paramethylbenzylamino, N-para. methoxybenzylamino, acetamide (-NHCOCH _3), propionamide (-NHCO
C ₂ H ₅ ), phenylacetamide (-NHCOCH ₂ C ₆ H ₅ ), parameparamethylphenylacetamide (-NHCOCH ₂ C ₆ H ₄ -pC
H ₃ ), benzamide (-NHC ₆ H ₅ ), parachlorobenzamide
(-NHCOC ₆ H ₄ -p-Cl), -NHCOCH ₂ NH ₂ , -NHCOCH (CH ₃ ) NH ₂ , -N
Such _{HCOCH (CH 2 C 6 H 5} ) NH 2 and the like.

In the above formula [I], R ₂ represents a carboxyl group which may be amidated or esterified. Examples of the amidated carboxyl group include -CONR ₃ R ₄ [R ₃ and R ₄ are lower alkyl groups each optionally substituted with a hydrogen atom or an aromatic ring group; the aromatic ring group is, for example, phenyl or naphthyl. May be further substituted with a halogen atom, a lower alkyl group or a lower alkoxy group; the lower alkyl group is, for example, a C _1-3 alkyl group, methyl, ethyl, propyl, etc .; The halogen atom is, for example, fluoro, chloro, Brom, etc .; the lower alkoxy group is, for example, a C _1-3 alkoxy group, such as methoxy, ethoxy, propoxy, etc.] or —COR ₅ [R ₅ is α-amino acid α-
A group formed by removing one hydrogen atom from an amino group; α-amino acids are glycine, alanine, valine, norvaline, leucine, isoleucine, tertiary leucine, norleucine, methionine, 2-aminobutyric acid, serine, threonine, phenylalanine, aspartic acid. , Glutamic acid, asparagine, glutamine, lysine, tryptophan, arginine, tyrosine, proline, and other commonly known α-amino acids, which may be D, L, or DL]
Etc., and as the esterified carboxyl group, for example, —COOR ₆ [R ₆ is a lower alkyl group which may be substituted with an aromatic ring group; the aromatic ring group is, for example, phenyl, naphthyl, etc .; For example, it is a C _1-3 alkyl group such as methyl, ethyl, propyl, etc.]. Specific examples of the amidated carboxyl group include carbamoyl, N-methylcarbamoyl, N, N-dimethylcarbamoyl, N-benzylcarbamoyl, N-parachlorobenzylcarbamoyl, N-paramethylbenzylcarbamoyl and N-paramethoxybenzyl. Carbamoyl, -CONHCH ₂ COOH, -CONHCH (CH ₃ ) COOH, -CONHCH (CH ₂ C ₆ H
₅ ) COOH, etc. are also esterified carboxyl groups
Specific examples of (-COOR ') include methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl and the like.

In the above formula [I], m and n each represent 1 or 2. The carbon atom to which R ₁ is bonded is an asymmetric carbon except when R ₁ = H, but may be D, L or DL. On the other hand, the carbon atom to which R ₂ is bonded is also an asymmetric carbon and may be D, L or DL, but D is more preferred.

The pharmacologically acceptable salt of the peptide derivative [I] of the present invention is a metal salt such as sodium salt, potassium salt, calcium salt, magnesium salt, or an inorganic salt such as hydrochloride, sulfate or phosphate. Examples thereof include acid addition salts, acetates, propionates, citrates, tartrates, malates, oxalates and other organic acid salts.

The peptide derivative [I] of the present invention is generally produced by oxidizing the peptide [II] of the following formula, which is obtained in the final step of peptide synthesis described in detail below.

[0020]

[Chemical 4]

[The symbols in the formula are the same as above] The above peptide [II] can be produced by a conventional means for peptide synthesis. That is, either the liquid phase synthesis method or the solid phase synthesis method may be used, but the liquid phase synthesis method may be preferable in some cases. The means for synthesizing such a peptide may be according to any known method,
For example, M. Bodansky and MA Ondetti, Peptide Synthesis, Interscience, New York, 1966; FM Finn and K.
Hofmann, The Proteins, No. 2
Vol., H. Nenrath, RL Hill, Academic Press, New York, 1976; Nobuo Izumiya et al., "Basics and Experiments of Peptide Synthesis" Maruzen Co., Ltd. 1985; Haruaki Yajima, Shunpei Sakakibara et al., Biochemistry Laboratory. 1. Biochemical Society of Japan, edited by Tokyo Kagaku Dojin, 1977; Shun Kimura et al., Sequel to Biochemistry Experiment Course 2, edited by Biochemical Society of Japan, Tokyo Kagaku Doujin, 1987; JM
Solid Phases by Stewart and JD Young
Peptide Synthesis (Solid Phase Peptide Synthesi
s), Pierce Chemical Company, Illinois, 1984, etc., for example, azide method, chloride method,
Acid anhydride method, mixed acid anhydride method, DCC method, active ester method,
Examples include a method using Woodward reagent K, a carbonylimidazole method, a redox method, a DCC / HONB method, and a method using a BOP reagent.

The above-mentioned peptide [II] is a raw material having a reactive carboxyl group corresponding to one of the two fragments divided into two at any position of the peptide bond, and a reactive amino group corresponding to the other fragment. It can be produced by condensing the starting material having ## STR1 ## by a conventional means for peptide synthesis and, if the resulting condensate has a protecting group, removing the protecting group by a conventional means.

Particularly in the solid phase synthesis method, an amino acid having a functional group which should not participate in the reaction and an insoluble carrier such as Pam resin are bound through the carboxyl group of the amino acid to remove the amino protecting group. Then, condense amino acids with protected functional groups that should not be involved in this, and repeat this operation until the desired protected peptide is obtained.
Then, the protective group can be removed by a conventional means such as hydrogen fluoride treatment, trifluoromethane sulfonic acid treatment, trifluoroacetic acid treatment and the like, and at the same time, the bond with the insoluble carrier can be cleaved.

The protection of a functional group which should not be involved in the reaction of the starting material, the protection of the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction and the like can also be appropriately selected from known ones or means.

Examples of the protective group for the amino group of the raw material include benzyloxycarbonyl, tert-butyloxycarbonyl, tert-amyloxycarbonyl,
Isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, 9-fluorenyl Examples include methyloxycarbonyl. Examples of the protective group for the carboxyl group include alkyl esters (for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl,
Ester groups such as cycloheptyl, cyclooctyl, 2-adamantyl, etc.), benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, phenacyl ester, benzyloxycarbonyl hydrazide, tersha Ributyloxycarbonyl hydrazide, trityl hydrazide and the like.

Examples of the thiol-protecting group for cysteine include 4-methoxybenzyl, 4-methylbenzyl, benzyl, tert-butyl, adamantyl, trityl,
Acetamidomethyl, carbomethoxysulfenyl, 3
Examples include -nitro-2-pyridinesulfenyl and trimethylacetamidomethyl.

The hydroxyl group of serine can be protected by, for example, esterification or etherification. Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethyloxycarbonyl group.
Further, groups suitable for etherification include, for example, benzyl group, tetrahydropyranyl group, tert-butyl group and the like. However, the hydroxyl group of serine does not necessarily need to be protected.

Examples of the protective group for the phenolic hydroxyl group of tyrosine include benzyl, 2,6-dichlorobenzyl, 2-nitrobenzyl, 2-bromobenzyloxycarbonyl and tert-butyl, but they are not always protected. No need. Methionine may be protected in the form of sulfoxide.

Examples of the protecting group for imidazole of histidine include paratoluenesulfonyl, 4-methoxy-2,3,6-
Trimethylbenzenesulfonyl, 2,4-dinitrophenyl, benzyloxymethyl, tert-butoxymethyl, tert-butoxycarbonyl, trityl, 9-
Examples thereof include fluorenylmethyloxycarbonyl, but it is not always necessary to protect.

The protecting group for the indole of tryptophan includes formyl, 2,4,6-trimethylbenzenesulfonyl, 2,4,6-trimethoxybenzenesulfonyl, 4-
Methoxy-2,3,6-trimethylbenzenesulfonyl,
Examples include 2,2,2-trichloroethyloxycarbonyl and diphenylphosphinothioyl oil, but they are not necessarily protected.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitro, etc. Ester with phenol, cyanomethyl alcohol, para-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxysuccinide, N-hydroxyphthalimide, N-hydroxybenztriazole), etc. can give. The activated amino group of the raw material includes, for example, the corresponding phosphoric acid amide.

The condensation reaction can be carried out in the presence of a solvent. The solvent can be appropriately selected from those known to be usable in the peptide condensation reaction. For example, anhydrous or hydrous dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, acetonitrile,
Examples thereof include ethyl acetate, N-methylpyrrolidone or an appropriate mixture thereof.

The reaction temperature is appropriately selected from the range known to be applicable to peptide bond-forming reactions, and is usually selected in the range of about -20 ° C to 30 ° C.

The protecting group can be removed by catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, or anhydrous hydrogen fluoride, methanesulfonic acid or trifluoromethanesulfone. Acid treatment with an acid, trifluoroacetic acid or a mixed solution thereof, and reduction with sodium in liquid ammonia are also included.
The elimination reaction by the acid treatment is generally carried out at a temperature of -20 ° C to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol are used. The addition of a cation scavenger such as 1,2-ethanedithiol is effective. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above-mentioned 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.

The peptide [II] obtained by removing the protecting group of the protected peptide in this manner is subjected to an oxidation reaction to obtain the object compound [I] of the present invention. Examples of the method of oxidizing [II] to [I] include a method of oxidizing with air, potassium ferricyanide, iodine, diiodoethane or the like in a solvent such as water. It is desirable that the oxidation reaction is generally carried out at a temperature of about 0 ° C. to about 40 ° C., a pH of about 6-8.5, and a high dilution method. Peptide of the present invention [I]
Is often easily obtained from [II] by air oxidation.

After the reaction, the peptide derivative [I] thus produced is collected by means of peptide separation and purification, such as extraction, partitioning, reprecipitation, recrystallization, column chromatography, high performance liquid chromatography and the like. It

The peptide derivative [I] of the present invention is a metal salt (eg sodium salt, potassium salt, calcium salt, magnesium salt etc.), a salt with a base or a basic compound (eg ammonium salt, arginine salt) according to a method known per se. Etc.), an acid addition salt, especially a pharmacologically acceptable acid addition salt.
(For example, hydrochloric acid, sulfuric acid, phosphoric acid) or organic acids (for example, acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, methanesulfonic acid) and the like.

In the present specification, when an abbreviation is used for an amino acid, a peptide, etc., IUPAC-IUB Com
It is based on the abbreviations used in the mission on Biochemical Nomenclature or the abbreviations commonly used in this field. Examples are given below.

Gly: glycine Ala: alanine Val: valine Nva: norvaline Ile: isoleucine aIle: alloisoleucine Leu: leucine tLeu: tertiary leucine γMeLeu: gammamethylleucine Met: methionine Arg: arginine Lys: lysine Hisp: histidin Asp. Acid Glu: Glutamic acid Ser: Serine Thr: Threonine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Cys: Cysteine Cyt: Cystine mTrp: 5-methyltryptophan Phg: Phenylglycine Nal (1): 1-naphthylalanine Nal (2): 2 -Naphtylalanine βAla: betaalanine γAbu: gammaaminobutyric acid The protecting groups and reagents commonly used in the present specification are represented by the following abbreviations.

Boc: tertiary butoxycarbonyl Bzl: benzyl BrZ: 2-bromobenzyloxycarbonyl ClZ: 2-chlorobenzyloxycarbonyl Tos: paratoluenesulfonyl For: formyl OBzl: benzyl ester OPac: phenacyl ester ONB: HONB ester TFA : Trifluoroacetic acid TEA: triethylamine IBCF: isobutylchloroformate DMF: N, N-dimethylformamide DCC: N, N'-dicyclohexylcarbodiimide DCU: N, N'-dicyclohexylurea HONB: N-hydroxy-5-norbornene-2 , 3-Dicarboximide HOBt: 1-hydroxybenzotriazole DCM: Dichloromethane THF: Tetrahydrofuran MeBzl: 4-methylbenzyl Acm: Acetamidomethyl MBzl: 4-methoxybenzyl Next, the drug of the peptide derivative [I] of the present invention The operation will be described.

[0041]

[Experimental Example] Receptor binding assay Membrane fraction prepared from pig heart was diluted to 0.15 mg / ml with assay buffer, and 100 µl of this was dispensed into assay tubes for use in the assay. 2 μl of endothelin-1 solution labeled with 5 μM radioiodine and 3 μl of the test peptide solution were added to this membrane fraction suspension to give 1
The temperature was kept at 25 ° C. The membrane fraction suspension was then diluted with 900 μl of ice-cold assay buffer, and then diluted with 12,000 × g to 10
It was centrifuged for a minute and separated into a supernatant and a precipitate. The sediment contains the cell membrane and the endothelin receptor embedded therein, and the radioactive iodine-labeled endothelin bound to the receptor is also collected in the sediment. Therefore, the amount of radioiodine-labeled endothelin bound to the endothelin receptor was quantified by measuring the radioactive iodine in this sink with a gamma ray counter. The results are as follows. The control compound is the compound described in Japanese Patent Application No. 2-413828.

Example No. Binding activity (* 1) Control (* 3) 1.0 (* 2) 1 6.2 2 3.5 5 14 6 12 7 6 8 10 9 4.6 * 1 Porcine myocardial membrane fraction * 2 IC ₅₀ = 1.10 × 10 ^-6 M * 3 Control compound

[0043]

[Chemical 5]

As described above, the novel peptide derivative of the present invention
[I] or a pharmaceutically acceptable salt thereof has a property as an endothelin receptor antagonist and can be used as a circulatory function improving agent, a vasodilator or a therapeutic agent for asthma.

[0045]

[Action and effect] Novel peptide derivative of the present invention [I]
Has a remarkable effect as an endothelin receptor antagonist on suppressing the vasoconstrictor activity of endothelin. Therefore, the novel peptide derivative of the present invention or a salt thereof can be used as a circulatory function improving agent, a therapeutic agent for myocardial infarction, acute renal failure or the like, or an asthma therapeutic agent.

When the peptide derivative of the present invention is used as the above-mentioned therapeutic agent, it may be powdered, granulated, tablets, capsules, injections, suppositories, or may be mixed with pharmacologically acceptable carriers, excipients and diluents. It can be safely administered orally or parenterally in the form of a drug, an ointment, a sustained release preparation and the like. The peptide derivative of the present invention is mainly administered parenterally (for example, intravenous or subcutaneous injection, intraventricular or intraspinal injection, nasal administration, rectal administration), but in some cases, it may be orally administered.

Since the peptide of the present invention is stable as a substance, it can be stored as a solution of physiological saline, but it can also be dissolved at the time of use by adding mannitol and sorbitol to make a freeze-dried ampoule. The peptide derivative of the present invention can be administered as a free form or as a base salt or acid addition salt thereof. The dose of the free form, base salt and acid addition salt of the peptide derivative is generally in the range of 1 μg to 100 mg per kg of body weight. More specifically, the dose varies depending on the target disease, symptom, administration subject, administration method, etc., but when administered by injection to an adult hypertensive patient, for example, the medicinal component (peptide [1]) 1 1 μg / kg to 100 mg /
It is convenient to administer about kg body weight, more preferably about 100 μg / kg to 20 mg / kg body weight once to three times a day.
In addition, infusion is also effective, and the total dose in the case of infusion is the same as in the case of injection.

When this peptide derivative is used as a therapeutic agent, it must be carefully purified so that bacteria and pyrogens do not exist.

[0049]

EXAMPLES The present invention will be described in more detail with reference to the following examples. In addition, D and L of α-amino acids in the examples are L-forms unless otherwise specified.

The thin-phase chromatography plate used in the examples was a product of Merck (SILICAGE L60F-254), and the developing solvent was Rf ₁ : chloroform-methanol (19:
1), Rf _2: chloroform - methanol - acetic acid (9: 1: 0.
5) was used.

Example 1

[0052]

[Chemical 6]

Boc-D-Cys (MeBzl) -Merifield resin (0.
5 mmol, manufactured by Watanabe Chemical Co., Ltd.) as a starting material, Boc-amino acid derivative cartridge and Boc-D-Leu, Boc-D-Trp (CHO), Boc-D-Glu (OBzl) of Applied Biosystems.
For the product, a powder (manufactured by Peptide Institute Co., Ltd.) enclosed in a dedicated cartridge (2.0 mmol) was used. After elimination of the Boc group by trifluoroacetic acid, peptide chains were sequentially added from the C-terminal side by the HOBt active ester method. Extended. Thus Boc-Cys (MeBzl) -D-Leu-Leu-D-Trp (CHO) -D-Glu (OBz
A protected peptide resin represented by l) -D-Cys (MeBzl) -resin was obtained. This peptide resin contains 0.1% indole 50
B by treatment with% TFA / dichloromethane at room temperature for 20 minutes
The oc group was removed. The peptide resin (500 mg) thus obtained was treated with anhydrous hydrogen fluoride (5 ml) in the presence of para-cresol (500 mg) and 1,4-butanediol (0.75 ml) to give 0
The peptide was cleaved from the resin while being treated at 1 ° C for 1 hour to remove all protecting groups. Hydrogen fluoride was distilled off under reduced pressure, ethyl ether was added to the residue, and the precipitate was collected by filtration. TFA (30 ml) was added to and dissolved in this, the resin was filtered off, the filtrate was concentrated, ethyl ether was added to the residue, and the precipitate was collected by filtration and dried under reduced pressure. This is 0.1M ammonium acetate water containing 25% ethanol and 25% normal butanol (pH 8.5, 500 ml).
Dissolved in water, stirred at room temperature for 15 hours, and air-oxidized. Then, acetic acid was added to adjust the pH to 5.0, the solvent was distilled off under reduced pressure, and the residue was freeze-dried. This was dissolved in 60% acetic acid (20 ml), applied to a Sephadex G-25 column (2 cm x 100 cm), eluted with 60% acetic acid, and the target fractions were collected and lyophilized. Finally, this is a column of YMC-D-ODS-5 (2cm x 25cm).
The product was purified by preparative liquid chromatography (manufactured by YMC) to obtain the desired product.

Amino acid analysis value [6N-HCl, 110 ° C., 24 hours hydrolysis; () indicates theoretical value]: Glu 1.00 (1); Cyt 0.37
(1); Leu 2.07 (2) LSIMS (M + H ⁺ ) = 763 (theoretical value = 763) Example 2

[0055]

[Chemical 7]

By the same operation as in Example 1, Boc-D-Cys (M
eBzl) -D-Leu-Leu-D-Trp (CHO) -D-Glu (OBzl) -D-Cys (MeBz
l) -resin was obtained. However, Boc-D-Cys (MeBzl) was used after encapsulating a powder manufactured by Peptide Institute Co., Ltd. in a dedicated cartridge. This was further deprotected / oxidized / similar to Example 1.
The desired product was obtained by purification.

Amino acid analytical value [6N-HCl, 110 ° C., 24 hours hydrolysis; () indicates theoretical value]: Glu 1.00 (1); Cyt 0.35
(1); Leu 2.01 (2) LSIMS (M + H ⁺ ) = 763 (theoretical value = 763) Example 3

[0058]

[Chemical 8]

Boc-Cys (MeBzl) -OCH ₂ -pam resin (0.5 mmol) was used as a starting material, and Boc-
Cys (MeBzl) -D-Leu-Leu-D-Trp (CHO) -D-Glu (OBzl) -Cys (Me
Bzl) -OCH ₂ -pam resin was obtained. This is further described in Example 1.
The target product was obtained by the same deprotection, oxidation, and purification as in.

Amino acid analytical value [6N-HCl, 110 ° C., hydrolysis for 24 hours; () indicates theoretical value]: Glu 1.00 (1); Cyt 0.45
(1); Leu 1.97 (2) LSIMS (M + H ⁺ ) = 763 (theoretical value = 763) Example 4

[0061]

[Chemical 9]

By the same operation as in Example 3, Boc-D-Cys (M
eBzl) -D-Leu-Leu-D-Trp (CHO) -D-Glu (OBzl) -Cys (MeBzl)-
OCH ₂ -pam resin was obtained. However, Boc-D-Cys (MeBzl) is
A powder manufactured by Peptide Institute Co., Ltd. was used after being enclosed in a dedicated cartridge. This is further deprotected in the same manner as in Example 1.
The desired product was obtained by oxidation and purification.

Amino acid analytical value [6N-HCl, 110 ° C., hydrolysis for 24 hours; () indicates theoretical value]: Glu 1.00 (1); Cyt 0.44
(1); Leu 2.02 (2) LSIMS (M + H ⁺ ) = 763 (theoretical value = 763) Example 5 The following compound was synthesized by the same operation as in Example 1.

(1)

[0065]

[Chemical 10]

LSIMS (M + H ⁺ ) = 750 (theoretical value = 750) (2)

[0067]

[Chemical 11]

LSIMS (M + H ⁺ ) = 750 (theoretical value = 750) (3)

[0069]

[Chemical 12]

LSIMS (M + H ⁺ ) = 764 (theoretical value = 764) (4)

[0071]

[Chemical 13]

LSIMS (M + H ⁺ ) = 823 (theoretical value = 823) (5)

[0073]

[Chemical 14]

LSIMS (M + H ⁺ ) = 790 (theoretical value = 79
0)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // C07K 99:00

Claims (2)

[Claims] 1. The formula: [In the formula, A is a D-acidic-α-amino acid residue, and B is a D-neutral-
α-amino acid residue, C is L-α-amino acid residue, D is a D-α-amino acid residue having an aromatic ring group, R ₁ is a hydrogen atom or an optionally substituted amino group, R ₂ is a carboxyl group which may be amidated or esterified,
m and n each represent 1 or 2] or a salt thereof. 2. An endothelin receptor antagonist comprising the peptide derivative according to claim 1 or a pharmacologically acceptable salt thereof.