JPH0770183A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To obtain a new peptide or peptide derivative of high metastasis inhibitory activity. CONSTITUTION:A peptide, peptide derivative or pharmacologically permissible salt thereof ; the peptide is selected from (A) peptides formula [X]-Tyr-Ile-Gly-Y- Arg [] means that the residue in the [] may be present or absent; if present, X is Glu or Asp; Y is amino acid residue selected from Gly, Thr, Asp, Glu, Asn and Gin}, (B) amides thereof, (C) conjugates of the peptide A with other appropriate organic molecule(s), (D) cyclic peptides containing the peptide A and (E) polymers of the peptide A.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide having a specific constitution, a derivative thereof, or a pharmaceutically acceptable salt thereof, and uses thereof.

[0002]

BACKGROUND OF THE INVENTION Fibronectin, laminin, vitronectin and the like are proteins involved in the binding between cells and connective tissues and having various biological activities related to the cell function of animal cells, and are collectively referred to as cell adhesive proteins. For example, fibronectin is biosynthesized in the liver and about 0.3 mg in human plasma.
It is a glycoprotein present at a concentration of / ml.

The primary structure of fibronectin has been determined using molecular cloning (Koarnblihtt, A.
R. et al., EMBO Journal, Vol. 4, 2519 (1985)), a polypeptide having a molecular weight of about 250K and a chain of about 240K having a disulfide bond near the C terminus. Has been revealed. Regarding laminin, Sasaki et al. (Sasaki, M. et al., Proc. Natl. Acad.
Sci. USA., 81, 935 (1987), Sasaki, M. et al.,
1 by J. Biol. Chem., 262, 17111 (1987)).
The following structure has been determined. Laminin is composed of three polypeptide chains called A, B1 and B2, and is known to have a cruciform structure.

The binding site involved in cell adhesion was also studied, and it was reported in 1984 that the core sequence involved in cell adhesion of fibronectin was the tripeptide Arg-Gly-Asp (RGD) ( Pierschbache
r, MD et al., Nature 309, 30 (1984)). The core sequence of the cell adhesion site of laminin is Tyr-Ile-Gl.
It has also been elucidated to be a pentapeptide represented by y-Ser-Arg (YIGSR) (Graf, J. et al.,
Cell 48, 989 (1987)).

[0005] These fibronectin and laminin transmit various information to cells by binding to cell receptors through the above core sequences, and also bind to cells by binding to biopolymers such as heparin, collagen and fibrin. It is considered to be involved in adhesion to tissues, cell differentiation and proliferation.

[0006] As described above, since the cell adhesion protein has various biological activities, vigorous studies have been made using the active site sequence peptide. For example, in order to use the core sequence of the cell-binding portion of fibronectin, R
A method in which a peptide having a GD sequence is covalently bound and used as a substrate for artificial organs or a substrate for animal cell culture (JP-A-1-
309682, JP-A-1-305960, WO 90/05036 A
Patent), a method of attaching a peptide of interest to a solid surface by linking a hydrophobic region to a peptide having an RGD sequence, and utilizing it for a dental implant or a tissue culture substrate (WO 90 / 11297A patent), RGD sequence Method for inhibiting platelet aggregation or method for preventing or treating thrombosis using various cyclic and chain oligopeptides having the following or analogs thereof (Proceedings of the Polymer Society of Japan, Vol. 38, 3149 (1989), JP-A-2)
-174797, JP-A-3-118330, JP-A-3-11833
No. 1, JP 3-118398 JP, JP 3-118397 JP, JP 3-118333 JP, WO 91/01331 patent, WO 91/0
7429 patent, WO 91/15515 patent, WO 92/00995 patent), RG
A method of regulating platelet aggregation using a compound in which D peptide and hyaluronic acid are covalently bonded (JP-A-4-134096), and a method of using a peptide having an RGD sequence as a cell migration regulator (JP-A-2-4716). ), A method of using a membrane on which a peptide having an RGD sequence is immobilized as a cell adhesion membrane (Proceedings of the Polymer Society of Japan, Vol. 37, 705 (1988)), RG
A method of using a polypeptide having a DS sequence as a platelet protecting agent for extracorporeal blood (JP-A-64-6217), which is used as an artificial functional polypeptide by adding a peptide having cell adhesion activity into the polypeptide molecule The method (Japanese Patent Laid-Open No. 3-34996) and the like are disclosed.

Furthermore, in recent years, cell adhesion proteins have been attracting attention as biomolecules involved in cancer metastasis. During a series of stages of cancer metastasis, cancer cells come into contact with various host cells and biopolymers. At this time, if cell adhesion molecules such as fibronectin and laminin are present, the cells form a multicellular mass, and the growth and survival of cancer cells become easier. However, it has been reported that, for example, when the tripeptide RGD, which is the core sequence of the adhesion site of fibronectin, coexists, it competitively binds to the fibronectin receptor on the cancer cells and blocks the cell adhesion, showing a cancer metastasis suppressive action ( Science 238, 467 (1986)).

[0008] However, since the RGD peptide alone does not have sufficient cell adhesion activity, cancer metastasis is caused by using an oligopeptide having the sequence, a cyclic oligopeptide, or a polypeptide having a repeating sequence thereof for the purpose of enhancing the effect. How to control (Int. J. Biol. Macromo
l., Volume 11, 23 (1989), ibid, Volume 11, 226 (1989), Jpn.
J. Cancer Res., 60, 722, (1989), JP-A-2-174798.
No.) or a method for preventing tumor recurrence (JP-A-2-
No. 240020) is disclosed. In addition, a method for suppressing cancer metastasis using a cell adhesion polypeptide in a fibronectin molecule and a polypeptide having a heparin-binding polypeptide as a constitutional unit (JP-A-3-127742) has also been reported.

On the other hand, the adhesion site core sequence of laminin has also been investigated, and a method for controlling cell adhesion and cancer metastasis using a peptide derivative having a YIGSR sequence or an oligo (poly) peptide having a YIGSR repeat sequence (WO 88/06039 Patent, U.S. Patent Application 87-13919, U.S. Patent Application 88-221982, European Patent Application No. 0278781 Publication, JP-A
2-174798, JP 3-2196, Iwamoto, I. Sc
ience 238, 1132 (1987), Graf, J. Biochemistry 2
Volume 6, 6896 (1987), Mayumi, T. et al., Biochem. Biop
hys. Res. Commun., Volume 174, 1159 (1991), Mayumi, T.
et al., Peptide chemistry 145 (1991), Tanaka, N.
G. et al., Cancer Res., Volume 51, 903 (1991), Tokuyohei 3-5
06039), a method of using an ethylene-acrylic acid copolymer having YIGSR peptide immobilized thereon as a cell adhesion film, etc. (Nakajima A. et al., Polymer Journal 24, 465).
(1992)) is disclosed. It is believed that they exhibit activity by inhibiting cell adhesion to laminin. In addition, we performed a conformational calculation of a YIGSR peptide derivative and described the relationship between its three-dimensional structure and biological activity (McKelvey, DR et al., J. Prote.
in Chem., Volume 10, 265 (1991)) and CDPGYIGSR
-The glycine residue on the C-terminal side of the NH ₂ peptide is L- and D-
A paper describing the relationship between the three-dimensional structure and biological activity of analogs substituted with alanine residues (Ostheimer, GJ
et al., J. Biol. Chem., 267, 25120 (1992)), but nothing about their specific cancer metastasis-suppressing action.

[0010]

As described above, there have been active studies on the use of cell adhesive proteins such as laminin or peptide fragments thereof as pharmaceuticals and related medical materials. In particular, these are used as cancer metastasis inhibitors. Attempts to apply as are active. However, since the cell adhesion activity of the core sequence is still insufficient, their cancer metastasis inhibitory action was not satisfactory for practical medical application. Therefore, the object of the present invention is to retain a cell adhesive protein-like activity sufficiently, a novel cancer metastasis inhibitory ability enhanced than Tyr-Ile-Gly-Ser-Arg which is an adhesion core sequence of laminin. Contains a peptide sequence as an essential building block,
Another object of the present invention is to provide a novel peptide derivative which is highly stable in blood and can be synthesized by a simple means. Another object of the present invention is to provide a drug composition containing the above peptide derivative.

[0011]

[Means for Solving the Problems] For the above object, the present inventors have
Laminin adhesive core sequence Tyr-Ile-Gly-Ser-Arg, especially Ser
Part and C-terminal side amide part were changed, and various analogs in which various amino acid residues were added to the N-terminal side were systematically synthesized, and as a result of detailed examination of these biological activities, the following general formula ( Finding a peptide derivative selected from the group consisting of a peptide represented by I), an amide of the peptide, a complex of the peptide with another suitable organic molecule, a cyclic peptide containing the peptide, and a polymerized peptide. By the above, the above-mentioned subject was achieved and the present invention was completed. General formula (I) [X] -Tyr-Ile-Gly-Y-Arg (I) Here, Tyr, Ile, Gly, and Arg are tyrosine,
Represents isoleucine, glycine and arginine residues, respectively. These amino acid residues (excluding glycine residues) are D
It may be any of the -form, L-form and racemic form, but the L-form is preferred.

In general formula (I), [] represents that the residue in [] may or may not be present, and when it is present, X represents a Glu or Asp residue. Glu,
Asp represents glutamic acid and aspartic acid residues, respectively. These amino acid residues may be D-form, L-form or racemic form, but are preferably L-form.

Y is Gly, Thr, Asp, Glu, A
It represents an amino acid residue selected from the group consisting of sn and Gln. Here, Thr, Asn, and Gln represent threonine, asparagine, and glutamine residues, respectively. Among these amino acid residues, Y is Asp, Glu or T
Those having hr are particularly preferable in the present invention.

The peptide used in the present invention may be the peptide sequence itself represented by the above general formula (I), but is preferably an amide, a complex with another suitable organic molecule, a cyclic peptide and a polymer. It is a peptide derivative selected from the group consisting of modified peptides. First, as the amide form of the peptide represented by the general formula (I), a peptide derivative represented by the following general formula (II), (III) or (IV) can be particularly preferably mentioned. General formula (I
I) R- [X] -Tyr-Ile-Gly-Asp-Arg-NR ¹ R ² (II) General formula (III) R- [X] -Tyr-Ile-Gly-Glu-Arg-NR ¹ R ² (III) General formula (IV) R- [X] -Tyr-Ile-Gly-Thr-Arg-NR ¹ R ² (IV) In general formulas (II), (III) and (IV), [X] is It has the same meaning as above. R represents a hydrogen atom or an acyl group having 1 to 8 carbon atoms. Specific acyl groups used as R include an acetyl group, a propionyl group, an isobutyryl group, a benzoyl group, a succinyl group, and the like, and preferable acyl groups are an acetyl group and a benzoyl group. R
¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 8 carbon atoms. R ¹ and R ² may be the same or different, but R ^{1
One of 1} and R ² is preferably an alkyl group, and when it has 3 or more carbon atoms, it preferably has a branched structure. Further, R ¹ and R ² may be connected to each other to form a ring. Specific examples of -NR ¹ R ² include -N
H _2, -NHCH _3, it is possible to preferably exemplified -NHiC ₃ H ₇ or the like.

The organic molecule forming a complex with the peptide represented by the general formula (I) is pharmaceutically acceptable, and exhibits useful biological activity of the peptide represented by the general formula (I). As long as the water solubility of the entire molecule is not hindered without decreasing, it can be appropriately used. Specific organic molecules include organic acids having 1 to 20 carbon atoms (substituents, unsaturated bonds, cyclic structures, —O—, —NH—, —S (O) _n — [n is an integer of 0 to 2 ], Ester bond, amide bond, urea bond and the like may be included), monosaccharide, oligosaccharide (preferably having 2 to 8 sugar residues), polysaccharide derivative, polyvalent carboxylic acid (preferably the number of carboxyl groups) 2-12), a polyvalent amine (preferably a primary or secondary amino group having 2-12 amino groups), and a polymer formed by polymerizing a compound having an ethylenically unsaturated bond (preferably acrylic acid, It is preferably selected from the group consisting of methacrylic acid, polymers derived from allylamine), polyethylene glycol derivatives, and proteins of biological origin (eg albumin or globulin).

The binding mode for forming a complex between the peptide represented by the general formula (I) and the organic molecule should be appropriately selected according to the properties of the organic molecule forming the complex with the peptide. It may be either covalent bond or non-covalent bond (for example, ionic bond), but in the present invention, the binding mode when the peptide and the organic molecule form a complex is preferably covalent bond. .

As the cyclic peptide containing the peptide sequence represented by the general formula (I), the number of amino acid residues including the peptide sequence represented by the general formula (I) is 6 to 20 residues, more preferably Is a cyclic peptide having 6 to 12 residues. As a mode of forming a ring structure,
It is preferable that the α-amino acid is cyclized by linking it with a peptide bond, but in addition to the peptide bond, -S-CH ₂
-CO- bond may contain or ester bond (e.g. -SH groups of cysteine and BrCH ₂ CO- can be be formed by reaction). Further, a derivative cyclized by a disulfide bond is also a preferable derivative.

As the polymerized peptide containing the peptide sequence represented by the general formula (I), a poly (oligo) peptide having a repeating structure represented by the following general formula (V) can be preferably mentioned. ([X] -Tyr-Ile-Gly-Y-Arg) _n General formula (V) In general formula (V), [X] and Y have the same meanings as described above. n represents an integer of 2 to 20.

The ionic groups present in the peptides of the present invention, as well as the peptide derivatives, may form salts with suitable counterions. Even in the salt state, its biological activity is sufficiently maintained. However, the salt must be physiologically and pharmacologically acceptable. Specifically, salts with inorganic acids such as hydrochlorides, sulfates, nitrates, phosphates,
Examples thereof include salts with organic acids such as acetates, lactates and tartrates, and sodium salts and potassium salts. Among them, hydrochlorides and sodium salts are particularly preferable. Conversion to such salts can be accomplished by conventional means.

Specific examples of the peptide derivative of the present invention are shown below, but the present invention is not limited thereto. When the C-terminal of the peptide derivative is -OH, the description is omitted according to the custom in the art. When the amino acid residue is in the D-form, it is indicated as D-, but when it is in the natural L-form, it is not particularly indicated. Compound 1 Ac-Tyr-Ile-Gly-Asp-Arg-NH
iPr compound 2 Ac-Tyr-Ile-Gly-Glu-Arg-NH
iPr compound 3 Ac-Asp-Tyr-Ile-Gly-Thr-Ar
g-NHiPr here Ac- is CH ₃ CO- (acetyl group), - NHi
Pr represents -NHiC ₃ H ₇ (isopropylamino group).

Next, the method for synthesizing the peptide of the present invention and the peptide derivative will be described. Although the synthesis method is not particularly limited, it is preferable to use a liquid phase method or a solid phase method and a synthetic method using an automatic peptide synthesizer utilizing a solid phase method depending on the structure and properties of the target peptide or its derivative. Regarding the solid phase method and the method of synthesizing by the automatic peptide synthesizer using the solid phase method, Biochemistry Experiment Course / Protein Chemistry IV p.207 (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin), Zokusei Chemistry Experiment Course / Protein Chemistry (below) p.6
41 (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin). The peptide obtained by solid phase synthesis can be directly derivatized, but it is of course possible to appropriately modify it before cleavage from the resin.

When the peptide of the present invention and the peptide derivative are synthesized by the liquid phase method, starting from the amino acid component serving as the C-terminal component, the protected amino acid is sequentially condensed in the state of having an appropriate protecting group to proceed the synthesis. In general, the procedure is such that the protecting group at the modification site is removed if necessary for modification, and then all the protecting groups are removed. Further, a method of performing fragment condensation between the [X] -Tyr-Ile-Gly residue and the Y-Arg residue is also effective. Note that [X] and Y have the same meaning as above. The method for condensing the protected amino acid or the protected peptide can be appropriately selected from known methods, for example, the method described in "Basics and Experiments of Peptide Synthesis" (Maruzen) edited by Nobuo Izumiya et al. Although various methods are known for the condensation reaction,
DC using 1-hydroxybenzotriazole and DCC
The C-Additive method and the condensation method using carbonyldiimidazole or diphenylphosphoryl azide gave the best results.

The conditions for removing the protecting group depend on the type of protecting group used. Commonly used methods are hydrogenolysis,
HF treatment, trifluoromethane sulfonic acid / thioanisole / m-cresol / trifluoroacetic acid mixed system treatment and the like, but needless to say, various methods can be used depending on the type of the protecting group. The peptide of interest, as well as the peptide derivative, are deprotected and known by
For example, it can be purified by ion exchange chromatography, gel filtration chromatography and the like.

Next, the action and use of the peptide of the present invention and the peptide derivative will be described. Although the mechanism of the potent cancer metastasis inhibitory action of the peptide of the present invention and the peptide derivative in vivo is not completely clear, Tyr-Ile-Gly-Ser-, which is an adhesion core sequence peptide of laminin
By acting on the laminin receptor on malignant cells by a mechanism similar to Arg and inhibiting the binding to laminin, it is thought to prevent malignant cell adhesion, colonization, and destructive erosion. The peptide of the present invention, as well as the peptide derivative,
Breast cancer, epidermal cancer, muscle line melanom
a), epidermal neuroblastoma x glioma (epidermal line n
euroblastoma x glioma), chondrocytes, and fibrosarcoma, which are effective in inhibiting adhesion and metastasis of various cells.

Furthermore, the peptides and peptide derivatives of the present invention were found to have a wide range of biological activities such as a wound healing action. In addition, when the peptide of the present invention and the peptide derivative were subjected to a toxicity test using mice, no toxicity was observed at all.

The peptide of the present invention, as well as the peptide derivative or a pharmaceutically acceptable salt thereof, can be used by an administration method generally used for peptide drugs, and is usually administered as a drug composition containing an excipient. To be done. This drug composition is based on Remington's Phar
It may be prepared by any known method, as disclosed in maceutical Sciences, Merck, 16, (1980)). Excipients include distilled water, physiological saline, buffer solutions containing buffer salts such as phosphates or acetates, and antioxidants such as sodium chloride and sucrose as osmotic pressure regulators or ascorbic acid. , Or an acceptable combination of these.

Such a drug composition may be in various forms such as a solution and a tablet. The dosage form can be appropriately selected from oral, nasal, parenteral (intravenous injection, subcutaneous injection, intraperitoneal administration, etc.). For example, it may be dissolved in physiological saline to prepare an injectable preparation, or
It may be dissolved in an acetic acid buffer solution of about 0.1 N and then used as a freeze-drying agent. Further, it can be used in the form of microcapsules or microspheres encapsulated in liposomes.

The peptides and peptide derivatives of the present invention can also be used in combination with other compounds having pharmacological action, more specifically, anticancer compounds, and these are included in the scope of the present invention. It is what is done. The anticancer compound that can be used in combination with the peptide of the present invention and the peptide derivative can be appropriately selected from known anticancer agents usually used in cancer chemotherapy. Can include adriamycin, cisplatin, mitomycin and the like.
It is generally known that in conventional chemotherapy, the risk of side effects of anticancer agents is an unavoidable problem. Therefore, it seems to be very useful to reduce the dose of the anticancer drug and reduce the side effects of the anticancer drug by the combined administration of the peptide of the present invention and the peptide derivative. This also means that the effect of suppressing cancer metastasis is further improved. The peptide of the present invention and the peptide derivative are contained in the composition in an amount of 0.1 to 99% (by weight), and the dose is usually in the range of 0.2 μg / kg to 200 mg / kg per day. It is determined by sex, weight, symptoms, and administration method.

[0029]

The present invention will be described in more detail with reference to the following examples. The following abbreviations are used in the notation of commonly used solvents, reagents, and protecting groups.

Boc: t-butoxycarbonyl Bn: benzyl Mts: mesitylenesulfonyl HOBt: 1-hydroxybenzotriazole DCC: dicyclohexylcarbodiimide iPr ₂ NEt: diisopropylethylamine DMF: dimethylformamide CDI: carbonyldiimidazole TFA: trifluoroacetic acid TFA: trifluoroacetic acid Romethanesulfonic acid

Example 1 Synthesis of Compound 1 The liquid phase synthesis method of Compound 1 will be described in detail. The synthetic route of Compound 1 is shown below.

[0032]

[Chemical 1]

[0033]

[Chemical 2]

1) Synthesis of Intermediate 1 Boc-Arg (Mts) -OH (48.0 g, 110 mmol), isopropylamine (6.30 g, 110 mmol), 1-hydroxybenzotriazole monohydrate (16.1 g, 110 mmol) ) Was dissolved in a mixed solvent of DMF (100 ml) and methylene chloride (100 ml), and DCC (21.9 g, 110 mmol) was added while cooling with ice. The reaction mixture was stirred under ice cooling for 1 hour and further stirred while warming to room temperature overnight, and then filtered through Celite to remove the formed precipitate. The filtrate is diluted with an appropriate amount of ethyl acetate, water, 1 M citric acid solution,
The mixture was washed with a 5% sodium carbonate solution and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 51.0 g (quantitative) of the target Intermediate 1 as a colorless powder. FAB-MS: (M
+ H) ⁺ 457.

2) Synthesis of Intermediate 2 Intermediate 1 (51.0 g, 110 mmol) in dioxane (100 ml)
Hydrogen chloride / dioxane solution (4 M, 150 ml) was added to the solution and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was distilled off and the residue was crystallized from ether to give the hydrochloride salt.
2.6 g was obtained as a colorless powder. On the other hand, Boc-Asp (Bn) -OH (6.5
8 g, 20.4 mmol) was dissolved in DMF (20 ml), and CDI (3.30 g, 20.5 mmol) DMF (40 ml) was dissolved in this with ice cooling.
The solution was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the hydrochloride (8.86 g, 20.4
mmol) and diisopropylethylamine (2.84 g, 22 mm
ol) in DMF (40 ml) was added. The reaction mixture was stirred under ice-cooling for 2 hours and further stirred overnight while warming to room temperature, and then the solvent was evaporated under reduced pressure. The residue was diluted with an appropriate amount of ethyl acetate, washed with water, 1 M citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the desired intermediate 2 as a colorless powder. 12.76 g (89.1%)
Obtained. FAB-MS: (M + H) ⁺ 703.

3) Synthesis of Intermediate 3 Intermediate 2 (12.5 g, 17.8 mmol) in dioxane (50 ml)
Hydrogen chloride / dioxane solution (4 M, 60 ml) was added to the solution and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was distilled off and the residue was crystallized from ether to give the hydrochloride salt 1.
1.3 g was obtained as a colorless powder. Meanwhile, Boc-Gly-OH (3.10 g,
17.7 mmol) was dissolved in DMF (20 ml), and a DMF (40 ml) solution of CDI (2.87 g, 18.0 mmol) was added to this while cooling with ice. The reaction mixture was stirred for 1 hour while cooling with ice, and then the hydrochloride obtained by the above operation (11.3 g, 17.7 mmo
l) and diisopropylethylamine (2.45 g, 19.0 mmo
l) in DMF (40 ml) was added. The reaction mixture was stirred under ice-cooling for 2 hours and further stirred overnight while warming to room temperature, and then the solvent was evaporated under reduced pressure. The residue is diluted with an appropriate amount of ethyl acetate, washed with water, 1 M citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the desired intermediate 3 as a colorless powder. 12.36 g (92.0%)
Obtained. FAB-MS: (M + H) ⁺ 760.

4) Synthesis of Intermediate 4 Intermediate 3 (12.3 g, 16.2 mmol) in dioxane (60 ml)
Hydrogen chloride / dioxane solution (4 M, 60 ml) was added to the solution and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off and the residue was crystallized from ether to give the hydrochloride salt 1.
1.2 g was obtained as a colorless powder. Meanwhile, Boc-Ile-OH (3.72 g,
16.1 mmol) was dissolved in DMF (20 ml), and a DMF (30 ml) solution of CDI (2.61 g, 16.1 mmol) was added thereto while cooling with ice. The reaction mixture was stirred for 1 hour while cooling with ice, and then the hydrochloride obtained by the above operation (11.2 g, 16.1 mmo
l) and diisopropylethylamine (2.32 g, 18.0 mmo
l) in DMF (40 ml) was added. The reaction mixture was stirred under ice-cooling for 2 hours and further stirred overnight while warming to room temperature, and then the solvent was evaporated under reduced pressure. The residue was diluted with an appropriate amount of ethyl acetate, washed with water, 1 M citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
The residue was crystallized from ether to obtain 11.4 g (81.2%) of the target Intermediate 4 as a colorless powder. FAB-MS: (M + H) ⁺ 873.

5) Synthesis of Intermediate 5 Intermediate 4 (12.0 g, 13.8 mmol) in dioxane (80 ml)
Hydrogen chloride / dioxane solution (4 M, 70 ml) was added to the solution and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off and the residue was crystallized from ether to give the hydrochloride salt 1.
0.9 g was obtained as a colorless powder. On the other hand, Boc-Tyr (Bn) -OH (2.7
8 g, 7.5 mmol) was dissolved in DMF (12 ml), and a CDF solution (1.22 g, 7.5 mmol) in DMF (15 ml) was added thereto while cooling with ice. The reaction mixture was stirred for 1 hour while cooling with ice, and then the hydrochloride (6.08g, 7.5 mmo
l) and diisopropylethylamine (1.03 g, 8.0 mmol)
Of DMF (25 ml) was added. The reaction mixture was stirred under ice-cooling for 2 hours and further stirred overnight while warming to room temperature, and then the solvent was evaporated under reduced pressure. The residue is ether / ethyl acetate (9 /
Crystallization from 1) yielded 8.33 g (quantitative) of the desired Intermediate 5 as colorless crystals. FAB-MS: (M + H) ⁺ 1126.

6) Synthesis of Intermediate 6 Intermediate 5 (8.0 g, 7.1 mmol) in chloroform (40 ml)
To the solution was added trifluoroacetic acid (30 ml) and the reaction mixture was stirred at room temperature for 1 hour. After the reaction is completed, the solvent is distilled off,
The residue was crystallized from ether to give trifluoroacetate 7.
7 g was obtained as a colorless powder. The obtained trifluoroacetic acid salt (1.35 g, 1.2 mmol) was added to DMF (2 ml) and pyridine (2 m
l) dissolved in a mixed solvent of acetic anhydride (1
ml) was added. After leaving the reaction mixture at room temperature overnight,
The solvent was distilled off under reduced pressure. The residue was crystallized from a mixed solvent of ether / ethyl acetate (9/1) to obtain 1.03 g (80.4%) of the target Intermediate 6 as a colorless powder. FAB-MS: (M + H) ⁺ 1068. (M + Na) ⁺ 1090.

7) Synthesis of Compound 1 Intermediate 6 (1.03 g, 1.0 mmol) of trifluoroacetic acid (7
ml) solution, a mixed solution of trifluoromethanesulfonic acid (6 g), thioanisole (4 ml), m-cresol (3.5 ml), and trifluoroacetic acid (18 ml) was added with ice cooling, and the reaction mixture was added. The mixture was stirred under ice cooling for 2 hours. The reaction solution was added dropwise to ether (700 ml) and stirred slowly for 1 hour.
The crude product precipitated was dissolved in a small amount of water and subjected to ion exchange chromatography (Amberlite IRA-400,
Counterion OAc ^-) Purification, 510 mg (71.0% of the compound 1 of interest as a colorless powder by freeze-drying) was obtained. FAB-MS: (M + H) ⁺ 706.

Example 2 Synthesis of Compound 2 In the synthesis of Intermediate 2 described in Example 1, Boc-Asp
Boc-Glu (Bn) -OH was used instead of (Bn) -OH for condensation by the CDI method to synthesize Boc-Glu (Bn) -Arg (Mts) -NHiPr. Thereafter, synthesis was carried out in the same manner as in the method described in Example 1 to obtain the target compound 2 as a colorless powder. FAB-MS: (M + H) ⁺ 720.

Example 3 Synthesis of Compound 3 In the synthesis of Intermediate 2 described in Example 1, Boc-Asp
Boc-Thr (Bn) -OH was used in place of (Bn) -OH to carry out condensation by the CDI method to synthesize Boc-Thr (Bn) -Arg (Mts) -NHiPr. Thereafter, synthesis was carried out in the same manner as in the method described in Example 1, and Boc-Tyr
It was induced to (Bn) -Ile-Gly-Thr (Bn) -Arg (Mts) -NHiPr. Boc-Asp (Bn) -OH was condensed on the N-terminal side of this product by the CDI method to give Boc-Asp (Bn) -Tyr (Bn) -Ile-Gly-Thr (Bn) -Arg (Mts).
-NHiPr was synthesized. Thereafter, acetylation, deprotection and purification of the N-terminal amino group were carried out in the same manner as in Example 1 to obtain the target compound 3 as a colorless powder. FAB-MS: (M +
H) ⁺ 807.

Example 4 Study on Cancer Metastasis Inhibitory Action by Experimental Lung Metastasis Model System Using Melanoma Cells The tumor metastasis inhibitory action of the peptide derivative of the present invention was examined by an experimental lung metastasis model system. Compounds 1 and 3 of the present invention described in Examples and JP-A-3-21 as a comparative example
Ac-Tyr-Ile-Gly-Ser-Arg-NH ₂ described in JP-A-96 was used. 1000 μg of these peptides and B16-BL6 melanoma cells that are highly metastatic cancer cells (logarithmic growth phase 5
x 10 ⁴ ) were mixed in PBS, and each was mixed with 5 C57 per group.
BL / 6 female mice were administered by tail vein injection. 14 after administration
On the day, the mice were sacrificed and dissected, the number of cancer colonies metastasized to the lung was counted, and compared with the control PBS-administered group. The results are shown in Table 1 below.

[0044]

[Table 1] Table 1 ──────────────────────────────── Administered compound Lung metastases Mean ± SD (range ) ──────────────────────────────── PBS (untreated) 118 ± 29 (84-153) Ac-Tyr- Ile-Gly-Ser-Arg-NH ₂ 82 ± 25 (64-117) Compound 1 46 ± 22 (14-82) * Compound 3 44 ± 11 (25-60) * ─────────── ────────────────────── * P <0.001 compared to untreated plot by t-test

According to this result, it is a known peptide.
Ac-Tyr-Ile-Gly-Ser-Arg-NH ₂ is 1000 per mouse
Although a decrease in the number of cancer metastases was observed at the dose of μg, the cancer metastasis-suppressing effect judged to be significant at a risk rate of 2% was not observed compared to the untreated group. On the other hand, when the compound 1 or 3 of the present invention was administered, cancer metastasis to the lung was significantly suppressed. Therefore, the cancer metastasis inhibitory effect of the peptide derivative of the present invention, and its usefulness and superiority are obvious.

Although the present invention has been described with reference to particular examples by way of examples, it should not be construed as limiting. It will be apparent that various changes and modifications may be made without departing from the spirit and scope of the invention. And it is considered that such an invention is included in the present invention.

[0047]

EFFECT OF THE INVENTION As described above, the peptide derivative of the present invention has a greater cancer metastasis-inhibiting action than the core sequence peptide of laminin, which is a cell adhesive protein, and has almost no toxicity problem. Further, since its structure is simple, it can be easily synthesized and is highly valuable as a medicine.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ichiro Higashi 5-3-2 Kamikomachi, Makomanai, Minami-ku, Sapporo-shi, Hokkaido

Claims (8)

[Claims] 1. A peptide represented by the following general formula (I):
An amide of the peptide, a complex of the peptide with another suitable organic molecule, a peptide or a peptide derivative selected from the group consisting of a cyclic peptide containing the peptide and a polymerized peptide, or a pharmaceutically acceptable salt thereof. General formula (I) [X] -Tyr-Ile-Gly-Y-Arg (I) In general formula (I), [] means that the residue in [] may or may not be present. Represents, and if present X is Gl
represents a u or Asp residue. Y is Gly, Thr, As
It represents an amino acid residue selected from the group consisting of p, Glu, Asn and Gln. 2. The compound according to claim 1, wherein Y is Asp. 3. The compound according to claim 1, wherein Y is Glu. 4. The compound according to claim 1, wherein Y is Thr. 5. A peptide derivative represented by the following general formula (II) or a pharmaceutically acceptable salt thereof. General formula (II) R- [X] -Tyr-Ile-Gly-Asp-Arg-NR ¹ R ² (II) In general formula (II), [] is present even if the residue in [] is present. X is Gl
represents a u or Asp residue. R represents a hydrogen atom or an acyl group having 1 to 8 carbon atoms. R ¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 8 carbon atoms. R ¹ and R ² may be the same or different, and these two groups may be linked to each other to form a ring. 6. A peptide derivative represented by the following general formula (III) or a pharmaceutically acceptable salt thereof. General formula (III) R- [X] -Tyr-Ile-Gly-Glu-Arg-NR ¹ R ² (III) In general formula (III), [] is present even if the residue in [] is present. X is Gl
represents a u or Asp residue. R represents a hydrogen atom or an acyl group having 1 to 8 carbon atoms. R ¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 8 carbon atoms. R ¹ and R ² may be the same or different, and these two groups may be linked to each other to form a ring. 7. A peptide derivative represented by the following general formula (IV) or a pharmaceutically acceptable salt thereof. General formula (IV) R- [X] -Tyr-Ile-Gly-Thr-Arg-NR ¹ R ² (IV) In general formula (IV), [] is present even if the residue in [] is present. X is Gl
represents a u or Asp residue. R represents a hydrogen atom or an acyl group having 1 to 8 carbon atoms. R ¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 8 carbon atoms. R ¹ and R ² may be the same or different, and these two groups may be linked to each other to form a ring. 8. A pharmaceutically acceptable excipient, and claims 1 to 1.
A cancer metastasis inhibitor comprising the compound according to any one of 7 as an active ingredient.