JPH05331188A - Tripeptide, its production and endothelin antagonist - Google Patents

Abstract

PURPOSE:To provide a new tripeptide useful as a therapeutic agent for hypertension, bronchial asthma, etc., because of exhibiting an endothelin-antagonistic action and of inhibiting blood vessel contraction reactions caused by endothelin-1. CONSTITUTION:The compound of formula I [A is five to eight-membered ring cycloalkyl; B is CO, CS, etc.; D is NR<1> (R<1> is H, 1-6C alkyl); E is CHR<2> (R<2> is aryl, l-7C alkyl, etc.,); F is group of formula II, III (R<3> is H, CHO, etc.,); G is group of formula IV (R<5> is nitrogen-containing aromatic heterocyclic ring); Q is H, alkyl metal, etc.,], e.g. N-(N-cyclohexylcarbonyl-L-leucyl)-N<1n>-formyl-D- tryptophyl-D-tryptophan. The compound is obtained by carrying out condensation reactions of fragments of formula: A-B-Z (Z is hydroxyl, halogeno), formula: H-D-E-COOH and formula: H-G-OQ e.g. by an azide method, an N,N- dicyclohexylcarbodiimide method, etc. The reactions are preferably performed in a liquid phase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel tripeptide useful as an antagonist for an endothelin receptor, a method for producing this tripeptide, and an endothelin antagonist containing this tripeptide as an active ingredient.

[0002]

Since the discovery of the endothelium-dependent vasodilation response in 1980, the role of the vascular endothelium in the regulation mechanism of vascular tone has been emphasized, and from about 1982, endothelium-dependent contraction response to various stimuli. Alternatively, enhanced contraction has been reported. Recently, a strong peptide vasoconstrictor released in the culture supernatant of porcine aortic endothelial cells has been isolated, purified, and structurally determined. It is a 21-amino acid peptide called endothelin (ET). [M.Ya
nagisawa et al., Nature, 332, 411-415 (1988)]. Later, it was found that human endothelin has the same amino acid sequence [Y. Itoh et al., FEBS lett., 231,440-4].
44 (1988)].

As endothelin, ET-1, ET-
2 and ET-3 have been found, and their receptors are also ET-1 selective ET _A receptors and ET-1 and ET-3 non-selective due to the difference in binding affinity for each ET. two kinds of specific ET _B receptors have been found. When endothelin acts on the ET _A receptor ubiquitously distributed on vascular smooth muscle cells, it strongly contracts blood vessels.

Several compounds are known as substances that suppress the contractile reaction of blood vessels caused by ET-1. For example, the present applicant has applied for a patent for N-pentanoyltryptophan (Japanese Patent Laid-Open No. 3-251).
562), and New Current 3 (9) 19.
April 10, 1992, pp. 4-5, (R) 2-[(R)
-2-[(S) -2-[[1- (hexahydro-1H-
Azepinyl)] carbonyl] amino-4-methylpentanoyl] amino-3- [3- (1-methyl-1H-indolyl)] propionyl] amino-3- (2-pyridyl) propionic acid antagonizes ET _A receptors It is described as an agent.

[0005]

The object of the present invention is to provide ET
A novel tripeptide that suppresses the vasoconstriction reaction caused by -1, and is useful as a therapeutic agent for hypertension, bronchial asthma, renal failure, stroke attack after subarachnoid hemorrhage, etc.,
And a method for producing the same, and an endothelin antagonist containing the same.

[0006]

The present invention has the general formula (I):

[0007]

[Chemical 7]

[In the formula, A represents a 5- to 8-membered cycloalkyl group, B represents a group represented by -CO-, -CS- or -SO _2- , and D represents -NR ^1-. (Wherein R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), E is —CHR ² — (wherein R ² is substituted with an aryl group or an aromatic heterocyclic group). Represents an optionally substituted alkyl group having 1 to 7 carbon atoms or a sulfur atom-containing heterocyclic group), and F represents the following formula:

[0009]

[Chemical 8]

(In the formula, R ³ is a hydrogen atom, a —CHO group, an alkyl group having 1 to 6 carbon atoms, a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms, or a formula —COOR ⁴
(Wherein R ⁴ is a group represented by an alkyl group having 1 to 6 carbon atoms or a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms), and G is ,
The following formula:

[0011]

[Chemical 9]

(Wherein R ⁵ represents a nitrogen atom-containing aromatic heterocyclic group), Q is a hydrogen atom, an alkali metal atom, an alkyl group having 1 to 6 carbon atoms, or 1 carbon atom.
Represents a phenylalkyl group having an alkylene group of 4 to 4].

Another aspect of the present invention is a method for producing a tripeptide represented by the above general formula (I), which is a fragment A-B-Z which is a fragment optionally having a protecting group.
(However, Z is a hydroxyl group or a halogen atom), HDE-COOH, HF-OH and HG.
-OQ (these fragments may be reactive derivatives thereof) are condensed in any order to give the tripeptides of general formula (I), or the four fragments And a compound obtained by pre-condensing two or three kinds of the above in any combination with two kinds (may be pre-condensed) or one of the remaining fragments,
It is a production method which comprises removing a protecting group if desired.

Still another aspect of the present invention is an endothelin antagonist containing a tripeptide represented by the above general formula (I) as an active ingredient.

The preferred embodiments of the present invention are as follows. (1) In the general formula (I), B represents a group represented by -CO-, D represents a group represented by -NH-, and E represents -C.
HR ³ — (wherein R ³ represents a straight-chain or branched alkyl group having 1 to 5 carbon atoms), and G represents the following formula:

[0016]

[Chemical 10]

(In the formula, R ⁶ is a hydrogen atom, a —CHO group, an alkyl group having 1 to 6 carbon atoms, a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms, or a formula —COOR ⁸
(In the formula, R ⁸ represents an alkyl group having 1 to 6 carbon atoms or a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms), and R ⁷ is a hydrogen atom or 1 carbon atom. ~
6 alkyl groups, aralkyl groups and having a 1-3 phenyl groups having an alkylene group having 1 to 4 carbon atoms, 2 to 6 carbon atoms alkoxyalkyl group or the formula - (CH _{2) q} O
-R ⁹ (in the formula, q is an integer of 1 to 3 and R ⁹ is a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms). A tripeptide represented by the general formula (I):

The compound of the present invention represented by the general formula (I) is an α-amino acid unit represented by DE-CO and an α-amino acid unit represented by F, as is apparent from the general formula (I). And G are α-amino acid unit tripeptides, and the structure of the unit represented by AB in the general formula (I) is as described in New Current 3 (9) 1992 4
It is a tripeptide which is completely different from the compounds described on pages 10 to 10 of the month.

In the general formula (I), A is preferably a group such as cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. Also, B is -CO-
It is preferable that it is a group shown by, and it is preferable that D is a group shown by -NH-.

In the general formula (I), E is the following formula

[0021]

[Chemical 11]

It is preferably a group represented by

[0023]

[Chemical formula 12]

It is preferably a group represented by:

In the general formula (I), among the groups represented by F, R ³ is a hydrogen atom, a —CHO group, or an alkyl group having 1 to 4 carbon atoms (eg, methyl, ethyl, propyl, butyl, etc.). Or (f-1) which is a group such as a benzyl group or (f-1)
The group represented by -2) is preferable.

In the general formula (I), G is the following formula:

[0027]

[Chemical 13]

(In the formula, R ⁶ is a hydrogen atom, a —CHO group, an alkyl group having 1 to 6 carbon atoms, a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms, or a formula —COOR ⁸
(In the formula, R ⁸ represents an alkyl group having 1 to 6 carbon atoms or a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms), and R ⁷ is a hydrogen atom or 1 carbon atom. ~
6 alkyl groups, aralkyl groups and having a 1-3 phenyl groups having an alkylene group having 1 to 4 carbon atoms, 2 to 6 carbon atoms alkoxyalkyl group or the formula - (CH _{2) q} O
A group represented by —R ⁹ (wherein q is an integer of 1 to 3 and R ⁹ is a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms) is preferable.

Of the groups represented by the above (g-1), R
⁶ is preferably a group showing a hydrogen atom, a -CHO group, an alkyl group having 1 to 4 carbon atoms (for example, methyl, ethyl, propyl, butyl, etc.) and a benzyl group, and the above (g-2 ), R ⁷ is a hydrogen atom, a benzyloxymethyl group, a benzyl group, a benzhydryl group,
It is preferably a group showing a group such as a trityl group. The pyridyl group represented by the above (g-3) is preferably a 2-pyridyl group.

In the tripeptide of the present invention, the α-amino acid unit represented by DECO in the general formula (I) is L-type, the amino acid unit represented by F and the amino acid unit represented by G are Both are preferably D-type tripeptides.

In the present specification, amino acids, peptides,
Protecting groups, solvents and the like are abbreviations commonly used in the art,
Alternatively, the abbreviations adopted by the IUPAC-IUB naming committee are used. For example, the following abbreviations are used.
In the notation of amino acids, amino acids mean the L-form unless otherwise specified.

EDTA: ethylenediamine tetraacetic acid EGTA: ethylene glycol bis (2-aminoethyl ether) tetraacetic acid PMSF: phenylmethylsulfonyl fluoride Leu: L-leucine Trp: L-tryptophan D-Trp: D-tryptophan His: L- Histidine D-His: D-histidine Bzl: benzyl DMAP: 4-dimethylaminopyridine WSC.HCl: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride HOBt: 1-hydroxybenzotriazole TsOH: paratoluene sulfone Acid Pd-C: Palladium-carbon THF: Tetrahydrofuran

The tripeptide of the present invention can be produced by another production method of the present invention. In the production method of the present invention, A-B-Z (where Z is a hydroxyl group or a halogen atom, preferably chlorine), H-D-E-COOH, H-F-OH used as a raw material in the production method of the present invention. And H-
Each fragment of G-OQ may be as it is, or may have a protecting group on a carboxyl group, an amino group or the like that does not participate in the reaction, if necessary. The following groups can be illustrated as this protecting group.

Examples of the protecting group for the carboxyl group include methyl, ethyl, benzyl, p-nitrobenzyl,
Esters such as t-butyl and cyclohexyl can be mentioned.

Examples of the amino group-protecting group include benzyloxycarbonyl group, t-butoxycarbonyl group,
Examples thereof include an isobornyloxycarbonyl group and a 9-fluorenylmethyloxycarbonyl group.

Examples of the protecting group for the indole group include:
Formyl group, benzyloxycarbonyl group, 2,4-dichlorobenzyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, 4-methoxy-2,3
Examples thereof include a 6-trimethylbenzenesulfonyl group, a 2,4,6-trimethoxybenzenesulfonyl group and a mesitylene-2-sulfonyl group.

Examples of the protective group for the imidazole group include benzyl group, 2-nitrobenzyl group, phenacyl group, benzyloxymethyl group, benzhydryl group, 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, 2 , 2,2-trifluoro-1-t-butoxycarbonylaminoethyl group, 2,4-dinitrophenyl group,
2,4,6-trinitrophenyl group, trityl group, (4
-Pyridyl) diphenylmethyl group, piperidinocarbonyl group, benzyloxycarbonyl group, t-butoxycarbonyl group, adamantyloxycarbonyl group, 4-toluenesulfonyl group, 4-methoxybenzenesulfonyl group, 9-fluorenylmethoxycarbonyl group , T-butoxymethyl group and the like.

In the production method of the present invention, as a condensation method for forming a peptide bond, an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, an N, N'-dicyclohexylcarbodiimide method, Examples thereof include N, N'-dicyclohexylcarbodiimide-additive method, active ester method, carbonyldiimidazole method, redox method, method using Woodward reagent K, and the like.

Before carrying out the condensation reaction, a carboxyl group, an amino group or the like involved in the reaction may be activated as a reactive derivative thereof by means known per se.

As the reactive derivative of the carboxyl group,
For example, the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,
3-dicarboximide, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with 1-hydroxybenzotriazole), and the like. Reactive derivatives of amino groups include, for example, the corresponding phosphoric acid amides.

The reaction can be carried out by a liquid phase method or a solid phase method, but it is usually preferable to carry out in a liquid phase in a solvent, for example, chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide, It can be carried out in a solvent such as dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol, or a mixture thereof.

The reaction temperature is generally about -30 ° C.
Can be carried out in the range of about 50 ° C.

When the protecting group is removed in the production method of the present invention, it is necessary to remove the protecting group without affecting the peptide bond, although it depends on the kind of the protecting group used. Is.

Examples of the method for removing the protecting group include acid treatment with formic acid, hydrogen chloride, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Other examples include reduction with sodium in liquid ammonia and palladium on carbon. In the deprotection group reaction by the acid treatment, it is effective to add a cation scavenger such as anisole, phenol or thioanisole.

After completion of the reaction, the tripeptide of the present invention thus produced can be obtained by a peptide separation means known per se, such as extraction, partitioning, reprecipitation, recrystallization, column chromatography and the like. You can

The tripeptide of the present invention can be converted into a carboxylic acid ester or a pharmaceutically acceptable salt thereof by a method known per se.

In the production method of the present invention, each fragment used as a starting material can be obtained as a commercially available product, or can be prepared by a person skilled in the art by a known synthetic method or by referring to a known synthetic method. It can be easily synthesized. Also, a condensate of two or three kinds of fragments can be easily produced as described above.

For example, the following formula (X-1), which is one of the condensates of the above A-B-Z fragment and the H-D-E-COOH fragment:

[0049]

[Chemical 14]

The compound represented by the formula (wherein, m is an integer of 4 to 7) has the following formula in the presence of a solvent which does not participate in the reaction:

[0051]

[Chemical 15]

A cyclic carboxylic acid represented by the formula (wherein, m is an integer of 4 to 7) is combined with a leucine having a protected carboxyl group in a solvent that does not participate in the reaction to form an amide bond known per se. It can be synthesized by reacting according to the method of formation to obtain an amide, and then removing the protective group of the carboxyl group. The compound represented by the above formula (X-1) can also be synthesized by a known Schotten-Baumann reaction using the acid chloride of the above-mentioned cyclic carboxylic acid and leucine.

The following formula (X-2), which is one of the condensates of the above A-B-Z fragment and the H-D-E-COOH fragment:

[0054]

[Chemical 16]

The compound represented by the formula (wherein, m is an integer of 4 to 7) has the following formula in the presence of a solvent which does not participate in the reaction:

[0056]

[Chemical 17]

The sulfochloride represented by the formula (wherein m is an integer of 4 to 7) is reacted with leucine having a protected carboxyl group, and then the protecting group of the carboxyl group is eliminated to synthesize the compound. be able to.

The tripeptide of the present invention has an excellent inhibitory effect on the binding of endothelin to the endothelin receptor, and suppresses the vasoconstriction reaction of endothelin, as is clear from the following test results. It is an excellent endothelin receptor antagonist because it has a large action.

[Test for Endothelin Binding to Endothelin Receptor] A membrane fraction was prepared from fresh porcine aorta by the following method. Adipose tissue was removed and minced vascular tissue was frozen in liquid nitrogen and powdered using a Waring blender. Then buffer [25 mM Hepes, pH 7.
4, 0.25M sucrose, 5mM EDTA · Na ₂ , 1mM
EGTA, 0.4 mM phenylmethylsulfonyl fluoride (PMSF)] in a homogenizer (YAMATO U
It was homogenized using a ltra-Dispercer). The homogenate was centrifuged at 1,475 xg and 2,700 xg for 10 minutes each and the supernatant was further centrifuged at 10,500 xg for 6 minutes.
Centrifuge for 0 minutes. The resulting pellet is buffered (25
mM Hepes, pH 7.4, 150 mM NaCl, 1 m
M EDTA.Na ₂ , 0.4 mM PMSF, 5 μg
/ Ml aprotinin), washed once with 0.25 mg /
Resuspend in the same buffer plus ml bacitracin -80
Stored at ° C.

The membrane preparation (40 μg protein / assay) was
¹²⁵ I] Endothelin (1 × 10 ⁻¹¹ M) (Amersham, specific activity 74 TBq / mmol) and 1 × 10 ⁻⁷ M test substance with a final dose of 250 μl of buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM). MgC
( ₁₂ , 0.5 mg / ml bacitracin, 1 mg / ml bovine serum albumin) for 90 minutes at 25 ° C. After reaction, Whatmann treated with 0.3% polyethyleneimine using a cell harvester (Brandel M-48R)
It was suction filtered with a GF / B filter. The filter is 3 ml of washing buffer (25 mM Tris / HCl, pH
It was washed 3 times with 7.4, 150 mM NaCl, 5 mM MgCl ₂ ) and the radioactivity captured on the filter was measured with a gamma counter (Aloka ARC-301B).

The inhibition rate (A) of the test substance was calculated by the following formula. A (%) = 100− (B−C) / (D−C) × 100 However, B: radioactivity in the presence of the test substance 1 × 10 ⁻⁷ M C: in the presence of 1 × 10 ⁻⁷ M endothelin Radioactivity D: Radioactivity of 1 × 10 ^-11 M [ ¹²⁵ I] endothelin alone

Table 1 shows the inhibition rates when the compounds obtained in the following Examples were used as test substances.

[0063]

[Table 1] Table 1 Inhibition rate ────────────────────────────── Compound 1 × 10 ⁻⁷ M (%) ── ──────────────────────────── Example 1 72 Example 2 72 ─────────────── ────────────────

Since the tripeptide of the present invention has an excellent endothelin antagonism, for example, hypertension, angina, cardiomyopathy, arteriosclerosis, myocardial infarction, Raynaud's disease, cerebral artery spasm, cerebral ischemia, It can be used as a therapeutic agent for stroke attacks such as late brain spasm after subarachnoid hemorrhage, asthma such as bronchoconstriction, and renal failure such as acute renal failure.

The tripeptide of the present invention can be used in the form of a pharmaceutical composition suitable for parenteral administration, oral administration and external administration by mixing with a known solid or liquid excipient. The dosage form may be any of injections, inhalants, syrups, emulsions, powders, capsules, granules, tablets, ointments, suppositories, and the like. In this pharmaceutical formulation, adjuvants, stabilizers, wetting agents, emulsifiers, absorption enhancers, surfactants, bulking agents, binders, disintegrants, which are usually used for preparing a drug according to the form of use, Lubricants and the like may be included. Distilled water for injection, physiological saline, Ringer's solution, glucose, sugar syrup, gelatin, vegetable oil, cocoa butter, ethylene glycol, hydroxypropyl cellulose, lactose, sucrose, corn starch, magnesium stearate, talc, etc. are used as additives. it can.

The dose of the tripeptide of the present invention as an endothelin antagonist will differ depending on the administration method, the degree of symptoms of the patient, the age of the patient, the body weight, etc. The daily dose for oral administration is about 0.1 to 100 mg / kg body weight, and the daily dose for parenteral administration is about 0.01 to 10 mg / kg.
It's weight.

[0067]

EXAMPLES Next, the present invention will be described in more detail by way of examples.

[Example 1] N- (N-cyclohexylcarbonyl-L-leucyl)
Synthesis of -N ⁱⁿ -formyl-D-tryptophyll-D-tryptophan

(1) Nt-butoxycarbonyl-D
Synthesis of tryptophan benzyl ester Nt-butoxycarbonyl-D-tryptophan 3.
04 g (10.0 mmol) of dry methylene chloride 40 m
It was dissolved in 1 and 122 mg (1.0 mmol) of 4-dimethylaminopyridine (hereinafter abbreviated as DMAP) and 1.19 g (11.0 mmol) of benzyl alcohol were added. 1-Ethyl-3 was added to this solution under ice cooling.
-(3-Dimethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as WSC.HCl) 2.10 g (1
(1.0 mmol) was added and the mixture was stirred at room temperature overnight. The reaction solution was washed with water and concentrated, and the residue was dissolved in ethyl acetate. The solution was washed with 10% citric acid, water, saturated sodium hydrogen carbonate solution, water and saturated saline solution in this order, and then anhydrous sodium sulfate was added to dry the solution. , Concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 2/1) to quantitatively obtain the title compound as a white crystalline powder.

(2) Synthesis of D-tryptophan benzyl ester hydrochloride Nt-butoxycarbonyl-D obtained in the above (1)
-Tryptophan benzyl ester (theoretical amount: 10 mmol) was dissolved in 60 ml of 90% formic acid and stirred for 6 hours while returning to room temperature from an ice bath. Formic acid was distilled off under reduced pressure, 1N-hydrochloric acid / dioxane (10 ml) was added to the residue, and the mixture was allowed to stand at room temperature for 15 minutes.
After stirring for a minute, ether was added to crystallize. The crystals were collected by filtration, washed with ether, and dried under reduced pressure to give the title compound as a white crystalline powder (2.68 g, yield: 8).
1.2%) was obtained.

(3) Nt-butoxycarbonyl-N
Synthesis of ⁱⁿ -formyl-D-tryptophyll-D-tryptophan benzyl ester 198 mg (0.6 mmol) of D-tryptophan benzyl ester hydrochloride obtained in the above (2) was dissolved in 5 ml of dry methylene chloride, and N- t- butoxycarbonyl -N ⁱⁿ - formyl -D- tryptophan 199m
g (0.6 mmol), triethylamine 0.08 ml
(0.6 mmol), and 1-hydroxybenzotriazole (hereinafter abbreviated as HOBt) monohydrate 92 m
g (0.7 mmol) was added and the mixture was stirred on an ice bath. Further, 126 mg (0.7 mmol) of WSC.HCl was added, and the mixture was stirred at room temperature overnight. The reaction solution was treated in the same manner as in the above (1) to quantitatively obtain 360 mg of the title compound.

(4) Synthesis of N ⁱⁿ -formyl-D-tryptophyll-D-tryptophan benzyl ester Nt-butoxycarbonyl-N obtained in the above (3)
9% ⁱⁿ -formyl-D-tryptophyll-D-tryptophan benzyl ester (theoretical amount: 0.3 mmol)
It was dissolved in 6% of 0% formic acid and stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in ethyl acetate and washed with saturated aqueous sodium hydrogen carbonate. The organic layer was dried over anhydrous sodium sulfate and then concentrated, and the obtained residue was directly used in the next reaction.

(5) N-cyclohexylcarbonyl-
Synthesis of L-leucine benzyl ester 5.9 g (15.0 mmol) of L-leucine benzyl ester / p-toluenesulfonic acid was dissolved in 40 ml of dry methylene chloride, and 4.2 ml of triethylamine (30. 0 mmol) and 2.2 g (15.0 mmol) of cyclohexylcarbonyl chloride were added, and the mixture was stirred for 2 hours. The reaction solution is water, 1N HCl,
The extract was washed successively with water, saturated aqueous sodium hydrogen carbonate, water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain 5.0 g (quantitative) of the title compound as white crystals. ¹ H NMR (CDCl ₃ ) δ; 0.9-1.0 (6H, m), 1.2-
2.0 (13H, m), 2.1-2.2 (1H, m), 4.6-4.7 (1H, m), 5.1-5.2 (2
H, m), 5.90 (1H, d, J = 8Hz), 7.3-7.4 (5H, m)

(6) N-cyclohexylcarbonyl-
Synthesis of L-leucine N-cyclohexylcarbonyl-obtained in the above (5)
L-leucine benzyl ester (4.9 g, 14.8 mmol) was dissolved in 100 ml of methanol, and 10 g of this was dissolved.
% Pd-C (1.4 g) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature overnight. The reaction solution was filtered and washed with methanol. The filtrate and the washing solution were combined and concentrated under reduced pressure to obtain 3.22 g (yield: 90.2%) of the title compound as white crystals. ¹ H NMR (CDCl ₃ ) δ; 0.9-1.0 (6H, m), 1.2-1.
9 (13H, m), 2.1-2.2 (1H, m), 4.5-4.6 (1H, m), 6.2-6.3 (1H,
m), 10.63 (1H, br s)

[0075] (7) N-(-L-N-cyclohexylcarbonyl-leucyl) -N ⁱⁿ - formyl -D- tryptophyl -D- tryptophan benzyl ester N ⁱⁿ obtained in the above (4) of the - formyl -D -Tryptophyll-D-tryptophan benzyl ester 195
20 mg of dry methylene chloride (mg (0.38 mmol))
And N-cyclohexylcarbonyl-L-leucine obtained in (6) above (92 mg, 0.38 mmol) and HOBt monohydrate (233 mg, 0.42 mmol) were added thereto, and the mixture was placed on an ice bath. Stir for 15 minutes. further,
319 mg (0.42 mmol) of WSC.HCl was added, and the mixture was stirred at room temperature overnight. The precipitated white solid was collected by filtration,
The crystals were washed successively with 10% citric acid, water, saturated aqueous sodium hydrogen carbonate and water, and dried under reduced pressure to give the title compound as a white solid (241 mg, yield: 85.8%). ¹ H NMR (DMSO- _d6 ) δ; 0.6-0.7 (6H, m), 1.1-
1.6 (13H, m), 2.1-2.2 (1H, m), 2.8-3.3 (4H, m), 4.1-5.0 (5
H, m), 7.0-10.8 (19H, m)

(8) Synthesis of N- (N-cyclohexylcarbonyl-L-leucyl) -N ⁱⁿ -formyl-D-tryptophyll-D-tryptophan N- (N-cyclohexylcarbonyl-L obtained in (7) above. -Leucyl) -N ⁱⁿ -formyl-D-tryptophyll-D-tryptophan benzyl ester 50 m
g (0.07 mmol) in 50 ml of tetrahydrofuran
Was dissolved in, and 10 mg of Pd-C (20 mg) was added thereto, and the mixture was stirred under a hydrogen atmosphere at room temperature for 5 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol / acetic acid = 200/1).
0/1) to give the title compound (18 mg, yield: 41.1%) as a white crystalline powder. ¹ H NMR (DMSO- _d6 ) δ; 0.6-0.7 (6H, m), 1.1-
1.6 (13H, m), 2.0-2.1 (1H, m), 2.8-3.4 (4H, m), 4.2-4.6 (3
H, m), 6.9-10.7 (16H, m), FAB MS (m / z); 642 (M + 1)

[0077] [Example 2] N- [N- (N- cycloheptyl carbonyl -L- leucyl) -N ⁱⁿ - formyl-D-tryptophyl]-D-
Synthesis of tryptophan

(1) N-cycloheptylcarbonyl-
Synthesis of L-leucine benzyl ester 3.94 g (10.0 mmol) of L-leucine benzyl ester / p-toluenesulfonic acid was added to methylene chloride 1
Dissolve in 00 ml, and under stirring with ice cooling, triethylamine 1.39 ml, cycloheptanecarboxylic acid 1.42 g
(10.0 mmol), and 2.11 g of WSC · HCl
(11.0 mmol) was added, and the mixture was stirred at room temperature for 4 days.
The reaction solution is water, 10% citric acid, water, saturated aqueous sodium hydrogen carbonate, water,
The extract was washed successively with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off, and the residue was purified by silica gel column chromatography (chloroform / methanol = 200/1) to give 2.75 g of the title compound (yield:
79.7%). ¹ H NMR (CDCl ₃ ) δ; 0.8-1.0 (6H, m), 1.4-2.
0 (15H, m), 2.2-2.3 (1H, m), 4.6-4.8 (1H, m), 5.13 (1H, d, J
= 12Hz), 5.17 (1H, d, J = 12Hz), 5.73 (1H, d, J = 9Hz), 7.3-7.
4 (5H, m)

(2) N-cycloheptylcarbonyl-
Synthesis of L-leucine N-cycloheptylcarbonyl-obtained in (1) above
2.41 g (7.0 mmol) of L-leucine benzyl ester was dissolved in 60 ml of tetrahydrofuran, 240 mg of 10% Pd-C was added thereto, and the mixture was stirred under a hydrogen atmosphere at room temperature overnight. The catalyst was filtered and washed with tetrahydrofuran. The filtrate and washings were combined and evaporated, and the obtained residue was recrystallized from ethyl acetate-hexane to give the title compound (8%).
45 mg (yield: 47.5%) was obtained. ¹ H NMR (CDCl ₃ ) δ; 0.9-1.0 (6H, m), 1.4-
2.0 (15H, m), 2.2-2.3 (1H, m), 4.5-4.7 (1H, m), 5.8-6.0 (1
H, m), 7.8 (1H, br s) FAB MS (m / z); 256 (M + 1)

(3) N-cycloheptylcarbonyl-
L-leucyl-N ⁱⁿ -formyl-D-tryptophyll-
Synthesis of D-tryptophan benzyl ester N ⁱⁿ -formyl-D-obtained in (4) of Example 1 above
Tryptophyll-D-tryptophan benzyl ester 153 mg (0.3 mmol) was dried with methylene chloride 1
It was dissolved in 0 ml, and 77 mg (0.3 mg of N-cycloheptylcarbonyl-L-leucine obtained in (2) above was dissolved therein.
(Mmol) and added with stirring under ice cooling, and further added with WSC.HCl6.
3 mg (0.33 mmol) was added, and the mixture was stirred at room temperature for 7 days. The precipitate was filtered, washed successively with water, 10% citric acid, water, saturated aqueous sodium hydrogen carbonate and water, and dried to give white crystals. This was recrystallized from dimethylformamide-ether to obtain 37 mg (yield: 16.5%) of the title compound. ¹ H NMR (DMSO- _d6 ) δ; 0.5-0.8 (6H, m), 1.0-
2.4 (16H, m), 2.8-3.5 (4H, m), 4.0-5.1 (5H, m), 6.9-9.8 (1
9H, m), 10.86 (1H, s) FAB MS (m / z); 746 (M + 1)

(4) Synthesis of N- [N- (N-cycloheptylcarbonyl-L-leucyl) -N ⁱⁿ -formyl-D-tryptophyll] -D-tryptophan N-cycloheptyl obtained in the above (3) Carbonyl-
L-leucyl-N ⁱⁿ -formyl-D-tryptophyll-
28 mg of D-tryptophan benzyl ester (0.
(038 mmol) was dissolved in tetrahydrofuran (7 ml), 10% Pd-C (5 mg) was added thereto, and the mixture was stirred under a hydrogen atmosphere at room temperature overnight. The catalyst was filtered and washed with tetrahydrofuran. The filtrate and washings were combined and the solvent was evaporated to obtain 25 mg (quantitative) of the title compound as white crystals. ¹ H NMR (DMSO-d6) δ; 0.5-0.8 (6H, m),
1.0-2.4 (16H, m), 2.8-3.5 (4H, m), 4.0-4.8 (3H, m), 6.6-
9.7 (14H, m), 10.79 (1H, s), 12.5 (1H, br s) FAB MS (m / z); 656 (M + 1)

[0082]

INDUSTRIAL APPLICABILITY The tripeptide of the present invention is a novel tripeptide and a substance having an excellent endothelin antagonism.

Claims (3)

[Claims] 1. A compound represented by the general formula (I): [In the formula, A represents a 5- to 8-membered cycloalkyl group, and B
Is, -CO -, - CS- or -SO ₂ - represents a group represented by, D is, -NR ¹ - represented by (wherein, R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms) Represents a group
E is -CHR ² - (where, R ² represents an aryl group or an aromatic heterocyclic alkyl group carbon atoms which may be have 1-7 optionally substituted with a group, or a sulfur atom-containing heterocyclic group) represented by Represents a group, and F represents the following formula: (In the formula, R ³ is a hydrogen atom, a —CHO group, an alkyl group having 1 to 6 carbon atoms, a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms, or a formula —COOR ⁴ (wherein, R ⁴ is Represents an alkyl group having 1 to 6 carbon atoms or a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms), and G is a group represented by the following formula: ] (Wherein R ⁵ represents a nitrogen atom-containing aromatic heterocyclic group), Q is a hydrogen atom, an alkali metal atom,
Represents a phenylalkyl group having an alkyl group having 1 to 6 carbon atoms or an alkylene group having 1 to 4 carbon atoms]. 2. General formula (I): [In the formula, A represents a 5- to 8-membered cycloalkyl group, and B
Is, -CO -, - CS- or -SO ₂ - represents a group represented by, D is, -NR ¹ - represented by (wherein, R ¹ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms) Represents a group
E is -CHR ² - (where, R ² represents an aryl group or an aromatic heterocyclic alkyl group carbon atoms which may be have 1-7 optionally substituted with a group, or a sulfur atom-containing heterocyclic group) represented by Represents a group, and F represents the following formula: (In the formula, R ³ is a hydrogen atom, a —CHO group, an alkyl group having 1 to 6 carbon atoms, a phenylalkyl group having an alkylene group having 1 to 4 carbon atoms, or a formula —COOR ⁴ (wherein, R ⁴ is Represents a phenylalkyl group having an alkyl group having 1 to 6 carbon atoms or an alkylene group having 1 to 4 carbon atoms), and G is a group represented by the following formula: ] (Wherein R ⁵ represents a nitrogen atom-containing aromatic heterocyclic group), Q is a hydrogen atom, an alkali metal atom,
Represents a phenylalkyl group having an alkyl group having 1 to 6 carbon atoms or an alkylene group having 1 to 4 carbon atoms], and each may have a protecting group. Fragment A-B-Z (where Z is a hydroxyl group or a halogen atom), H
-DE-COOH, HF-OH and HG-OQ
(These fragments may be their reactive derivatives) may be condensed in any order to give the tripeptides of general formula (I), or the two of the above four fragments may be condensed. A compound obtained by pre-condensing one or three kinds in any combination with two (or may be pre-condensed) or one of the remaining fragments to condense and, if desired, removing a protecting group. Manufacturing method. 3. An endothelin antagonist comprising the tripeptide according to claim 1 as an active ingredient.