JPH06263798A - Cyclic pentapeptide containing beta turn and gamma turn, its production and use thereof - Google Patents

Abstract

PURPOSE:To form beta turn and gamma turn irrespective of the kind of residue only by controlling optical activity of a main chain of a cyclic pentapeptide and to obtain a cyclic pentapeptide containing beta turn and gamma turn. CONSTITUTION:In a cyclic pentapeptide of the formula cyclo(-A1-A2-A3-A4-A5-), A1, A3 and A5 are D-alpha-amino acid residues and A2 and A4 are L-alpha-amino acid residues or A1, A3 and A5 are L-alpha-amino acid residues and A2 and A4 are D-alpha- amino acid residues to give the objective cyclic pentapeptide having a steric structure of combination of beta turn at the 1-2-3 positions with gamma turn at the 3-4-5-1 positions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for producing a cyclic pentapeptide having β-turns and γ-turns, a cyclic pentapeptide having β-turns and γ-turns, and uses thereof.

[0002]

2. Description of the Related Art β-turn and γ-turn are known as one of the ordered structures of protein and peptide molecules and have a structure in which they are bent at 4 and 3 amino acid residues, respectively. Normally, a β-turn forms a hydrogen bond between the carbonyl oxygen at the i-position and the amide proton at the i + 3 position, resulting in 2 bonds at the i + 1-position and the i + 2-position.
It is classified into types I, II, etc. according to the surface angles φ and ψ, and the γ-turn forms hydrogen bonds at the i-position and the i + 2 position. Such a turn structure is often present on the surface of the molecule, and is often a projecting structure, so that it is often related to recognition and interaction between molecules. With the recent development of X-ray crystallographic analysis and NMR analytical methods, the three-dimensional structures of many physiologically active peptides have been elucidated. As an example of a peptide containing a β-turn in the active site, enkephalin [Sudha, TS & Balaram, P. (1983) International Journal of the Peptide and Protein Research (Int.J.Pept.Protein Res.) ; twenty one
(4) 381-388; Goodman, M. et al. (1987) Biopolymers, 26 (Suppl.) S25-S32] and somatostatin [Nagai, U. et al. (1988) Peptide (P
ept.): Chemistry Biology Procedures American Peptide Symposium (Chem.Bio
l., Proc.Am.Pept.Symp.) 10th, 129-130.], and attempts have been made to mimic its β-turn part with a non-peptide compound [Ball, JB & Alewood, P.
F. (1990) Journal of Molecular Recognition (J.Mol.Recognit.), 3 (2) 55-64; Olson, GL
et al. (1990) Journal of American Chemical Society (J. Am. Chem. Soc.), 112 (1) 323-33.
3]. It has also been reported that an RGD (Arg-Gly-Asp) -related peptide having vitronectin-sensitive cell adhesion activity takes a γ turn at the adhesion site [Muller, G. et al.
(1992) Angewante Chemie International Edition (Angew.Chem.Int.Ed.Engl.), 31 (3) 32
6-328]. Recently, Banyu Pharmaceutical reported a cyclic pentapeptide BQ123 as an endothelin antagonist (US Pat. No. 5,114,918), but the stereostructure of the compound is not mentioned here. In addition, it has been reported that certain cyclic pentapeptides that act as endothelin receptor antagonists or endothelin antagonists take β and γ turns [Atkinson, RA & Pe.
lton, JT (1992) FEBS Lett.,
296 (1) 1-6; Krystek Jr., SR et al. (1992) FEBS
Lett., 299 (3) 255-261), which is believed to be the unique conformation of the amino acids that make up BQ123.

[0003]

If β and γ turns, which are important for physiological activity, can be created at will, it is considered to make a great contribution to drug development. Further, it becomes possible to synthesize a compound in which a desired amino acid residue is introduced into a β-turn or γ-turn site which is important for physiological activity, which is considered to provide an effective means for designing a compound having physiological activity. It is the object of the present invention to provide such a means.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies on a method for synthesizing a compound having β-turn and γ-turn, and as a result of synthesizing various cyclic pentapeptides, the β-turn has been obtained regardless of the substituent. The inventors have found a novel production method by which a compound having γ-turn and γ-turn can be freely synthesized. The present invention can control β by simply controlling the optical activity of the main chain of a cyclic pentapeptide, regardless of the type of amino acid residue.
A method for synthesizing a compound having a turn and a γ turn, β
The present invention relates to a cyclic pentapeptide having a turn and a γ-turn and its use.

Namely, the present invention _{is, (1) cyclo (-A 1} -A 2 -A 3 -A 4 -A 5 -) (I) _{wherein, A 1, A 2, A} 3, A 4, A ₅ is α-
Indicates an amino acid residue. ] In the cyclic pentapeptide represented by the following, A ₁ , A ₃ , A ₅ are D-α-amino acid residues and A ₂ , A ₄ are L-α-amino acid residues,
Alternatively, A ₁ , A ₃ , and A ₅ are L-α-amino acid residues, and A ₂ and A ₄ are D-α-amino acid residues, and a γ-turn and a _1-2 turn at position 1-2-3. 3-4-5-1
A cyclic pentapeptide having a three-dimensional structure in which β-turns are combined at positions (2) A ₁ , A ₃ , and A ₅ are D-α-amino acid residues, and A ₂ and A ₄ are L-α-amino acid residues Where A ₁ is D-Asp, A ₃ is D-Val, and A ₄ is L-.
When A ₅ is D-Trp in Leu, A ₂ is L-Pr
(3) A ₁ , A ₃ , and A ₅ are L-α, which is an L-α-amino acid residue other than o.
A cyclic pentapeptide according to (1) above, which is an amino acid residue, and A ₂ and A ₄ are D-α-amino acid residues,
(4) A ₁ is a D-α-amino acid residue, A ₂ is an L-α-amino acid residue having a protecting group, and A ₃ is D-α.
An amino acid residue, A ₄ is an L-α-amino acid residue, and A ₅ is a D-α-amino acid residue, (5) The above-mentioned (1) cyclic pentapeptide. To (4) an NK2 antagonist containing any of the cyclic pentapeptides, (6) an NK2 antagonist according to (5) above, which is an anti-asthma agent, an anti-inflammatory agent or an anti-arthritic agent, (7)
Wherein _{cyclo (-A 1 -A 2 -A 3} -A 4 -A 5 -) in Peptide Synthesis (I) _{wherein, A 1, A 2, A} 3, A 4, A 5 each α-
Indicates an amino acid residue. ] In the method for producing a cyclic pentapeptide represented by the following formula, A ₁ , A ₃ , A ₅ are D-α-amino acids, A ₂ , A ₄ are L-α-amino acid residues, or A ₁ , A ₃ , the L-alpha-amino acid residues a _5, a _2, a ₄
A method for producing a cyclic pentapeptide having a steric structure in which a γ-turn at the 1-2-3 position and a β-turn at the 3-4-5-1 position are combined, characterized in that a D-α-amino acid residue is arranged at , (8) _{cyclo (-A 1 -A 2 -A 3} -A 4 -A 5 -) (I) _{wherein, A 1, A 2, A} 3, A 4, A 5 each α-
Indicates an amino acid residue. ] In the method for producing a cyclic pentapeptide, a linear pentapeptide is produced by condensing two kinds of fragments bisected at any position of the cyclic pentapeptide represented by
D-α-amino acid residues at ₁ , A ₃ and A ₅ are replaced by A ₂ and A ₄
L-α-amino acid residues, or A ₁ , A ₃ , A ₅ with L-α-amino acid residues, and A ₂ , A ₄ with D-α-amino acid residues. The present invention relates to a method for producing a cyclic pentapeptide having a steric structure in which a γ-turn is combined at the 1-2-3 position and a β-turn is combined at the 3-4-5-1 position.

In the cyclic pentapeptide described in (2) above, A ₁ is D-alanine, D-valine, D-norvaline, D-leucine, D-norleucine, D-isoleucine, D-alloisoleucine, D-phenylalanine, D
-Tyrosine, D-tryptophan, D-serine, D-threonine, D-ornithine, D-lysine, D-arginine, D-histidine or D-methionine, A ₂
Is an L-α-amino acid residue, A ₃ is a D-α-amino acid residue, A ₄ is an L-α-amino acid residue, A
₅ is a D-α-amino acid residue, A ₁ is D-aspartic acid, D-glutamic acid or D-cysteic acid, A ₂ is an L-α-amino acid residue, and A ₃ is D- α-amino acid residue, A ₄ is L-α-amino acid residue, A ₅ is D-valine, D-norvaline, D-
Leucine, D-norleucine, D-isoleucine, D-
Alloisoleucine, D-serine, D-threonine, D-
Aspartic acid, D-glutamic acid, D-ornithine,
D-lysine, D-arginine, D-histidine, D-methionine or D-cysteine, A ₁ is D
-Aspartic acid, D-glutamic acid or D-cysteic acid, A ₂ is an L-α-amino acid residue, A ₃
Is D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-aspartic acid, D-glutamic acid, D-ornithine, D-lysine, D-arginine, D-histidine, D-methionine or D-cysteine. , A ₄ is an L-α-amino acid residue, A ₅
Preferred examples include those in which is D-phenylalanine, D-tyrosine, D-alanine or D-tryptophan. The protecting group (4) includes a hydrophobic group, and among them, a benzyl group is preferable.
As a specific example of (4), cyclo (-D-Glu-S
er (Bzl) -D-Leu-Leu-D-Trp) and cyclo (-D-Glu-Thr (Bzl) -D-L.
eu-Leu-D-Trp). In the general formula (I), the α-amino acid residue represented by A ₁ , A ₂ , A ₃ , A ₄ , and A ₅ is, for example, alanine,
Valine, norvaline, leucine, norleucine, isoleucine, alloisoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartic acid, glutamic acid, ornithine, lysine, arginine, histidine, methionine, cysteine, cysteic acid and the like can be mentioned. Furthermore, A ₂ also includes proline. Moreover, the side chain of each amino acid may be substituted. The side chain substituents of this amino acid include amino group protecting groups, carboxyl group protecting groups, thiol group protecting groups, guanidino group protecting groups, and hydroxyl group protecting groups described below.
Protecting group for phenolic hydroxyl group, protecting group for imidazole,
Indole protecting groups and the like are used.

In this specification, when amino acids and peptides are represented by abbreviations, IUPAC-IUB Co
It is based on the abbreviations used in mmision on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. Gly: glycine Ala: alanine Val: valine Nva: norvaline Ile: isoleucine aIle: alloisoleucine Leu: leucine tLeu: tert-leucine: asphine lysine: para lysine: arginine lysine: arginine lysine: arginine lysine: arginine lysine: arginine lysine: arginine lysine: arginine lysine: arginine lysine: lysine: lysine: lysine: lysine: lysine: lysine: lysine: lysine: lysine: lysine: lysine. Glutamic acid Asn: Asparagine Gln: Glutamine Ser: Serine Thr: Threonine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan mTrp: 5-Methyltryptophan Phg: Phenylglycine Nalina2-alna (2) -Nal (2) -Nal (2) -Nal (1) -Nal (1) -Nal (1) -Nal (2) -Nal (1) -Nal (1) -Nal (2) -Nal (1)) Also, the protecting groups and reagents commonly used in the text are indicated by the following abbreviations. That. Boc: t-butoxycarbonyl Bzl: benzyl BrZ: 2-bromobenzyloxycarbonyl ClZ: 2-chlorobenzyloxycarbonyl Tos: p-toluenesulfonyl For: formyl OBzl: benzyl ester OPac: phenacyl ester ONB: HONB ester TFA: Fluoroacetic 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.

DMSO: Dimethyl sulfoxide The cyclic pentapeptide (I) of the present invention can be produced by a conventional method known per se 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 a method known per se, for example, M. Bod
ansky and MA Ondetti, Peptide Synthesis, Interscience, New York, 1966; FM Finn and K. Hofmann.
Written by The Proteins, Volume 2, H.
Neurath, 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, Japan. Biochemical Society, edited by Tokyo Kagaku Dojin, 1977; Toshi Kimura et al., Sequential Biochemistry Experiment Course 2, edited by Biochemical Society of Japan, Tokyo Kagaku Dojin, 1987;
JM Stewart and JD Young, Solid Phase Peptide Syn
thesis), Pierce Chemical Company, Illinois, 19
84, etc., such as 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, DCC / HONB
Method, DCC / HOBt method, method using BOP reagent and the like.

The peptide relating to the present invention is cyclo (-A ₁ -A ₂ -A ₃ -A ₄ -A _5- ) (I) [wherein A ₁ , A ₂ , A ₃ , A ₄ , A ₅ are respectively α-
Indicates an amino acid residue. ] In the cyclic pentapeptide represented by the following, A ₁ , A ₃ , A ₅ are D-α-amino acid residues and A ₂ , A ₄ are L-α-amino acid residues,
Alternatively, A ₁ , A ₃ , and A ₅ are L-α-amino acid residues, and A ₂ and A ₄ are D-α-amino acid residues, and a γ-turn and a _1-2 turn at position 1-2-3. 3-4-5-1
It is a cyclic pentapeptide with a three-dimensional structure combining β-turns at positions. Since the peptide of the present invention relates to a cyclic peptide, cyclo (-A ₂ -A ₃ -A
_{4 -A 5 -A 1 -),} cyclo (-A 3 -A 4 -A 5
−A ₁ −A ₂ −), cyclo (−A ₄ −A ₅ −A ₁ −
A ₂ −A ₃ −), cyclo (−A ₅ −A ₁ −A ₂ −A
₃ -A ₄ -) also represents the same compound as in the above formula (I). The cyclic pentapeptide represented by the above formula (I) can be obtained by obtaining a linear peptide consisting of five amino acid residues constituting the pentapeptide and then cyclizing this linear peptide. . This linear peptide can be obtained by condensing a fragment consisting of two or more amino acids or by condensing amino acids one by one. One fragment has less than 5 amino acid residues that make up the cyclic peptide, the other fragment has the remaining amino acid residues, which can be a total of 5 amino acids. One fragment is 1 to 4
It can have amino acid residues. The cyclic peptide activates the carboxyl group of one of the two fragments, or the amino group of the other fragment, which is bisected at any position of the peptide bond, and does not participate in the reaction. The groups are protected and both fragments are condensed by conventional means of peptide synthesis to produce linear pentapeptides, followed by simultaneous or stepwise protection of the C-terminal α-carboxyl group and the N-terminal α-amino group protecting groups of the product. After removal, the C-terminal α-carboxyl group and the N-terminal α-amino group are intramolecularly condensed by a known ring-closing condensation method to obtain a cyclic pentapeptide, and when the product has a protecting group, it is optionally deprotected by conventional means. It can be manufactured by separating. Where A ₁ , A
₃ , A ₅ is a D-α-amino acid residue, and A ₂ and A ₄ are L-
α-amino acid residue, or L-α at A ₁ , A ₃ , and A ₅
-A three-dimensional structure in which an amino acid residue is combined with a γ-turn at the 1-2-3 position and a β-turn at the 3-4-5-1 position by arranging D-α-amino acid residues at A ₂ and A _4. Can be obtained. Cyclic pentapeptides prepared by the process of the present invention, specifically, the formula _{cyclo (-A 1 '-A 2'} -A 3 '-A 4' -A 5 '-) (II) wherein A ₁ ′, A ₃ ′ and A ₅ ′ each represent a D-α-amino acid residue, A ₂ ′ and A ₄ ′ each represent an L-α-amino acid residue, or A ₁ ′ and A ₃ ′. , a ₅ 'is an L-alpha-amino acid residues, respectively, a _2', a ₄ 'represents a D-alpha-amino acid residues, respectively. Here, "" represents that the case where the amino acid residue has a protecting group is included. ] Is represented. In the above formula, A ₁ ′, A ₂ ′,
The D-α-amino acid residues represented by A ₃ ′, A ₄ ′ and A ₅ ′ include the α-amino acid residues represented by A ₁ , A ₂ , A ₃ , A ₄ and A ₅ described above. Of the D form of A ₁ ′, A ₂ ′, A ₃ ′, A ₄ ′ and A
_The L-α-amino acid residue represented by ₅ ′ is the α represented by A ₁ , A ₂ , A ₃ , A ₄ and A ₅ described above.
-The L-form of the amino acid residues is used. More specifically, the present invention will be described in detail with reference to Examples below. For example, dipeptide X-A ₁ ′ -A ₂ ′ -O
H and the tripeptide H-A ₃ ′ -A ₄ ′ -A ₅ ′ -Y (provided that X and Y each represent a protecting group, A ₁ ′, A ₃ ′,
A ₅ ′ is a D-α-amino acid residue, A ₂ ′, A
₄ 'or respectively indicate a L-alpha-amino acid residue, or _{A 1', A 3 ',} A 5' , respectively, a L-alpha-amino acid residue, A ₂ ', A _4' are each D-alpha -Indicating an amino acid residue] with H-A ₁ ′ -A ₂ ′ -A ₃ ′-
A linear pentapeptide represented by A ₄ ′ -A ₅ ′ -OH is produced, and then this is subjected to ring closure condensation to give 1
It is possible to produce a cyclic pentapeptide (II) having a steric structure in which a γ-turn is located at the 2-3 position and a β-turn is located at the 3-4-5-1 position.

The protection and protection of a functional group not involved in the reaction of the raw material, elimination of the protection group, activation of the functional group involved in the reaction and the like can also be appropriately selected from known ones or means. Below, the peptide (I) of the present invention,
The protective group for the functional group of the amino acid constituting (II) and the amino acid as the raw material will be described. Examples of the amino-protecting group include carbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, tri- Fluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, 9-fluorenylmethyloxycarbonyl and the like can be mentioned. Among them, carbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl are preferable. Examples of the protective group for the carboxyl group include alkyl esters (eg, methyl, ethyl,
Ester groups such as propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl), benzyl ester, 4
-Nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, phenacyl ester, carbobenzoxyhydrazide, t-butyloxycarbonylhydrazide, tritylhydrazide and the like can be mentioned. Among them, alkyl ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl), benzyl ester and phenacyl ester are preferable. Examples of the protecting group for the thiol group of cysteine include paramethoxybenzyl, 4-methylbenzyl, benzyl, t-butyl, adamantyl, trityl, acetamidomethyl, carbomethoxysulfenyl, 3-nitro-2-pyridinesulfenyl and the like. To be Of these, paramethoxybenzyl, 4-methylbenzyl, benzyl, t-butyl, adamantyl, trityl and acetamidomethyl are preferable. Examples of the protective group for the guanidino group of arginine include nitro, tosyl, p-methoxybenzenesulfonyl, mesitylenesulfonyl, pentamethylbenzenesulfonyl, 4-methoxy-2,3,6.
-Trimethylbenzenesulfonyl, carbobenzoxy,
Examples thereof include isobornyloxycarbonyl and adamantyloxycarbonyl. Among them, nitro, tosyl, p-
Methoxybenzenesulfonyl, mesitylenesulfonyl,
Pentamethylbenzenesulfonyl and 4-methoxy-
2,3,6-Trimethylbenzenesulfonyl is preferred. In addition, the guanidino group may be protected in the form of an acid (eg, benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid, etc.) salt.

The hydroxyl groups of serine and threonine can be protected by, for example, esterification or etherification. Examples of the group suitable for this esterification include a lower alkanoyl group such as acetyl group, an aroyl group such as benzoyl group, a group derived from carbonic acid such as benzyloxycarbonyl group and ethyloxycarbonyl group, among which acetyl and benzoyl groups. And benzyloxycarbonyl are preferred. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, a t-butyl group and the like, and among them, a benzyl group, a tetrahydropyranyl group and a t-butyl group are preferable. However, it is not always necessary to protect the hydroxyl groups of serine and threonine. Examples of the protective group for the phenolic hydroxyl group of tyrosine include benzyl, 2,6-dichlorobenzyl, 2-nitrobenzyl, 2-bromobenzyloxycarbonyl, t-butyl and the like, and among them, benzyl, 2,6-dichloro. Benzyl, 2-bromobenzyloxycarbonyl and t-butyl are preferred but not necessarily protected. Methionine may be protected in the form of sulfoxide. Examples of the protecting group for imidazole of histidine include p-toluenesulfonyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl,
2,4-dinitrophenyl, benzyloxymethyl, t
-Butoxymethyl, t-butoxycarbonyl, trityl, 9-fluorenylmethyloxycarbonyl and the like can be mentioned, among which 2,4-dinitrophenyl, benzyloxymethyl, t-butoxymethyl, t-butoxycarbonyl and trityl are preferable. , Not necessarily protected. Examples of the protecting group for indole of tryptophan include formyl, 2,4,6-trimethylbenzenesulfonyl, 2,4,6-trimethoxybenzenesulfonyl,
4-methoxy-2,3,6-trimethylbenzenesulfonyl, β, β, β-trichloroethyloxycarbonyl, diphenylphosphinothioyl t-butoxycarbonyl and the like are mentioned, among which formyl and 2,4,6-trimethylbenzene. Sulfonyl, 2,4,6-trimethoxybenzenesulfonyl and 4-methoxy-2,3,6
-Trimethylbenzenesulfonyl is preferred but not necessarily protected.

The carboxyl group is activated prior to the reaction due to the condensation reaction and ring-closing condensation reaction described above. Examples of activated carboxyl groups include the corresponding acid anhydrides, azides and active esters [alcohols. (Eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide, N- Esters with hydroxysuccinide, N-hydroxyphthalimide, N-hydroxybenztriazole), and the like. Further, the amino group may be activated instead of the activation of the carboxyl group, and examples of the activated amino group include 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 water-containing dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, acetonitrile, ethyl acetate, N-methylpyrrolidone or an appropriate mixture thereof can be used. The reaction temperature is appropriately selected from the range known to be applicable to peptide bond-forming reactions, and is usually about -20 ° C to
It is appropriately selected from the range of 30 ° C.

The intramolecular ring-closing condensation reaction can be carried out at any position of the peptide by a known method. For example, first, the C-α-carboxyl protecting group of the C-terminal amino acid of the protected pentapeptide is eliminated by a known method and then activated by a known method, and then the N-α-amino protecting group of the N-terminal amino acid is removed. It is carried out by elimination by a known method and ring-closing condensation in the molecule. Alternatively, after removing the C-α-carboxyl protecting group of the C-terminal amino acid of the protected pentapeptide and the N-α-amino protecting group of the N-terminal amino acid simultaneously or sequentially, known methods using condensing agents such as dicyclohexylcarbodiimide are known. You may cyclize in a molecule by a condensation reaction. In some cases, it may be preferable to carry out the intramolecular ring-closing condensation reaction under high dilution. When the peptide (I) of the present invention thus produced has a protecting group, the protecting group can be eliminated by a conventional method, but physiological activity can be exerted even if the protecting group is not eliminated. . Examples of the method for removing the protecting group include P
Catalytic reduction under a hydrogen stream in the presence of a catalyst such as d black or Pd carbon, and acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Moreover, reduction with sodium in liquid ammonia can also be mentioned. The leaving group reaction by the acid treatment is generally -20 ° C to 40 ° C.
Is performed at an appropriate temperature, but in acid treatment, anisole, phenol, thioanisole, m-cresol, p
-The addition of cation scavengers such as 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 1,2-ethanedithiol or 1,4-butane as described above. 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.

After the reaction, the cyclic pentapeptide (I) thus produced is collected by means of peptide separation such as extraction, partitioning, reprecipitation, recrystallization, column chromatography, high performance liquid chromatography and the like. It The cyclic pentapeptide (I) according to the present invention can also be obtained as a salt such as sodium salt, potassium salt, calcium salt, magnesium salt or the like, or an acid addition salt, particularly a pharmacologically acceptable acid addition salt, by a method known per se. Examples thereof include salts of inorganic acids (eg, hydrochloric acid, sulfuric acid, phosphoric acid) or organic acids (eg, acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, methanesulfonic acid).

Next, the three-dimensional structure of the cyclic pentapeptide according to the present invention will be described. The three-dimensional structure analysis method used in the present invention uses a pulse-Fourier transform two-dimensional NMR method developed by Ernst (Nobel Prize in Chemistry in 1991) and the like, and a three-dimensional structure of a biopolymer in solution developed by Wuethrich et al. The structure determination method is followed. To date, there are hundreds of peptides and proteins whose three-dimensional structures have been determined by this method, and they have become the main method of three-dimensional structure determination together with X-ray crystallographic analysis. (1) Cyclic pentapeptide cyclo (-D-Glu-
Three-dimensional structure of Ala-D-Phe-Leu-D-Trp-) (A) A (details described in Examples below) of DMSO-d ₆ synthesized by the method described above at a concentration of 6.7 mg / ml.
AM500 manufactured by Bruker
The following measurements were made with a spectrometer. One-dimensional at 30 ℃
After checking the signal separation by ¹ H NMR spectrum (Fig. 1), the method of Wuethrich et al. (Wuethrich, K. (198
6) NMR of proteins and nucleic acids, Wiley, New
York), DQF-COSY, HOHAHA (τ
_mix = 90 ms), ROESY (τ _mix = 20, 40,
(60, 80, 100, 120 ms) was measured and each proton signal was assigned by the chain assignment method (FIG. 2). ROES
In addition to the distance restrictions collected from Y, J _NH-αH , J
The range of dihedral angles φ and χ ¹ was limited by the _αH-βH value. Although not used as distance information, the temperature dependence of the amide protons was examined to determine the presence or absence of intramolecular hydrogen bonds. A summary of the above NMR information is shown in FIG. Distance geometry calculation was performed from the obtained distance information, and further energy minimization calculation with distance limitation was performed to determine the three-dimensional structure of FIG. The three-dimensional structure is characterized by 1 in relation to the main chain structure.
A γ-turn was formed at the 2-3 position, and a type II β-turn was formed at the 3-4-5-1 position. A hydrogen bond is formed between the amide proton at the 3-position and the carbonyl oxygen at the 1-position, and between the amide proton at the 1-position and the carbonyl oxygen at the 3-position. I am doing it.

(2) Cyclic pentapeptide c (-Trp-
Stereostructure of D-Leu-Leu-D-Ala-Glu-) (B) c (-D-Glu-Ala-) having the same main chain structure as A
A retro-inverso body B expected to have the same main chain structure and the same side chain type and arrangement as D-Leu-Leu-D-Trp-) (C) was synthesized according to the method described above. The retro-inverso body refers to the one in which the amino acid residue sequence and the D and L arrangements are reversed. As shown in FIG. 5, the retro-inverso body B was expected to have the same main chain structure and side chain arrangement only by replacing the amino group and the carbonyl group in the peptide bond of C. In this case, B is 5-1-2
A β-turn structure is formed at the 3-position and a γ-turn structure is formed at the 3-4-5 position, forming a hydrogen bond between the amide proton at the 5-position and the carbonyl oxygen at the 3-position and between the amide proton at the 3-position and the carbonyl oxygen at the 5-position. Will be done. B was dissolved in DMSO-d ₆ at a concentration of 5.5 mg / ml to prepare an NMR sample.
After checking the signal separation by one-dimensional 1H NMR spectrum (Fig. 6) at ° C, DQF-COSY, HOHA
HA (τ _mix = 90 ms), ROESY (τ _mix = 3)
(0, 60, 90, 120 ms) was measured, and each proton signal was assigned by the chain assignment method (FIG. 7). Distance limit collected from ROESY as in (1) and J
Restriction information of dihedral angles φ and χ ¹ was collected by _NH-αH and J _αH-βH values, and the temperature dependence of amide protons was investigated (Fig. 8). The three-dimensional structure was calculated from the obtained distance information in the same manner as (1), and the three-dimensional structure of FIG. 9 was determined. The obtained three-dimensional structure is different from the expected one, forming a γ-turn at the 1-2-3 position, a type-II β-turn at the 3-4-5-1 position, and a hydrogen bond at the 3-position. It was formed between the amide proton and the carbonyl oxygen at the 1-position and between the amide proton at the 1-position and the carbonyl oxygen at the 3-position (consistent with the temperature-dependent data). (3) Rule derived from the three-dimensional structure of A and B c (-Glu-D-Al in which the arrangements of D and L of A are exchanged
a-Phe-D-Leu-Trp-) (A ′) is shown in FIG.
As a result of this analysis, it was clarified that, as shown in 0, A has a three-dimensional structure having an enantiomer relationship with A, and B has the same main chain structure as A '. Since the types of side chains at the corresponding positions of B and A'are completely different, they have the same main chain structure, so it is concluded that the main chain structure is determined by the arrangement of D and L. That is, -LD-LD-
Cyclic pentapeptides having the L- and -D-L-D-L-D- configurations are γ-turns at the 1-2-3 position and type at the 3-4-5-1 position regardless of the type of side chain. It is thought to form the β-turn of II.

[0017]

By the production method of the present invention, a compound having a β-turn and γ-turn steric structure can be produced regardless of the type of amino acid residue, and the β-turn is important from the viewpoint of biological activity. And the desired amino acid residue can be introduced at the position of the γ turn. Therefore, the present invention is useful for designing various compounds having biological activity. The cyclic pentapeptide of the present invention has a β-turn in combination with a γ-turn, so that its structure is rigid and has an ability to bind to a receptor having a specific structure depending on the form of this peptide. Therefore,
The cyclic pentapeptide can be used for screening receptors and elucidating the structure and function of the receptors, and can also be used as a kind of antagonist or agonist depending on the relationship with these receptors. In particular, certain cyclic pentapeptides of formula I are for example NK2
It exhibits a pharmacological action like an antagonist to the receptor and endothelin receptor, and can be used as an anti-asthma agent, an anti-inflammatory agent, an anti-arthritic agent or the like. The cyclic pentapeptide of the present invention can be used as a reagent for measuring the reaction selectivity for NK2 receptors and the selectivity for endothelin A or B receptors in cell and tissue samples. Further, the cyclic pentapeptide of the present invention can also be used as a reagent for purification by affinity chromatography.

The cyclic pentapeptide (I) of the present invention is NK
It exerts the remarkable effect of suppressing the contraction of smooth muscle through the 2 receptor and having low toxicity. For this reason,
The cyclic pentapeptide or a salt thereof of the present invention is a mouse,
It can be used as a prophylactic agent and a therapeutic agent as an anti-asthma agent, an anti-inflammatory agent or an anti-arthritic agent, as an NK2 receptor antagonist for mammals such as rat, rabbit, cat, dog, monkey and human. it can. When the cyclic peptide of the present invention is used as the above-mentioned therapeutic agent, it is used as it is or mixed with a pharmacologically acceptable carrier (carrier, excipient, diluent), and 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. As the carrier, distilled water, physiological saline, an aqueous solvent such as Ringer's solution, an isotonicity agent (eg, glucose, D-sorbitol, D-mannitol, sodium chloride, etc.), a preservative (eg, benzyl alcohol, chloro). Butanol, methyl paraoxybenzoate, propyl paraoxybenzoate, etc.), buffering agents (eg, phosphate buffer, sodium acetate buffer, etc.) and the like are used. The cyclic peptide of the present invention is mainly administered parenterally (for example, intravenous or subcutaneous injection, intraventricular or intraspinal injection, nasal administration, rectal administration),
In some cases, it may be administered orally. 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 form a lyophilized 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 amount of the free form of the cyclic peptide, the base salt, and the acid addition salt is generally 1 dose body weight 1 kg as the free form.
An appropriate amount is in the range of 1 μg to 100 mg. 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 usual medicinal component (peptide [I]) 1 The dose is about 1 μg / kg to 100 mg / kg body weight, more preferably about 100 μg / kg to 20 mg / kg body weight, and even more preferably about 1 mg / kg to 20 mg / kg body weight once to three times a day. Is convenient. The injection is usually an intravenous injection. In addition, infusion is also effective, and the total dose for intravenous infusion is the same as for intravenous injection. When this cyclic peptide is used as a therapeutic agent, it must be carefully purified so that bacteria and pyrogens do not exist.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following examples. In addition, the D-form and the L-form are included in the α-amino acid
When a body is present, it is an L-body except where D and L are specifically noted. The thin-layer chromatography plate used in the examples is a product of Merck (SILICAGEL 60F-254),
As the developing solvent, Rf _1: chloroform - methanol (19: 1), Rf _2: chloroform - methanol - acetic acid (9: 1: 0.5) was used. Example 1 Cyclo (-D-Glu-Ala-DP
he-Leu-D-Trp- manufacture of) (I) Boc-Ala- OPac manufacturing Boc-Ala-OH 28.4g and Cs ₂ CO ₃ 2
Dissolve 4.5 g in 90% MeOH in water, concentrate, dissolve the residue in DMF (450 ml) and phenacyl bromide 32.9.
g and stirred overnight. The precipitate formed is filtered off,
The filtrate was concentrated, the residue was dissolved in AcOEt and this was 4%.
Wash with NaHCO ₃ water, 10% citric acid water, wash with water,
After drying over Na ₂ SO ₄ and concentrating, ether was added to the residue and the crystals were collected by filtration. Yield 42.4 g (yield 91.9%) m.p. p. 123 ° C Rf 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.I. 62.53; 6.89;
4.56 Found: C.I. 62.94; 6.87;
4.63 (II) Boc-D-Glu (OBzl) -Ala-OP
Production of ac Boc-D-Glu (OBzl) -OH (2.02 g) was dissolved in THF, and the mixture was cooled to -15 ° C while stirring. 0.66 ml of N-methylmorpholine was added, followed by 0.80 ml of IBCF. 2 minutes later HCl.H-
Ala-OPac and N-methylmorpholine 0.66 ml
DMF solution was added (HCl.H-Ala-OPac
Is Boc-Ala-OPac 1.84 g TFA20
ml was added to dissolve and then concentrated, 8N-HCl / dioxane 1.95 ml was added, ether was added and the precipitated crystals were collected by filtration and dried). 3 at -15 ° C
After stirring for 0 minutes, the temperature was returned to room temperature. After 30 minutes, the insoluble matter was filtered off and concentrated, the residue was dissolved in AcOEt, washed with 4% NaHCO ₃ water, 10% citric acid water, washed with water, dried over Na ₂ SO ₄ and concentrated, The crystals were collected by filtration from AcOEt-petroleum ether. Yield 2.63 g (yield 83.2%) m.p. p. 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.I. 63.87; 6.51;
5.32 Found: C.I. 63.88; 6.32;
5.18 (III) Boc-D-Trp-D-Glu (OBzl)
-Production of Ala-OPac Boc-D-Glu (OBzl) -Ala-OPac
TFA (20 ml) was added to 1.05 g to dissolve and then concentrated, 8N-HCl / dioxane (0.58 ml) was added, ether was added, and the precipitated crystals were collected by filtration and dried. This was dissolved in DMF (15 ml) and cooled with ice, and TEA 0.2
0 ml was added. To this, Boc-D-Trp-ONB
(Boc-D-Trp-OH 0.67 g, HONB
0.43 g and DCC 0.50 g) was added and the mixture was stirred overnight. The resulting insoluble matter was filtered off and concentrated, and the residue was dissolved in AcOEt, which was washed with 4% NaHCO ₃ water and 10% citric acid water, washed with water, dried over Na ₂ SO ₄ and concentrated to leave a residue. Ether was added to the product, and the precipitate was collected by filtration. Yield 1.39 g (yield 97.8%) m.p. p. 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.I. 65.72; 6.22;
7.86 Found: C.I. 65.62; 6.46;
7.78 (IV) Boc-Leu-D-Trp-D-Glu (OB
zl) -Production of Ala-OPac Boc-D-Trp-D-Glu (OBzl) -Ala
21.4 g of -OPac was suspended in dioxane, 5.1 ml of ethanedithiol was added, and the mixture was ice-cooled, and 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. This was dissolved in 150 ml of DMF and cooled with ice, and TEA 8.
42 ml was added. To this, Boc-Leu-ONB
(Boc-Leu-OH.H2O 8.22g, HON
B (prepared from 6.51 g and DCC 7.50 g) was added and the mixture was stirred overnight. The resulting insoluble matter is filtered off and then concentrated,
The residue was dissolved in AcOEt and this was added to 4% NaHCO ₃
Wash with water, 10% citric acid water, wash with water, and then wash with Na ₂ SO _4.
After drying and concentration with ether, ether was added to the residue and the precipitate was collected by filtration. Yield 23.0 g (yield 92.8%) m.p. p. 127-128 ° C Rf ₁ 0.35
Rf ₂ 0.63 [α] _D 28 −9.91 ° (c = 1.15 in DMF) Elemental analysis Calculated as C ₄₅ H ₅₅ N ₅ O ₁₀ : C.I. 65.44; 6.71;
8.48 Found: C.I. 65.39; 6.92;
8.30 (V) Boc-D-Phe-Leu-D-Trp-D-
Production of Glu (OBzl) -Ala-OPac Boc-Leu-D-Trp-D-Glu (OBzl)
-Ala-OPac (2.07 g) was suspended in dioxane, ethanedithiol (0.42 ml) was added, and the mixture was ice-cooled to give 8N.
-HCl / dioxane was added, and the mixture was stirred under ice cooling for 30 minutes and then concentrated. Ether was added, and the precipitated crystals were collected by filtration and dried. Dissolve this in DMF (15 ml) and chill with ice.
A 0.70 ml was added. To this, Boc-D-Ph
e-OH 0.73g, HOBt 0.41g, DCC
0.63 g was added and stirred overnight. The resulting insoluble matter was filtered off and concentrated, the residue was dissolved in AcOEt, washed with 4% NaHCO ₃ water, 10% citric acid water, washed with water, dried over Na ₂ SO ₄ and concentrated to leave a residue. Ether was added to the product, and the precipitate was collected by filtration. Yield 2.37 g (yield 97.4%).

(VI) Boc-D-Phe-Leu-D-
Production of Trp-D-Glu (OBzl) -Ala-OH Boc-D-Phe-Leu-D-Trp-D-Glu
90% of 1.46 g of (OBzl) -Ala-OPac
It was dissolved in 20 ml of AcOH water, 4.91 g of Zn powder 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, washed with 10% citric acid, 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 1.20 g (yield 93.6%) (VII) Cyclo (-D-Glu-Ala-D-Ph
e-Leu-D-Trp) Boc-D-Phe-Leu-D-Trp-D-Glu
0.43 g of (OBzl) -Ala-OH was added to DCM 2
Dissolve in 0 ml, cool with ice, HONB 0.18 g, DC
After 0.21 g of C was added and the mixture was stirred for 3 hours, the DCU formed was filtered off and concentrated, and ether was added to the residue, and the precipitate was collected by filtration. Under ice cooling, 0.09 ml of ethanedithiol and 20 ml of 8N-HCl / dioxane were added and dissolved, followed by stirring for 10 minutes and concentration. Ether was added to the residue, and the precipitate was collected by filtration and dried. This is D
Dissolved in 10 ml of MF and containing 0.7 ml of TEA D
The mixture was added dropwise to 90 ml of MF over 30 minutes, then stirred overnight and concentrated. Acetonitrile was added to the residue and the precipitate was collected by filtration and dried. 74 mg of this is DMF
It was dissolved in 15 ml, and catalytic reduction was carried out in a hydrogen stream using Pd-carbon as a catalyst. The catalyst was filtered off and then concentrated. The residue was dissolved in a small amount of AcOH, water was added, and the mixture was freeze-dried. Finally this is YMC-made YMC-D-O
The product was purified by preparative liquid chromatography using a DS-5 (2 cm x 25 cm) column to obtain the desired product. Yield 9.1 mg (yield 14.1%) Amino acid analysis value (hydrolysis for 24 hours at 110 ° C .; ()
The inside shows the theoretical value. ): Glu 1.00 (1); Al
a 0.98 (1); Leu 0.76 (1); Phe
0.81 (1) LSIMS (M + H +) = 647 (theoretical value) = 64
7.

Example 2 Cyclo (-Trp-D-L
eu-Leu-D-Ala-Glu-) (I) Production of Boc-D-Leu-Leu-OBzl H-Leu-OBzl pTos 21.6 g of DMF
Dissolve in 100 ml, cool with ice, TEA 7.7 ml, B
oc-D-Leu-ONB (Boc-D-Leu-OH
・ H ₂ O 12.5g, HONB 9.86g, DCC
(Prepared from 11.4 g) was added and stirred overnight. The DCU formed was filtered off, the filtrate was concentrated and the residue was treated with AcOEt.
This was dissolved in 4% NaHCO ₃ water, washed with 4% NaHCO ₃ water and 10% citric acid water, washed with water, dried over Na ₂ SO ₄ and concentrated. Ether was added to the residue, and crystals were collected by filtration. Yield 19.8 g (yield 91.3%) m.p. p. 94-95 ° C. Rf2 0.76 [α] _D 25 + 3.6 ° (c = 1.06 in DMF) Elemental analysis Calculated as C ₂₄ H ₃₈ N ₂ O ₅ : C.I. 66.33; 8.81;
6.45 Found: C.I. 66.38; 8.87;
6.53 (II) Production of Boc-D-Leu-Leu-OPac 6.0 g of Boc-D-Leu-Leu-OBzl was dissolved in 20 ml of methanol, and 10% Pd-carbon was used as a catalyst for catalytic reduction under a hydrogen stream. I did. The catalyst was filtered off and then concentrated, and the residue and Cs ₂ CO ₃ 2.1 g were added to 90% Me
It was dissolved in OH water and concentrated. The residue was dissolved in DMF (60 ml), phenacyl bromide (2.8 g) was added, and the mixture was stirred overnight. The formed precipitate was filtered off, the filtrate was concentrated, the residue was dissolved in AcOEt, and the residue was dissolved in 4% NaHCO ₃ water, 10%.
It was washed with citric acid water, washed with water, dried over Na ₂ SO ₄ and concentrated. Ether was added to the residue, and the crystals were collected by filtration. Yield 5.48 g (yield 85.8%) m.p. p. 98-99 ° C. Rf ₂ 0.66 [α] _{D 25 -3.9 ° (c = 1.09} , in DMF) Elemental analysis C ₂₅ H ₃₈ N ₂ O ₆ Calculated: C. 64.91; 8.28;
6.06 Found: C.I. 65.21; 8.54;
6.24 (III) Boc-Trp-D-Leu-Leu-OPa
Preparation of c Boc-D-Leu-Leu-OPac (9.25 g) was dissolved in dioxane (5.0 ml) and ice-cooled, and 10N-HC was used.
1 / dioxane (30.0 ml) was added and the mixture was stirred for 30 minutes. The solvent was distilled off at room temperature and dried under reduced pressure. This is D
It was dissolved in 20 ml of MF and neutralized with TEA while stirring under ice cooling. In addition to this, Boc-Trp-ONB (Boc-
Trp-OH 6.08 g, HONB 4.30 g, D
CC (prepared from 5.45 g) was added, and the mixture was stirred at room temperature overnight. The generated insoluble matter was removed by filtration, the filtrate was concentrated, the residue was dissolved in ethyl acetate, washed with 4% aqueous sodium hydrogen carbonate solution, 10% aqueous citric acid solution, washed with water, dried and concentrated with Na ₂ SO ₄ ,
Ether-petroleum ether was added to the residue, and the precipitate was collected by filtration. Yield 12.19 g (yield 93.9%) m. p. 98-100 ° C Rf ₁ 0.43 R
f ₂ 0.67 Elemental analysis Calculated as C ₃₆ H ₄₈ N ₄ O ₇ : C.I. 66.65; 7.46;
8.64 Found: C.I. 66.73; 7.41;
8.45.

(IV) Boc-Glu (OBzl) -Tr
Production of p-D-Leu-Leu-OPac Boc-Trp-D-Leu-Leu-OPac 3.
Dissolve 24 g in 2.0 ml dioxane and cool with ice,
10.0 ml of N-HCl / dioxane was added and stirred for 30 minutes. The solvent was distilled off at room temperature, ether was added, and the deposited precipitate was collected by filtration and dried under reduced pressure. DMF this
It was dissolved in 10 ml and neutralized with TEA while stirring under ice cooling. In addition, Boc-Glu (OBzl) -ONB (B
oc-Glu (OBzl) -OH 1.78g, HON
B 1.18 g and DCC 1.36 g) were added, and the mixture was stirred at room temperature overnight. The resulting insoluble matter was filtered off, the filtrate was concentrated, the residue was dissolved in ethyl acetate and
After washing with sodium bicarbonate water and 10% citric acid aqueous solution and washing with water, Na ₂
The mixture was dried and concentrated with SO ₄ , ether-petroleum ether was added to the residue, and the precipitate was collected by filtration.

Yield 3.91 g (yield 87.2%) m.p. p. 88-90 ° C Rf ₁ 0.37 Rf ₂
0.67 Elemental analysis C ₄₈ H ₆₅ N ₅ O ₁₂ Calculated: C. 63.77; 7.25;
7.75 Found: C.I. 63.93; 7.33;
7.57 (V) Boc-D-Ala-Glu (OBzl) -Tr
Production of p-D-Leu-Leu-OPac Boc-Glu (OBzl) -Trp-D-Leu-L
3.14 g of eu-OPac was dissolved in 2.0 ml of dioxane, ice-cooled, and 10N-HCl / dioxane 10.0
ml was added and stirred for 30 minutes. Distill off the solvent at room temperature,
Ether was added and the deposited precipitate was collected by filtration and dried under reduced pressure. This was dissolved in 10 ml of DMF and neutralized with TEA while stirring under ice cooling. To this, Boc-D-Ala
-ONB (Boc-D-Ala-OH 0.83 g, H
ONB (0.86 g, prepared from DCC 1.16 g) was added, and the mixture was stirred at room temperature overnight. The resulting insoluble matter was filtered off, the filtrate was concentrated, and the residue was dissolved in ethyl acetate.
% Sodium bicarbonate water, 10% citric acid aqueous solution, washed with water, and then Na
_The mixture was dried and concentrated with ₂ SO ₄ , ether-petroleum ether was added to the residue, and the precipitate was collected by filtration.

Yield 2.94 g (89.5% yield) m.p. p. 188-190 ° C Rf ₁ 0.29
Rf ₂ 0.67 Elemental analysis Calculated as C ₅₁ H ₆₆ N ₆ O ₁₁ : C.I. 65.23; 7.08;
8.95 Found: C.I. 65.98; 7.23;
8.87 (VI) Boc-D-Ala-Glu (OBzl) -Tr
Production of p-D-Leu-Leu-OH Boc-D-A
la-Glu (OBzl) -Trp-D-Leu-Le
u-OPac (1.41 g) was dissolved in 90% acetic acid (50 ml), and Zn powder (4.96 g) was added with stirring under ice cooling.
Furthermore, it stirred at room temperature for 4 hours. Zn powder is filtered off,
The filtrate was concentrated, the residue was dissolved in ethyl acetate, and washed successively with 10% aqueous citric acid solution and saturated brine. Na ₂ SO
After drying at ₄ , the solvent was distilled off, ether-petroleum ether was added to the residue, and the precipitate was collected by filtration and dried under reduced pressure.

Yield 1.05 g (yield 85.8%) m.p. p. 122.0-124.0 ° C. Rf ₁ 0.0
6 Rf ₂ 0.56 (VII) Cyclo (-Trp-D-Leu-Leu-
Preparation of D-Ala-Glu (OBzl)-) Boc-D-Ala-Glu (OBzl) -Trp-D
-Leu-Leu-OH (0.30 g) was dissolved in dichloromethane (3.5 ml), and HONB was added while stirring under ice cooling.
143 mg and DCC 165 mg were sequentially added, and the mixture was further stirred under ice cooling for 3 hours. The insoluble matter that forms is filtered off,
The solvent was distilled off. Acetonitrile was added to the residue to obtain a precipitate, which was collected by filtration and dried under reduced pressure. Dioxane 2
Dissolve in 10 ml of 10N-HCl /
10 ml of dioxane was added, and the mixture was further stirred for 10 minutes. The solvent was distilled off at room temperature, ether was added to the residue, and the precipitate was collected by filtration and dried under reduced pressure. This was dissolved in 5 ml of DMF, added dropwise to 80 ml of DMF containing 5.57 ml of TEA, and further stirred overnight. The solvent was distilled off, the residue was dissolved in a small amount of DMF, ethyl acetate was added, and the precipitate was collected by filtration and dried under reduced pressure.

Yield 195 mg (yield 75.9%) Rf ₁ 0.29 Rf ₂ 0.67 (VIII) Cyclo (-Trp-D-Leu-Leu-
Preparation of D-Ala-Glu-) Cyclo (-Trp-D-Leu-Leu-D-Al
a-Glu (OBzl)-) 100 mg in DMF 10
It was dissolved in ml, 100 mg of Pd black was added, and the mixture was vigorously stirred at room temperature for 1 hour under a hydrogen stream. The catalyst was removed by filtration, the filtrate was concentrated, ether was added to the residue, and the precipitate was collected by filtration and dried under reduced pressure.

Yield 85 mg (Yield 98.0%) Of this, 30.0 mg was subjected to reverse phase liquid chromatography (Column: YMCD-ODS-5 (2 cm × 25).
cm), Solvent: 40% acetonitrile / H ₂
O (0.1% TFA)).

Yield 22.5 mg Amino acid analysis value (6 N
-HCl, 110 [deg.] C., hydrolysis for 24 hours; () indicates theoretical value): Ala 1.05 (1); Glu 1.08
(1); Leu 2.20 (2) LSIMS (M + H +) = 613 (theoretical value) = 61
3.

Example 3 Cyclo (-D-Glu-S
er (Bzl) -D-Leu-Leu-D-Trp-)
Production of Boc-Ser (Bzl) -OH, Boc-D-Glu
(OBzl) -OH and Boc-D-Trp-OH are respectively Boc-Trp-OH and Boc-Glu (OBz
1) The above cyclic pentapeptide was synthesized in the same manner as in Example 2 except that it was used in place of -OH and Boc-D-Ala-OH. LSIMS (M + H +) = 719 (theoretical value) = 71
9. Example 4 Cyclo (-D-Glu-Thr (Bz
l) -Production of -D-Leu-Leu-D-Trp-) Boc-Thr (Bzl) -OH, Boc-D-Glu.
(OBzl) -OH and Boc-D-Trp-OH are respectively Boc-Trp-OH and Boc-Glu (OBz
1) The above cyclic pentapeptide was synthesized in the same manner as in Example 2 except that it was used in place of -OH and Boc-D-Ala-OH. LSIMS (M + H +) = 733 (theoretical value) = 73
3. Example 5 Cyclo (-D-Asp-Trp-DL
Preparation of eu-Leu-D-Trp-) Boc-D-Asp (OBzl) -OH and Boc-
D-Trp-OH was added to Boc-Glu (OBzl)
The above cyclic pentapeptide was synthesized in the same manner as in Example 2 except that it was used instead of -OH and Boc-D-Ala-OH. LSIMS (M + H +) = 714 (theoretical value) = 71
4.

Test Example 1 Receptor Binding Assay ... N
K2 receptor binding inhibitory activity Paul LM VAN Giersbergen et al. [Proc. Natl. Acad. Sc
i. USA, 88 1661 (1991)].
Receptors were prepared from the inner wall of bovine rumen [purchased from Kyoto Central Livestock By-product Wholesale Cooperative]. The bovine abomasum inner wall stored at -80 ° C was cut into 1 cm squares or less, and 3 liters per 1 kg of 120 mM sodium chloride, 5 mM potassium chloride, 0.02% B
50 mM Tris-HCl buffer containing SA and 5% sucrose (pH 7.4)
In Polytron Homogenizer [Kinemati
ka), Germany, ”and centrifuged at 1,000 × g for 10 minutes. The supernatant was further centrifuged at 45,000 xg for 20 minutes.
The precipitate was mixed with 200 ml of 300 mM potassium chloride, 10 mM ethylenediaminetetraacetic acid, 0.1 mM phenylmethylsulfonium fluoride and 0.02% BSA in 50 mM Tris-HCl buffer (pH 7.
It was suspended in 4) and gently stirred under ice cooling for 60 minutes. The suspension was centrifuged at 45,000 xg for 20 minutes, and the precipitate was washed with 200 ml of 50 mM Tris-HCl buffer (pH 7.4) and stored frozen (-40 ° C) as a receptor standard.

This preparation was suspended in a reaction buffer solution [50 mM Tris buffer solution (pH 7.4), 0.02% bovine serum albumin, 4 mM manganese chloride] so that the protein concentration was 0.7 mg / ml, and 100 μl volume was reacted. Used for. Sample and ¹²⁵ I-NKA [0.61
KBq, ¹²⁵ I-Neurokinin A, 81.4T
Bq / m mole, manufactured by Du Pont / NEN Research Products, USA] was also added, and the mixture was reacted in 0.2 ml of reaction buffer at 25 ° C. for 3 hours.
After the reaction, using a cell harvester [Model 290PHD, manufactured by Cambridge Technology Inc., USA], glass filter [GF / B, Whatman
(Whatman), USA] to stop the reaction by rapid filtration
The plate was washed three times with 250 μl of 50 mM Tris-HCl buffer (pH 7.4) containing 0.02% bovine serum albumin, and the radioactivity remaining on the filter was measured with a gamma counter. The results are shown in Table 1 below. The numerical values in Table 1 are the NK2 receptor binding inhibitory activity (IC ₅₀ , unit: μM).

Table 1 NK2 receptor binding inhibitory activity Compound IC ₅₀ (μM) Cyclo (-D-Glu-Ser (Bzl) -D-Leu-Leu-D-Trp-) 0.58 Cyclo (-D-Glu- Thr (Bzl) -D-Leu-Leu-D-Trp-) 1.5 Cyclo (-D-Asp-Trp-D-Leu-Leu-D-Trp-) 0.027

[0033]

EFFECTS OF THE INVENTION According to the production method of the present invention, a compound having β-turn and γ-turn can be synthesized regardless of the type of amino acid residue, and the β-turn and γ-turn sites important for physiological activity are synthesized. Since it becomes possible to synthesize a compound into which a desired amino acid residue is introduced, it is effective for designing a compound having physiological activity. In particular, the cyclic pentapeptide produced by the present invention exhibits a pharmacological action such as an NK2 receptor antagonistic action and is useful as a drug such as an anti-asthma drug, an anti-inflammatory drug, an anti-arthritic drug and the like.

[Brief description of drawings]

FIG. 1 Cyclic pentapeptide cyclo (-D-Glu
-Ala-D-Phe-Leu-D-Trp-) (A)
2 is a ¹ H NMR spectrum of

FIG. 2 is a ROESY spectrum (τ _mix = 120 ms) of cyclic pentapeptide (A). The horizontal lines show the correlation from the protons shown in the figure, and the hyphenated numbers show the chain correlation between the α-proton and the amide proton.

FIG. 3 is a diagram summarizing NMR information of cyclic pentapeptide (A). J _N α represents a coupling constant value between the amide proton and the α proton, and d _NN and d _αN represent a NOE correlation between the i-th amide proton and the α-proton and the i + 1-th amide proton. d _αN (i, i + 2) is the NOE between the i-th α-proton and the i + 2-th amide proton.
Show correlation. δNH / T indicates the temperature preservation property of the chemical shift of the amide proton.

FIG. 4 is a stereo diagram showing the three-dimensional structure of (A) determined by calculation from distance information obtained by NMR.

FIG. 5: Cyclic pentapeptide cyclo (-D-Glu
-Ala-D-Leu-Leu-D-Trp-) (C)
FIG. 3 is a schematic diagram of a three-dimensional structure of and a predicted three-dimensional structure of a retro-inverso body (B) designed based on the three-dimensional structure.

FIG. 6 is a cyclic pentapeptide cyclo (-Trp-D-Leu-Leu-corresponding to the retro-inverso body.
It is a < ¹ > H NMR spectrum of D-Ala-Glu-) (B).

FIG. 7 is a ROESY spectrum (τ _mix = 120 ms) of cyclic pentapeptide (B). The meanings of horizontal lines and numbers with hyphens are the same as in FIG.

FIG. 8 is a diagram summarizing the NMR information of cyclic pentapeptide (B). The meanings of the symbols are the same as in FIG.

FIG. 9 is a stereo diagram showing the three-dimensional structure of (B) calculated and determined from the distance information obtained as described above.

FIG. 10: Cyclic pentapeptide cyclo (-Glu-D-Ala-Phe in which the arrangements of D and L in (A) are interchanged.
FIG. 3 is a diagram showing that -D-Leu-Trp-) (A ') has a stereoscopic structure having a mirror image relationship with (A), and (B) has the same main chain structure as (A'). .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical display location // C07K 99:00 (72) Inventor Takashi Kikuchi 14-1, Namiki 4-chome, Tsukuba-shi, Ibaraki Building 404

Claims (8)

[Claims] 1. Cyclo (-A ₁ -A ₂ -A ₃ -A ₄
-A _5- ) (I) [In the formula, A ₁ , A ₂ , A ₃ , A ₄ , and A ₅ are each α-
Indicates an amino acid residue. ] In the cyclic pentapeptide represented by the following, A ₁ , A ₃ , A ₅ are D-α-amino acid residues and A ₂ , A ₄ are L-α-amino acid residues,
Alternatively, A ₁ , A ₃ , and A ₅ are L-α-amino acid residues, and A ₂ and A ₄ are D-α-amino acid residues, and a γ-turn and a _1-2 turn at position 1-2-3. 3-4-5-1
A cyclic pentapeptide having a three-dimensional structure in which β-turns are combined at positions. 2. A ₁ , A ₃ and A ₅ are D-α-amino acid residues, A ₂ and A ₄ are L-α-amino acid residues, and A ₁
Is D-Asp, A ₃ is D-Val, A ₄ is L-Leu
The cyclic pentapeptide according to claim 1, wherein when A ₅ is D-Trp, A ₂ is an L-α-amino acid residue other than L-Pro. 3. The cyclic pentapeptide according to claim ₁ , wherein A ₁ , A ₃ and A ₅ are L-α-amino acid residues and A ₂ and A ₄ are D-α-amino acid residues. 4. A ₁ is a D-α-amino acid residue, and A _{2 is}
Is a L-α-amino acid residue having a protecting group, A ₃ is a D-α-amino acid residue, A ₄ is an L-α-amino acid residue, and A ₅ is a D-α-amino acid. The cyclic pentapeptide according to claim 1, which is a residue. 5. An NK2 antagonist containing any one of the cyclic pentapeptides according to claim 1. 6. The NK2 antagonist according to claim 5, which is an anti-asthma agent, an anti-inflammatory agent or an anti-arthritic agent. 7. Formula cyclo in peptide synthesis _{(-A 1 -A 2 -A 3 -A} 4 -A 5 -) (I) _{wherein, A 1, A 2, A} 3, A 4, A 5 is Α-
Indicates an amino acid residue. ] In the method for producing a cyclic pentapeptide represented by the following formula, A ₁ , A ₃ , A ₅ are D-α-amino acids, A ₂ , A ₄ are L-α-amino acid residues, or A ₁ , A ₃ , the L-alpha-amino acid residues a _5, a _2, a ₄
A method for producing a cyclic pentapeptide having a steric structure in which a γ-turn at the 1-2-3 position and a β-turn at the 3-4-5-1 position are combined, characterized in that a D-α-amino acid residue is arranged at . 8. The formula cyclo (−A ₁ −A ₂ −A ₃ −A ₄ −A ₅ −) (I) [wherein A ₁ , A ₂ , A ₃ , A ₄ and A ₅ are each α-
Indicates an amino acid residue. ] In the method for producing a cyclic pentapeptide, a linear pentapeptide is produced by condensing two kinds of fragments bisected at any position of the cyclic pentapeptide represented by
D-α-amino acid residues at ₁ , A ₃ and A ₅ are replaced by A ₂ and A ₄
L-α-amino acid residues, or A ₁ , A ₃ , A ₅ with L-α-amino acid residues, and A ₂ , A ₄ with D-α-amino acid residues. A method for producing a cyclic pentapeptide having a steric structure in which a γ-turn is combined at the 1-2-3 position and a β-turn is combined at the 3-4-5-1 position.