JPH0559098A - Cyclic peptide and its use - Google Patents

Abstract

PURPOSE:To provide the title peptide having antagonistic activity to endoserine receptor, thus to be used for therapeutic agents for hypertension, cardiopathy, cerebrocirculatory diseases, or nephropathy, made up of D-acidic amino acid, D-neutral amino acid, L-tryptophan and aromatic amino acid, etc. CONSTITUTION:The carboxyl group of a L-alpha-amino acid (e.g. L-alanine) is first protected, followed by successively Wing to the resultant amino acid (A) a D-acidic alpha-amino acid (D-glutamic acid) with its alpha-amino group and, if needed, side chain functional group protected, (B) an aromatic group-carrying D-alpha-amino acid (e.g. D-tryptophan), (C) a (substituted) tryptophan, and (D) a D-neutral alpha-amino acid (e.g. D-leucine-). Thence, the terminal protecting group of the resultant peptide is removed, followed by treatment with a condensation agent in a solvent to effect cyclization, thus giving the objective cyclic peptide (salt) of the formula A is D-acidic alpha-amino acid residue; B is L-alpha-amino acid residue; C is Dneutral alpha-amino acid residue; D is (substituted) L-tryptophan residue; E is aromatic group-carrying D-alpha-amino acid residue.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cyclic peptide having an endothelin receptor antagonistic action 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 and a therapeutic agent for asthma. Regarding 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 conducted earnest research with the action of suppressing the strong vascular smooth muscle contractile activity of endothelins as an index, and have developed a novel cyclic peptide 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 an L-α-amino acid residue, C is a D-neutral-α-amino acid residue, D is an optionally substituted L-tryptophan residue, and E is an aromatic ring group-containing D-α- Indicates amino acid residue]
1. A cyclic peptide represented by or a salt thereof, and The present invention relates to an endothelin receptor antagonist containing a cyclic peptide represented by the formula [I] or a pharmacologically acceptable salt thereof.

The above-mentioned formula [I] indicates that the presence of five amide bonds including A to E forms a ring as a whole molecule, and in the present specification, cyclo [-A].
-B-C-D-E-].

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-
3- (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], L-α represented by B
-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 listed, and particularly L-alanine, L-proline, and L-asparagine. Acid and L-glutamic acid are preferred. In the above formula [I], the amino acid that is the base of the D-neutral-α-amino acid residue represented by C is, for example,
D-alanine, D-valine, D-norvaline, D-leucine, D-
Isoleucine, D-alloisoleucine, D-norleucine,
D-tertiary leucine, D-gamma methyl leucine, D-
Examples thereof include D-α-amino acids such as phenylglycine and D-phenylalanine, with D-leucine, D-alloisoleucine, D-tert-leucine, D-gammamethylleucine and D-phenylglycine being particularly preferred.

In the above formula [I], D represents an optionally substituted L-tryptophan residue. Here, the substituent of the indole ring of L-tryptophan is, for example, a lower alkyl group at the 1-position. Groups (eg, C _1-6 alkyl groups such as methyl, ethyl, n-propyl), alkanoyl groups (eg, C _1-6 alkanoyl groups such as formyl, acetyl, propionyl), carboxy lower alkyl groups (eg, carboxymethyl) Carboxy C such as 1-carboxyethyl and 2-carboxyethyl
_1-6 alkyl group), lower alkoxycarbonyl lower alkyl group (for example, C _1-6 alkoxycarbonyl C _1-6 alkyl group such as methoxycarbonylmethyl, ethoxycarbonylmethyl, tertiary butoxycarbonylmethyl) or carbamoyl lower alkyl group ( Examples thereof include carbamoylmethyl, 1-carbamoylethyl, carbamoylC _1-6 alkyl groups such as 2-carbamoylethyl), and the like, and examples of the substituent at the 2-position, 4-position, 5-position, 6-position or 7-position include lower alkyl. Group (eg, C _1-6 alkyl group such as methyl, ethyl, n-propyl), lower alkoxy group (eg, methoxy, ethoxy, n-)
Examples thereof include C _1-6 alkoxy groups such as propoxy), alkanoyl groups (eg, C _1-6 alkanoyl groups such as formyl, acetyl, propionyl), hydroxyl groups or halogen atoms (eg, fluoro, chloro, bromo). Therefore, specific examples of the amino acid which is the base of the optionally substituted L-tryptophan residue represented by D include L-tryptophan, LN ⁱⁿ -methyltryptophan, LN ⁱⁿ -formyl tryptophan, and LN ⁱⁿ -acetyltryptophan. , LN ⁱⁿ - carboxymethyl tryptophan, LN ⁱⁿ - tert-butoxycarbonyl-methyl-tryptophan, LN ⁱⁿ - carbamoylmethyl tryptophan, L-5-methyl-tryptophan, L-5-methoxy-tryptophan, L-5-acetyl-tryptophan, L-5- Hydroxytryptophan, L-5-Fluorotryptophan, L
Examples include -6-fluorotryptophan. Also,
Another example is L-3- (2,3-dihydro-2-oxoindol-3-yl) alanine. Among them, L-tryptophan, L-
N ⁱⁿ -formyltryptophan, L-5-methyltryptophan and L-5-fluorotryptophan are particularly preferable.

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 E 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.

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

The cyclic peptide [I] of the present invention can be produced by a conventional method for peptide synthesis. That is, the liquid phase synthesis method,
Although any of the solid phase synthesis methods may be used, 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. Bod
Ansky and MA Ondetti, Peptide Synthesis
(Peptide Synthesis), Interscience, New York, 1966; by FM Finn and K. Hofmann, The
The Proteins, Volume 2, H. Nenrath, R.
Edited by L. 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. Academic Society, Tokyo Kagaku Dojin 1977
Year; Shun Kimura et al., Continuation Biochemistry Laboratory 2, edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin 1987; JM Stewart and J.
D. Young, Solid Phase Peptide Synthesis
(Solid Phase Peptide Synthesis), Pierce Chemical
Company, Illinois, method described in 1984, etc.,
For example, azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, method using Woodward reagent K, carbonyl imidazole method, redox method,
Examples include the DCC / HONB method and the method using a BOP reagent.

The cyclic peptide [I] of the present invention comprises 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 reaction corresponding to the other fragment. A raw material having a reactive amino group is condensed by a conventional method for peptide synthesis, and then the protecting groups of the C-terminal α-carboxyl group and the N-terminal α-amino group of the product are removed simultaneously or stepwise. It can be produced by condensation in the molecule by a condensation method to obtain a cyclic compound, and when the product has a protecting group, the protecting group is eliminated by a conventional means.

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

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, phthalyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, 9-fluorenyl Examples include methyloxycarbonyl. Examples of the protective group for the carboxyl group include alkyl ester
(For example, ester groups such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl), benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4 -Chlorobenzyl ester, benzhydryl ester, phenacyl ester, benzyloxycarbonyl hydrazide, tertiary butyloxycarbonyl hydrazide, trityl hydrazide and the like.

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.
Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group and a tert-butyl group. 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, tert-butyl, and the like. There is no. 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). 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 intramolecular cyclization reaction can be performed at any position of the peptide by a known method. For example, first, the terminal α-carboxyl protecting group of the C-terminal amino acid of the protected peptide is eliminated by a known method and then activated by a known method, and then the terminal α-amino protecting group of the N-terminal amino acid is changed by a known method. It is also possible to desorb and to cyclize in the molecule. Alternatively, the terminal α-carboxyl protecting group of the C-terminal amino acid and the terminal α-amino protecting group of the N-terminal amino acid of the protected peptide may be simultaneously eliminated and then cyclized intramolecularly by a known condensation reaction. In some cases, it may be preferable to carry out the intramolecular cyclization reaction under high dilution.

As a method for removing the protecting group, for example, Pd
Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, and acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Also, reduction with sodium in liquid ammonia can be mentioned. The elimination reaction by the acid treatment is generally -20 ° C to 40 ° C.
Although it is carried out at a temperature of ℃, in the acid treatment, anisole, phenol, thioanisole, metacresol,
The addition of a cation scavenger such as para-cresol, dimethyl sulfide, 1,4-butanedithiol, 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.

Cyclic peptide produced in this way
[I] is collected after the reaction by means of peptide separation and purification, for example, extraction, partitioning, reprecipitation, recrystallization, column chromatography, high performance liquid chromatography and the like. The cyclic peptide [I] of the present invention can be prepared by a method known per se in the form of 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 etc.), It can also be obtained as an acid addition salt, especially a pharmacologically acceptable acid addition salt, for example, an inorganic acid (for example, hydrochloric acid, sulfuric acid, phosphoric acid) or an organic acid (for example,
Salts such as acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid and methanesulfonic acid) can be mentioned.

Since the cyclic peptide [I] of the present invention has an L-tryptophan residue which may be substituted as a constituent amino acid (D), it is remarkably effective as an endothelin receptor antagonist in suppressing the vasoconstrictor activity of endothelin. In addition to exhibiting the following characteristics, (1) it has excellent crystallinity, (2) it has good stability as a substance, and (3) it has low toxicity.

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 Ile: Isoleucine aIle: Alloisoleucine Leu: Leucine Met: Methionine Arg: Arginine Lys: Lysine His: Histidine Asp: Aspartic acid Glu: Glutamic acid Ser: Serine Thr: Phenylalanine Phe Tyr: Tyrosine Trp: Tryptophan Cys: Cysteine Cys (O ₃ Na): Cysteinic acid monosodium salt mTrp: 5-Methyltryptophan Phg: Phenylglycine The protecting groups and reagents commonly used in the present specification are represented by the following abbreviations. To do.

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: isobutyl chloroformate 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 AcOEt: ethyl acetate Next, the pharmacological action of the cyclic peptide of the present invention will be described.

[0034] The membrane fraction prepared from the heart of the receptor-binding assay i pigs using the assay buffer was diluted to 0.15 mg / ml, which is used in the assay and dispensed into the assay tube by 100 [mu] l. 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 Compound Binding Activity (* 1) No. Specific Activity Control cyclo [-D-Glu-Ala-D-aIle-Leu-D-Trp-] 1.00 (* 2) 1 cyclo [-D -Glu-Ala-D-Leu-Trp-D-Trp-] 12.2 * 1 Pig myocardial membrane fraction * 2 IC ₅₀ = 1.10 x 10 ^-6 M As described above, the novel cyclic peptide [I] of the present invention or its The pharmacologically acceptable salt 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.

[0036]

[Operation / Effect] The novel cyclic peptide [I] of the present invention is
As an endothelin receptor antagonist, it exerts a remarkable effect in suppressing the vasoconstrictor activity of endothelin. Therefore, the novel cyclic peptide of the present invention or a salt thereof is used as a circulatory function improving agent for mammals (eg, human, mouse, rat, pig, dog, etc.) or a therapeutic agent for myocardial infarction / acute renal failure or an asthma therapeutic agent. Can be used.

When the cyclic peptide of the present invention is used as the above-mentioned therapeutic agent, it may be used as it is, or may be mixed with a pharmacologically acceptable carrier, excipient or diluent to prepare powder, granules, tablets, capsules, injections, suppositories. It can be safely administered orally or parenterally in the form of a drug, an ointment, a sustained release preparation and the like. The cyclic peptide of the present invention is mainly administered parenterally
(For example, intravenous or subcutaneous injection, intracerebroventricular or intraspinal injection, nasal administration, rectal administration), but may be orally administered depending on the case. Since the peptide of the present invention is stable as a substance, it can be stored as a physiological saline solution, but it can also be dissolved at the time of use by adding mannitol or sorbitol to form a freeze-dried ampoule. The cyclic peptide 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 cyclic peptide free form, base salt, acid addition salt,
The amount of the free form is generally in the range of 1 μg to 100 mg per 1 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 as a batch
μg / kg to 100 mg / kg body weight, more preferably 100 μ / kg to
It is convenient to administer about 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 the cyclic peptide of the present invention is used as a therapeutic agent, it must be carefully purified so that bacteria and pyrogens do not exist. No toxicity is found in the purified cyclic peptide of the present invention.

[0038]

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-layer 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 cyclo [-D-Glu-Ala-D-Leu-Trp-D
-Trp-] (1) Production of Boc-Ala-OPac Boc-Ala-OH (28.4g) and Cs ₂ CO ₃ (24.5g) were dissolved in 90% methanol water and concentrated, and the residue was added to DMF ( 450 ml), phenacyl bromide (32.9 g) was added, and the mixture was stirred overnight. Generated T
EA hydrochloride was filtered off, the filtrate was concentrated, the residue was dissolved in AcOEt and this was washed with 4% NaHCO ₃ water, 10% citric acid water, washed with water, dried over Na ₂ SO ₄ and concentrated, Ether was added to the residue and the precipitate was collected by filtration.

Yield 42.4 g (yield 91.9%), melting point 123 ° C., R
f ₁ 0.72, Rf ₂ 0.74 [α] _D ²⁸ -46.1 ° (c = 1.36, in DMF) Elemental analysis Calculated as C ₁₆ H ₂₁ NO ₅ : C, 62.53; H, 6.89; N, 4.56 Experimental value: C , 62.94; H, 6.87; N, 4.63 (2) Production of Boc-D-Glu (OBzl) -Ala-OPac Dissolve Boc-D-Glu (OBzl) -OH (2.02g) in THF and stir it while stirring. Cooled to -15 ° C. To this was added N-methylmorpholine (0.66 ml) followed by IBCF (0.80 ml). Two
After a minute, HCl H-Ala-OPac and N-methylmorpholine (0.66 ml)
DMF solution was added [HCl ・ Ala-OPac is Boc-Ala-OPac (1.84g)
TFA (20 ml) was added to the solution to dissolve and then concentrated, 8N-HCl / dioxane (1.88 ml) was added, ether was added, and the precipitated crystals were collected by filtration and dried]. After stirring at -15 ° C for 30 minutes, the temperature was returned to room temperature. After 30 minutes, the insoluble matter was filtered off, the filtrate was concentrated, and the residue was dissolved in AcOEt.
Wash with 0% citric acid water, 4% NaHCO ₃ water, wash with water, and then wash with Na ₂ SO ₄
Dried over, concentrated and recrystallized from AcOEt-petroleum ether.

Yield 2.63 g (yield 83.2%), melting point 128 ° C., Rf
₁ 0.54, Rf ₂ 0.65 [α] _D ²⁸ -13.0 ° (c = 1.19, in DMF) Elemental analysis Calculated as C ₂₈ H ₃₄ N ₂ O ₈ : C, 63.87; H, 6.51; N, 5.32 Experimental value: C, 63.88; H, 6.32; N, 5.18 (3) Production of Boc-D-Trp-D-Glu (OBzl) -Ala-OPac Boc-D-Glu (OBzl) -Ala-OPac (1.05g) TFA (20 ml) was added to dissolve and then concentrated. 8N-HCl / dioxane (0.63 ml) was added, ether was added, and the precipitated crystals were collected by filtration and dried. Dissolve this in DMF (15 ml) and cool with ice, then TEA (0.20 ml)
Was added. In addition, Boc-D-Trp-ONB [Boc-D-Trp-OH (0.6
7g), HONB (0.43g), DCC (0.50g)] was added and stirred overnight. The resulting insoluble matter is filtered off and then concentrated,
The residue was dissolved in AcOEt and this was mixed with water, 10% citric acid water, 4
% NaHCO ₃ water, washed with water, dried over Na ₂ SO ₄ , concentrated,
Ether was added to the residue and the precipitate was collected by filtration.

Yield 1.39 g (yield 97.8%), melting point 127-129 ° C.,
Rf ₁ 0.36, Rf ₂ 0.62 [α] _D ²⁸ -4.04 ° (c = 1.04, in DMF) Elemental analysis Calculated as C ₃₉ H ₄₄ N ₄ O ₉ : C, 65.72; H, 6.22; N, 7.86 Experimental value : C, 65.62; H, 6.46; N, 7.78 (4) Production of Boc-Trp-D-Trp-D-Glu (OBzl) -Ala-OPac Boc-D-Trp-D-Glu (OBzl) -Ala- OPac (2.85 g) was suspended in dioxane, ethanedithiol (0.68 ml) was added, and the mixture was ice-cooled,
8N-HCl / dioxane was added, and the mixture was stirred under ice-cooling for 1 hour and then concentrated. Ether was added, and the precipitated crystals were collected by filtration and dried. Dissolve this in DMF (20 ml) and chill with ice, then use TEA (1.12 m
l) was added. Boc-Trp-ONB [Boc-Trp-OH (1.34g),
HONB (0.86 g) and DCC (0.99 g) were added] and the mixture was stirred overnight. The resulting insoluble matter was filtered off, concentrated, and the residue was dissolved in AcOEt. This was dissolved in water, 10% aqueous citric acid solution, 4% NaHCO 3.
₃ Washed with water, washed with water, dried over Na ₂ SO ₄ and concentrated, ether was added to the residue, and the precipitate was collected by filtration.

Yield 2.96 g (yield 82.3%), melting point 159-161 ° C,
Rf ₁ 0.45, Rf ₂ 0.70 [α] _D ²⁸ -9.96 ° (c = 1.21, in DMF) Elemental analysis Calculated as C ₅₀ H ₅₄ N ₆ O ₁₀ : C, 66.80; H, 6.05; N, 9.35 Experimental value : C, 66.47; H, 6.21; N, 9.36 (5) Boc-D-Leu-Trp-D-Trp-D-Glu (OBzl) -Ala-OPac Boc-Trp-D-Trp-D-Glu (OBzl) -Ala-OPac (2.25 g) was suspended in dioxane, ethanedithiol (0.42 ml) was added, and the mixture was ice-cooled, 8N-HCl / dioxane was added, and the mixture was stirred for 30 minutes under ice-cooling and then concentrated, ether was added. The precipitated crystals were collected by filtration and dried. This was dissolved in DMF (15 ml) and cooled with ice, and TEA (0.7
0 ml) was added. Boc-D-Leu-ONB [Boc-D-Leu-OH
(0.69 g), HONB (0.54 g) and DCC (0.63 g)] were added and stirred overnight. The resulting insoluble matter was filtered off and concentrated, the residue was dissolved in AcOEt, washed with water, 10% citric acid water, 4% NaHCO ₃ water, washed with water, dried over Na ₂ SO ₄ and concentrated. Ether was added to the residue, and the precipitate was collected by filtration.

Yield 2.20 g (yield 86.9%), melting point 119-121 ° C,
Rf ₁ 0.44, Rf ₂ 0.70 [α] _D ²⁸ -2.29 ° (c = 1.14, in DMF) Elemental analysis Calculated as C ₅₆ H ₆₅ N ₇ O ₁₁ : C, 66.45; H, 6.47; N, 9.69 Experimental value : C, 66.12; H, 6.55; N, 9.65 (6) Production of Boc-D-Leu-Trp-D-Trp-D-Glu (OBzl) -Ala-OH Boc-D-Leu-Trp-D-Trp -D-Glu (OBzl) -Ala-OPac (1.52g) 9
It was dissolved in 0% AcOH water (20 ml), Zn powder (4.91 g) was added, and the mixture was stirred for 3 hours. The Zn powder was filtered off and the filtrate was concentrated.
AcOEt was added to the residue to dissolve it, and the residue was washed with 10% citric acid water, washed with water, dried over Na ₂ SO ₄ , concentrated, and ether was added to the residue, and the precipitate was collected by filtration.

Yield 1.12 g (yield 83.5%), melting point 124-125 ° C.,
Rf ₁ 0.04, Rf ₂ 0.69 [α] _D ²⁸ + 12.0 ° (c = 0.94, in DMF) Elemental analysis Calculated as C ₄₈ H ₅₉ N ₇ O ₁₀ : C, 64.49; H, 6.65; N, 10.97 Experimental value : C, 64.67; H, 6.61; N, 10.64 (7) Production of cyclo [-D-Glu-Ala-D-Leu-Trp-D-Trp-] Boc-D-Leu-Trp-D-Trp-D -Glu (OBzl) -Ala-OH (0.45g) in DCM
Dissolve in (20 ml), cool with ice, add HONB (0.18 g), DCC (0.21 g) and stir for 3 hours, then filter off the DCU produced,
The mixture was concentrated, ether was added to the residue, and the precipitate was collected by filtration. Under ice cooling, ethanedithiol (0.09ml), 8N-
After adding HCl / dioxane (20 ml) to dissolve the mixture, the mixture was stirred for 10 minutes and concentrated, and ether was added to the residue to obtain a precipitate, which was dried. Dissolve this in DMF (10 ml) and add TEA (0.7
(ml) in DMF (90 ml) over 30 minutes, and the mixture was stirred overnight and concentrated. Acetonitrile was added to the residue and the precipitate was collected by filtration and dried. Of this, 78 mg was added to DMF (15
ml) and catalytically reduced under a hydrogen stream using Pd black as a catalyst. The catalyst was filtered off and concentrated, the residue was dissolved in a small amount of AcOH, water was added, and the mixture was freeze-dried. Finally, this was purified by preparative liquid chromatography using a YMC-D-ODS-5 (2 cm × 25 cm) column manufactured by Yamamura Chemical Co., Ltd. to obtain the desired product.

Yield 2.0 mg (yield 3.4%) Amino acid analytical value [6N-HCl, 110 ° C., hydrolysis for 24 hours; () indicates theoretical value]: Glu 1.00 (1); Ala 0.96 (1); Leu 0.8
5 (1) LSIMS (M + H ⁺ ) = 686 (theoretical value = 686) Example 2 Production of cyclo [-D-Asp-Ala-D-Leu-Trp-D-Trp-] According to the method of Example 1. According to the same manner, the title cyclic peptide is produced using Boc-Trp-OBzl as a starting material. Boc-D-Glu (OB
Use Boc-D-Asp (OBzl) -OH instead of zl) -OH.

Example 3 cyclo [-D-Asp-Asp-D-aIle-Trp-
Production of D-Trp-] According to the method of Example 1, the above-mentioned cyclic peptide is produced using Boc-Trp-OBzl as a starting material. Boc-Asp (OBzl) -OH was used instead of Boc-Ala-OH, Boc-D-Asp (OBzl) -OH was used instead of Boc-D-Glu (OBzl) -OH, and Boc-D-Leu -Instead of OH
Boc-D-aIle-OH is used.

Example 4 cyclo [-D-Asp-Asp-D-Leu-Trp-D
Preparation of -Trp-] According to the method of Example 1, the above cyclic peptide is prepared using Boc-Trp-OBzl as a starting material. Boc-Asp (OBzl) -OH is used instead of Boc-Ala-OH, and Boc-D-Asp (OBzl) -OH is used instead of Boc-D-Glu (OBzl) -OH.

Example 5 cyclo [-D-Asp-Glu-D-Leu-Trp-D
Preparation of -Trp-] According to the method of Example 1, the above cyclic peptide is prepared using Boc-Trp-OBzl as a starting material. In addition, Boc-Glu (OBzl) -OH is used instead of Boc-Ala-OH, and Boc-D-Asp (OBzl) -OH is used instead of Boc-D-Glu (OBzl) -OH.

Example 6 cyclo [-D-Asp-Pro-D-Leu-Trp-D
Preparation of -Trp-] According to the method of Example 1, the above cyclic peptide is prepared using Boc-Trp-OBzl as a starting material. Boc-Pro-OH is used instead of Boc-Ala-OH, and Boc-D-Glu (OBzl) -OH is used instead of Boc-Pro-OH.
Boc-D-Asp (OBzl) -OH is used.

Example 7 cyclo [-D-Cys (O ₃ Na) -Ala-D-Leu
Production of -Trp-D-Trp-] According to the method of Example 1, the above-mentioned cyclic peptide is produced using Boc-Trp-OBzl as a starting material. Boc-D-Glu (OB
zl) using the _{Boc-D-Cys (O 3} Na) -OH in place of -OH.

Example 8 cyclo [-D-Cys (O ₃ Na) -Pro-D-Leu
Production of -Trp-D-Trp-] According to the method of Example 1, the above-mentioned cyclic peptide is produced using Boc-Trp-OBzl as a starting material. Boc-Pro-OH is used instead of Boc-Ala-OH, and Boc-D-Glu (OBzl) -OH is used instead of Boc-Pro-OH.
Using the the _{Boc-D-Cys (O 3} Na) -OH.

Example 9 cyclo [-D-Cys (O ₃ Na) -Asp-D-Leu
Production of -Trp-D-Trp-] According to the method of Example 1, the above-mentioned cyclic peptide is produced using Boc-Trp-OBzl as a starting material. Incidentally, the Boc-Asp (OBzl) -OH in place of Boc-Ala-OH, using the the _{Boc-D-Cys (O 3} Na) -OH in place of Boc-D-Glu (OBzl) -OH.

Example 10 cyclo [-D-Cys (O ₃ Na) -Glu-D-Leu
Production of -Trp-D-Trp-] According to the method of Example 1, the above-mentioned cyclic peptide is produced using Boc-Trp-OBzl as a starting material. Incidentally, the Boc-Glu (OBzl) -OH in place of Boc-Ala-OH, using the the _{Boc-D-Cys (O 3} Na) -OH in place of Boc-D-Glu (OBzl) -OH.

Example 11 cyclo [-D-Asp-Asp-D-Leu-Trp (C
Production of HO) -D-Trp-] According to the method of Example 1, the above-mentioned cyclic peptide is produced starting from Boc-Trp (CHO) -OBzl. Boc-Al
Boc-Asp (OBzl) -OH instead of a-OH, Boc-D-Glu (OBzl)-
Use Boc-D-Asp (OBzl) -OH instead of OH.

─────────────────────────────────────────────────── ─── 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: [Wherein A is a D-acidic-α-amino acid residue, B is an L-α-amino acid residue, C is a D-neutral-α-amino acid residue, and D is an optionally substituted L -Tryptophan residue, E represents a D-α-amino acid residue having an aromatic ring group] or a salt thereof. 2. An endothelin receptor antagonist comprising the cyclic peptide according to claim 1 or a pharmacologically acceptable salt thereof.