JPH0641193A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To provide the new compound sufficiently keeping various bioactivities of laminin as a cell-adhesive protein, exhibiting a cancer metastasis inhibitory effect higher than in a known laminin core sequence, ready to synthesize and free from a serious side effect on the human body. CONSTITUTION:A peptide of the formula, its pharmaceutically permissible salt and a cancer metastasis inhibitor containing the same compound. R-X-Tyr-Ile- Gly-Ser-Arg-Y. (R is hydrogen atom or a 2 to 4C acyl group; X is Glu or Asp residue; Y is NR<1>R<2>. R<1> and R<2> are each hydrogen atom or a 1 to 4C alkyl group. Both R<1> and R<2> may be same or different, provided that both of these are not hydrogen atom).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an active site sequence peptide derivative of laminin, which is a cell adhesive protein, or a pharmaceutically acceptable salt thereof, and uses thereof.

[0002]

BACKGROUND OF THE INVENTION Laminin, fibronectin, vitronectin and the like are proteins that are involved in the binding between cells and connective tissues and have 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 of the cell-binding portion of fibronectin was a tripeptide called Arg-Gly-Asp (RGD) (Pierschbacher). , M.
D. et al., Nature 309, 30 (1984)). The core sequence of the cell adhesion site of laminin is Tyr-Ile-Gly-.
It has also been elucidated that it is a pentapeptide represented by Ser-Arg (YIGSR) (Graf, J. et al., Cel
l 48, 989 (1987)).

[0005] These fibronectins and laminins transmit their information to cells by binding to cell receptors through the above core sequences, and also bind to biopolymers such as heparin, collagen and fibrin to connect cells with connective tissue. It is considered to be involved in adhesion with the, 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 as a dental implant or a tissue culture substrate (WO 90/11297 A patent), RGD Methods for inhibiting platelet aggregation or methods for preventing and treating thrombosis using various cyclic and chain oligopeptides having sequences or analogs thereof (Proceedings of the Polymer Society of Japan, Vol. 38, 3149 (1989), JP-A 2-174797, JP3-118330, JP3-11
8331, JP-A-3-118398, JP-A-3-118397, JP-A-3-118333, WO 91/01331 patent, WO 91
/ 07429 patent, WO 91/15515 patent, WO 92/00995 patent),
A method of regulating platelet aggregation using a compound in which RGD 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 (Japanese Patent Laid-Open No. 6217/1988), and a peptide having cell adhesion activity is added to the inside of the polypeptide to be used as an artificial functional polypeptide. 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 adhesive 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 adhesion site core sequence of fibronectin, coexists, it competitively binds to the fibronectin receptor on the cancer cells and thus block the cell adhesion, showing an inhibitory effect on cancer metastasis ( Science 238, 467 (1986)).

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. Macrom
ol., 11 volumes, 23 (1989), ibid, 11 volumes, 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) has been tried. 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 studied, 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-A-3-2196, Iwamoto, I.
Science 238, 1132 (1987), Graf, J. Biochemistry
Volume 26, 6896 (1987), Mayumi, T. et al., Biochem. Bi
Ophys. Res. Commun., Volume 174, 1159 (1991), Mayumi,
T. et al., Peptide chemistry 145 (1991), Tanaka,
NG et al., Cancer Res., 51, 903 (1991), Tokuhei Hyohei 3
-506039), a method of using an ethylene-acrylic acid copolymer having a YIGSR peptide immobilized thereon as a cell adhesion film (Nakajima A. et al., Polymer Jourmnal 24, 4).
65 (1992)) and the like are disclosed. It is believed that they exhibit activity by inhibiting cell adhesion to laminin. Also, a paper describing the relationship between the three-dimensional structure of the laminin core peptide and the biological activity by performing the conformational calculation of the YIGSR peptide derivative has been reported (McKelvey, DR et al., J. Protein Chem., 10
Volume, 265 (1991)).

[0010]

As described above, since the active site core sequence of cell adhesive proteins such as laminin retains various biological activities, its application value is considered to be high. However, the biological activity of the core sequence is still insufficient as compared with the natural cell adhesive protein, and in this respect, the development of a more effective substance has been required. In addition, according to the research conducted by the present inventors, many laminin core sequence peptide derivatives have a high positive charge, and therefore, when administered to a large amount of mice, shock may occur. Was wanted.

Therefore, an object of the present invention is to sufficiently retain various biological activities possessed by laminin which is a cell adhesive protein,
It is an object of the present invention to provide a novel compound that is easy to synthesize and does not show serious side effects on the living body. Another object of the present invention is to provide a peptide derivative having a high cancer metastasis inhibitory activity. Another object of the present invention is to provide a drug composition containing the above peptide derivative.

[0012] The present inventors have conducted earnest studies for a novel compound that sufficiently retains various biological activities of laminin, which is a cell adhesive protein, is easy to synthesize, and does not show serious side effects in the living body. As a result, the inventors have found a novel peptide derivative that has a greater cancer metastasis-inhibiting ability than the known laminin core sequence and does not show serious side effects in the body, and completed the present invention.

[0013]

The present invention is a peptide derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof. Formula (I) R-X-Tyr-Ile-Gly-Ser-Arg-Y (I) Here, Tyr, Ile, Gly, Ser, and Arg represent tyrosine, isoleucine, glycine, serine, and arginine residues, respectively. . These amino acids (excluding glycine) may be D-form, L-form or racemic form, but are preferably L-form.

In the formula, R represents a hydrogen atom or an acyl group having 2 to 4 carbon atoms. However, when the carbon number is 4, it may have a branched structure. Examples of such an acyl group include an acetyl group, a propionyl group, an isobutyryl group, and a succinyl group, and a particularly preferable acyl group is an acetyl group.

X represents a Glu or Asp residue. Glu and Asp represent glutamic acid and aspartic acid residues, respectively.

Y represents --NR ¹ R ² . Here, R ¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. However, when R ¹ and R ² have 3 or 4 carbon atoms, it preferably has a branched structure. R ¹ and R ² may be the same or different, but these two are not hydrogen atoms at the same time. Such —NR ¹ R ² includes —NHCH ₃ , —NHC ₂ H ₅ , and
NH-i- C ₃ H _7, can be mentioned -N (CH _{3) 2} and the like, among others _{-NHCH 3, -NH-i-C} 3 H 7 are preferred.

The ionic group present in the peptide derivative of the present invention may form a salt with an appropriate counter ion (including a state of forming a salt in the molecule). The biological activity of the peptide is sufficiently maintained even in the salt state. However, the salt must be physiologically and pharmacologically acceptable. Specific examples include salts with inorganic acids such as hydrochlorides, sulfates, nitrates and phosphates, salts with organic acids such as acetates and lactates, and ammonium salts, sodium salts, potassium salts and the like. However, of these, the hydrochloride, acetate and sodium salt are preferable. Conversion to such salts can be accomplished by conventional means.

Specific examples of the compound of the present invention are shown below.
The present invention is not limited to these. Peptide 1 Ac-Asp-Tyr-Ile-Gly-Ser-Arg-N
HCH ₃ peptide 2 H-Asp-Tyr-Ile-Gly-Ser-Arg-NH
CH ₃ peptide 3 Ac-Asp-Tyr-Ile-Gly-Ser-Arg-N
H-i-C ₃ H ₇ Peptide 4 Ac-Glu-Tyr-Ile -Gly-Ser-Arg-N
H-i-C ₃ H ₇ peptide 5 C ₂ H ₅ CO-Asp-Tyr-Ile-Gly-Ser-Ar
In the g-N (CH ₃ ) ₂ formula, Ac represents an acetyl group.

Next, a method for synthesizing the compound of the present invention will be described. The method for synthesizing the peptide is not particularly limited, and examples thereof include a solid phase method and a synthetic method using an automatic peptide synthesizer utilizing the solid phase method. 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.641 (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin).

The peptide derivative of the present invention can also be synthesized by a liquid phase method. That is, this is a method in which starting from a protected arginine which is a C-terminal component, converting the C-terminal to an alkylamide or a dialkylamide, removing the N-terminal protecting group, and successively condensing the protected amino acid residue. Also, Asp-Tyr-Ile-Gly residue and Ser-
A method of performing fragment condensation between Arg residues is also effective. 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, 1-hydroxybenzotriazole and D
The DCC-Additive method using CC or the condensation method using carbonyldiimidazole 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 target peptide derivative is deprotected and then "basic and experimental of peptide synthesis"
It can be purified by a known method described in (Maruzen) and the like, for example, ion exchange chromatography or gel filtration chromatography.

Next, the action and use of the peptide derivative of the present invention will be described. Since the peptide derivative of the present invention has an alkyl amide or a dialkyl amide at the C-terminal, it is less susceptible to degradation by a protease or the like in vivo, and thus exhibits a remarkable cancer metastasis inhibitory activity. Furthermore, the peptide derivative of the present invention is characterized by containing an acidic amino acid residue such as glutamic acid or aspartic acid as an essential component. This acidic amino acid residue has a function of neutralizing an excessive positive charge without reducing the useful biological activity of the laminin core sequence peptide, and therefore the peptide derivative of the present invention causes harmful side effects such as shock to the living body. Not shown. The peptide derivative of the present invention acts on the laminin receptor on malignant cells and inhibits the binding to laminin, thereby preventing adhesion, colonization and destructive erosion of malignant cells.

Therefore, the peptide derivative of the present invention is
Epidermal cancer, muscle line melanoma, epidermal neuroblastoma x epidermal line neurobla
stoma x glioma), chondrocytes, and various cells including fibrosarcoma. Further, the peptide derivative of the present invention was found to have a wide range of biological activities such as a wound healing effect and an inhibitory effect on platelet aggregation by cancer cells occurring in capillaries.

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

Such a drug composition can be made into 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 single dose of the peptide derivative of the present invention is usually in the range of 0.2 μg / kg to 200 mg / kg, but it is determined according to the age, weight, symptom and administration method of the patient.

The present invention will be described in more detail with reference to the following examples. The method for synthesizing peptide 3 by the liquid phase method will be described in detail, but the other exemplified compounds were basically synthesized by the same method. The synthetic route of peptide 3 is shown below.
The following abbreviations that are commonly used are used to indicate solvents, reagents, and protecting groups.

Boc: t-butoxycarbonyl Bn: benzyl Ac: acetyl Mts: mesitylenesulfonyl HOBt: 1-hydroxybenzotriazole DCC: dicyclohexylcarbodiimide iRr ₂ NEt: diisopropylethylamine DMF: dimethylformamide CDI: carbonyldiimidazole TFA: TFMSA: trifluoromethanesulfonic acid THF: tetrahydrofuran

[0029]

[Chemical 1]

[0030]

[Chemical 2]

[0031]

【Example】

Example 1 Synthesis of Peptide 3 1) Synthesis of Intermediate 1 Boc-Arg (Mts) -OH (25.0 g, 55 mmol), isopropylamine (3.25 g, 55 mmol), 1-hydroxybenzotriazole monohydrate ( 8.42 g, 55 mmol) was dissolved in a mixed solvent of DMF (30 ml) and methylene chloride (30 ml), and DCC (11.3 g, 55 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, washed with water, 1 M citric acid solution, 5% sodium carbonate solution and saturated saline, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the desired intermediate 1. 29.0 g (quantitative) was obtained as a powder.

2) Synthesis of Intermediate 2 Intermediate 1 (29.0 g, 55 mmol) in methylene chloride (70 ml)
Trifluoroacetic acid (70 ml) was added to the solution, and the reaction mixture was stirred at room temperature for 1 hr. After completion of the reaction, the solvent was distilled off, and the residue was crystallized from ether to give trifluoroacetic acid salt 27.4 g.
(97.4%) was obtained as a colorless powder. On the other hand, Boc-Ser (Bn)-
Dissolve OH (15.93 g, 54 mmol) in DMF (30 ml) and chill it with CDI (8.76 g, 54 mmol) DMF (60 ml).
ml) solution was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetate salt obtained by the above operation
(27.4 g, 53.6 mmol) and diisopropylethylamine
A solution of (7.10 g, 55 mmol) in DMF (45 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. 3
6.14 g (quantitative) was obtained. FAB-MS: (M + H) ⁺ 675.

3) Synthesis of Intermediate 3 Intermediate 2 (36.14 g, 54 mmol) in methylene chloride (70 ml)
Trifluoroacetic acid (70 ml) was added to the solution, and the reaction mixture was stirred at room temperature for 1 hr. After the reaction was completed, the solvent was distilled off and the residue was crystallized from ether to give the trifluoroacetic acid salt 33.2.
g (89.3%) was obtained as a colorless powder. On the other hand, Boc-Gly-OH
(8.58 g, 49 mmol) was dissolved in DMF (30 ml), and CDI (7.95 g, 49 mmol) DMF (60 ml) was dissolved in this solution while cooling with ice.
The solution was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (33.
2 g, 48.3 mmol) and diisopropylethylamine (6.46
g, 50 mmol) in DMF (50 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. 34.87 g (9
8.8%) obtained. FAB-MS: (M + H) ⁺ 732.

4) Synthesis of Intermediate 4 Intermediate 3 (34.8 g, 47.7 mmol) of methylene chloride (80 m
l) Trifluoroacetic acid (80 ml) was added to the solution 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 the trifluoroacetate salt 3
4.6 g (97.4%) was obtained as a colorless powder. On the other hand, Boc-Ile
-OH (10.9 g, 47 mmol) was dissolved in DMF (30 ml), and while cooling with ice, CDI (7.62 g, 47 mmol) of DMF (60
ml) solution was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetate salt obtained by the above operation
(34.6 g, 46.4 mmol) and diisopropylethylamine
A solution of (6.33 g, 48 mmol) in DMF (50 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, water, 1 M citric acid solution, saturated aqueous sodium hydrogen carbonate,
The extract is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the desired intermediate 4 as colorless powder (38.8 g).
(Quantitative) obtained. FAB-MS: (M + H) ⁺ 845.

5) Synthesis of Intermediate 5 Intermediate 4 (38.8 g, 47.7 mmol) in methylene chloride (60 m)
l) Trifluoroacetic acid (60 ml) was added to the solution 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 the trifluoroacetate salt 3
8.1 g (95.6%) was obtained as a colorless powder. On the other hand, Boc-Tyr
Dissolve (Bn) -OH (16.5 g, 44.4 mmol) in DMF (35 ml) and add CDI (7.30 g, 45 mmol) to it while cooling with ice.
DMF (50 ml) solution was added. After stirring the reaction mixture for 1 hour while cooling with ice, a solution of the trifluoroacetic acid salt (38.1 g, 44.4 mmol) and diisopropylethylamine (5.94 g, 46 mmol) obtained in the above operation in DMF (50 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 recrystallized from ethyl acetate to obtain 36.51 g (75.0%) of the target Intermediate 5 as colorless crystals. FAB-MS: (M + H) ⁺ 1098.

6) Synthesis of Intermediate 6 Intermediate 5 (15.25 g, 13.9 mmol) was treated with methylene chloride (30 m
l) and trifluoroacetic acid (30 ml) were dissolved in a mixed solvent, and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off, and the residue was crystallized from ether to obtain 15.5 g (quantitative) of trifluoroacetic acid salt as a colorless powder. Meanwhile, Boc-Asp (Bn) -OH (4.52 g, 14.0 mmol) was added to DMF.
Dissolve it in (15 ml) and cool it with CDI (2.27
A solution of g, 14.0 mmol) in DMF (25 ml) was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (15.5 g, 14.0 mmol) obtained by the above operation and diisopropylethylamine (1.94 g, 15 mmol) in DMF (3
5 ml) solution 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 recrystallized from ethyl acetate / ether (1/1) to give the desired intermediate 6 as colorless crystals, 16.6 g.
(91.1%) was obtained. FAB-MS: (M + H) ⁺ 1303.

7) Synthesis of Intermediate 7 Intermediate 6 (4.69 g, 3.6 mmol) was added to methylene chloride (40 ml).
And dissolved in a mixed solvent of trifluoroacetic acid (40 ml), and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off, and the residue was crystallized from ether to obtain trifluoroacetic acid salt 4.7 g (quantitative) as a colorless powder. This was dissolved in a mixed solvent of DMF (20 ml) and pyridine (5 ml), acetic anhydride (1.0 g) was added, and the reaction mixture was left at room temperature for 4 hours. Most of the solvent was distilled off under reduced pressure, and the residue was crystallized from ether to give the desired intermediate 7.
As a colorless powder, 4.17 g (93.1%) was obtained. FAB-MS: (M + H) + 1245.

8) Synthesis of peptide 3 Intermediate 7 (2.68 g, 2.2 mmol) of trifluoroacetic acid (15
ml) solution, trifluoromethanesulfonic acid (12 g),
A mixed solution of thioanisole (10 ml), m-cresol (8.7 ml) and trifluoroacetic acid (45 ml) was added under ice cooling, and the reaction mixture was stirred under ice cooling for 1 hour. The reaction solution was added dropwise to ether (1000 ml) and stirred slowly for 30 minutes. The precipitated crude peptide was collected, dissolved in a small amount of water, purified with ion exchange chromatography (Amberlite IRA-400, OAc-) after treatment with activated carbon, and freeze-dried to give the desired peptide 3 as a colorless powder, 950 mg.
(54.5%) was obtained. FAB-MS: (M + H) ⁺ 793, (M + Na) ⁺ 815.

Example 2 Synthesis of Peptide 1 1) Synthesis of Boc-Arg (Mts) -NHCH ₃ Boc-Arg (Mts) -OH (20.0 g, 44 mol) and p-nitrophenol (6.12 g, 44 mol) DMF (15 ml) and methylene chloride
The (15 ml) solution was ice-cooled, and DCC (9.29 g, 45 mol) was added thereto. The reaction mixture was stirred under ice-cooling for 2 hours, further stirred for 6 hours while warming to room temperature, and then filtered through Celite to remove the formed precipitate. Dilute the filtrate with an appropriate amount of ethyl acetate,
The extract was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude p-nitrophenyl ester. This was dissolved in THF (100 ml), 40% methylamine solution (6 ml) was added, and the reaction mixture was stirred at room temperature for 18 hours. The solvent was distilled off under reduced pressure, and the residue was crystallized from ether to give the desired Boc-Arg (Mts) -NHCH ₃ as a colorless powder.
Obtained 9.4 g (94%). FAB-MS: (M + H) ⁺ 470.

2) According to the method described in Example 1 below,
Protected amino acid residues were sequentially condensed from Boc-Arg (Mts) -NHCH ₃ to the N-terminal side to extend the peptide chain. The N-terminal was acetylated, deprotected and purified to obtain 780 mg of the desired peptide 1 as a colorless powder. FAB-MS: (M + H) ⁺ 765.

Example 3 Peptides 2, 4 and 5 were synthesized in the same manner as in Examples 1 and 2. Peptide 2 FAB-MS: (M + H) ⁺ 723. Peptide 4 FAB-MS: (M + H) ⁺ 807. Peptide 5 FAB-MS: (M + H) ⁺ 793.

Example 4 Experiment on Cancer Metastasis Inhibitory Action Regarding the cancer metastasis inhibitory action of the peptide derivative of the present invention,
It was examined by an experimental lung metastasis model system. Peptides 1 to 5 of the present invention, as a comparative example Tyr-Ile-Gly-Ser-Arg-NH ₂
And Ac-Tyr-Ile-Gly-Ser-A described in JP-A-3-2196.
rg-NH ₂ was used. 1000 μg of each of these peptides and B16-BL6 melanoma cells (5 × 10 ^{4 in} logarithmic growth phase), which are highly metastatic cancer cells, were mixed in PBS, and 0.2 ml of the mixture was used for 5 animals per group. C57BL / 6 female mice were injected by tail vein. On the 14th day after the administration, the mice were sacrificed and dissected, and the number of colonies of cancer metastasized to the lung was counted and compared with the control PBS administration group. The results are shown below.

[0043]

[Table 1] ────────────────────────────────── Administered compound Lung metastases Mean ± SD (range ) ────────────────────────────────── PBS (untreated) 148 ± 28 (118-189) Peptide 1 23 ± 7 (16-31) ** Peptide 2 31 ± 7 (23-40) ** Peptide 3 25 ± 2 (23-28) ** Peptide 4 30 ± 11 (14-42) ** Peptide 5 44 ± 7 (35-52) ** Tyr-Ile-Gly-Ser-Arg-NH ₂ 128 ± 5 (123-125) Ac-Tyr-Ile-Gly-Ser-Arg-NH ₂ 70 ± 13 (57-88) * ────────────────────────────────── * P <0.01 compared to untreated control by t-test ** P <0.001

As can be seen from the above results, cancer metastasis to the lung was significantly suppressed by the administration of peptides 1 to 5 of the present invention. Among them, the peptides 1 to 4 of the present invention are particularly effective and at least twice as effective as the known peptide Ac-Tyr-Ile-Gly-Ser-Arg-NH ₂ . Therefore, the cancer metastasis inhibitory effect of the peptide derivative of the present invention and its usefulness are clear.

Example 5 Results of Acute Toxicity Test of Peptide Derivatives of the Present Invention Using Mice Peptides 1 and 3 of the present invention as test peptides, and Ac-Tyr-Ile-Gly containing no aspartic acid residue as a comparative example
_{-Ser-Arg-NHCH 3 (J.} Protein Chem., 10 , pp. According to 265 (1991)), was used Ac-Tyr-Ile-Gly- Ser-Arg-NHiC 3 H 7. These peptides were dissolved in PBS, and 10 mice / mouse of three C57BL / 6 mice per group were intravenously injected into the tail vein to observe the appearance of the mice. As a result, no remarkable changes were observed in the groups to which the peptides 1 and 3 of the present invention were administered, showing a general form considered to be normal. On the other hand, Ac-Tyr-Ile-Gly-Ser-Arg-NHCH ₃ or Ac containing no aspartic acid residue
In -Tyr-Ile-Gly-Ser- Arg-NHiC 3 groups treated with H _7, shows a convulsive state or crouching state after a few minutes of tail vein injection, followed by gradually led to death as they become prone state. From the above experimental results, the usefulness and superiority of the peptide derivative of the present invention are clear.

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]

INDUSTRIAL APPLICABILITY As described above, the peptide derivative of the present invention has greater cell adhesiveness as compared with the conventionally known laminin core sequence peptide, and exhibits various biological activities such as cancer metastasis inhibitory action. It retains and shows almost no serious side effects on the living body. Further, since its structure is simple, it can be easily synthesized and is highly valuable as a medicine.

Continued Front Page (72) Inventor Ichiro Higashi 5-3-2 Makomanaikamimachi, Minami-ku, Sapporo-shi, Hokkaido

Claims (2)

[Claims] 1. A peptide derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof. General formula (I) R-X-Tyr-Ile-Gly-Ser-Arg-Y (I) In formula, R represents a hydrogen atom or a C2-C4 acyl group. X represents a Glu or Asp residue. Y is -NR ¹
Represents R ² . Here, R ¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ¹ and R ² may be the same or different, but these two are not hydrogen atoms at the same time. 2. A cancer metastasis inhibitor comprising a pharmaceutically acceptable excipient and the peptide derivative according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.