JPH05163300A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To provide a nontoxic new peptide derivative high in cell adhesion level, useful as e.g. metastasis inhibitor, having, as the essential constituent units, acidic sugar chain residue and minimal unit of Arg-Gly-Asp tripeptide sequence involving cell adhesion. CONSTITUTION:A C-terminated amino acid is esterified to protect the side chain followed by successive reaction of both alpha-amino group and side chain functional group-protected amino acids to produce a protected peptide with cell adhesion nature of formula I (Bn is benzyl; Z is benzyloxycarbonyl), which is, in turn, made to react with an alpha-D-glucopyranosylcarboxylic acid derivative of formula III which has been prepared by reaction of succinic anhydride with an amino-deprotected compound of reaction product from a protected alpha-D- glucopyranosyl bromide derivative of formula II (Ac is acetyl; Me is methyl) and a protected aminoethoxyethanol; the resulting reaction product is then deprotected, thus affording the objective peptide derivative of formula IV (Q is acidic sugar residue; W is 1-15C alkylene, etc.; X is bond or Gly; Y is bond, Ser, etc.; R is OH, etc.).

Description

Detailed Description of the Invention

[0001]

The present invention relates to Arg-Gly-As.
The present invention relates to a peptide derivative having a tripeptide sequence p and an acidic sugar residue as essential constituent units or a pharmaceutically acceptable salt thereof, and uses thereof.

[0002]

BACKGROUND OF THE INVENTION Laminin, fibronectin, 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 chain, which is a polypeptide having a molecular weight of about 250K, and a B chain, which is about 240K, are disulfide-bonded proteins 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 part 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 to be a pentapeptide represented by Ser-Arg (YIGSR) (Graf, J. et al., C
ell 48, 989 (1987)).

[0005] These fibronectins and laminins transmit their information to cells by binding to cellular receptors through the above core sequences, and also bind to biopolymers such as heparin, collagen and fibrin to form cells. It is considered to be involved in adhesion to connective tissue, cell differentiation and proliferation.

[0006] As described above, since the cell adhesion protein has various biological activities, studies using its active site sequence peptide have been vigorously carried out. For example, as the utilization of the core sequence of the cell-binding portion of fibronectin, a method in which a peptide having an RGD sequence is covalently bound to a polymer and used as a substrate for artificial organs or a substrate for culturing animal cells (Japanese Patent Application Laid-open No. HEI-H10)
1-309682, JP-A-1-305960, WO 90/05036
A patent), a method of attaching a target peptide 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 / 11297A patent), RGD Method for inhibiting platelet aggregation by using various cyclic and chain oligopeptides having sequence or its analogs (Proceedings of the Polymer Society of Japan, 38, 31)
49 (1989), JP-A-2-174797, JP-A-3-18330, JP-A-3-118331, JP-A-3-118398, JP-A-3-118397, JP-A-3-118333 Publication, WO91 / 013
31 patent, WO 91/07429 patent), a method of using a peptide having an RGD sequence as a cell migration control agent (JP-A-2-4716).
Japanese Patent Publication), 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, Proc. 37).
Vol. 705 (1988)), a method of using a polypeptide having an RGDS sequence as a platelet protective agent for extracorporeal blood (JP-A-64)
No. 6217), a method of using a peptide having cell adhesion activity in the polypeptide molecule to utilize it as an artificial functional polypeptide (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 adhesion core sequence of fibronectin, coexists, it competitively binds to the fibronectin receptor on the cancer cells and thus the cell adhesion is blocked, showing a cancer metastasis inhibitory 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. Macrom
ol., Volume 11, 23 (1989), Ibid, Volume 11, 226 (1989), Jpn.
J. Cancer Res., Volume 60, 722, (1989), JP-A-2-174798.
Or a method for preventing tumor recurrence (JP-A-2-
No. 240020) has also been tried. 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. GR for the purpose of enhancing blood life and efficacy
A compound in which sialic acid is linked to a GDS peptide (JP-A-2-
No. 233696), but nothing is said about the actual effect.

[0009]

As described above, the ability of the adhesive core sequences such as fibronectin and laminin to suppress cancer metastasis is considered to be highly valuable as a drug, and many studies have been conducted to date. .. However, these cancer metastasis-suppressing actions are not yet satisfactory for practical application to medical treatment, and the development of more effective substances has been desired. In particular, it is thought that there are multiple patterns (eg, sugar chains and sugar chains, sugar chains and adhesion proteins) in the adhesion factors used by cancer cells during metastasis, and the RGD peptide alone cannot block the receptor. It is highly predictable that the ability to suppress cancer metastasis is insufficient.

As a result of intensive studies in view of the above situation, the present inventors have found a novel peptide derivative which is expected to enhance the ability to bind to a receptor by multipoint binding,
The present invention has been completed. That is, the object of the present invention is to
It is intended to provide a novel peptide derivative having a useful biological activity. Another object of the present invention is to provide a drug composition containing the above peptide derivative.

[0011]

Means for Solving the Problems The above problems have been achieved by finding a peptide derivative represented by the following general formula (I). Therefore, the present invention is a peptide derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof, and a cancer metastasis inhibitor containing the compound as an active ingredient. General formula (I) Q-W-CO- [X] -Arg-Gly-Asp- [Y] -R (I) Here, Arg, Gly, and Asp represent an arginine, glycine, and aspartic acid residue, respectively. These amino acid residues may be in the D-form, L-form or racemic form, but are preferably the natural L-form.

In the formula, W represents a straight-chain or substituted alkylene group having 1 to 15 carbon atoms or an arylene group,
It may be bonded via -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond. When W is an alkylene group, it preferably has 1 to 11 carbon atoms, and may have an unsaturated bond such as a double bond or a cyclic structure such as a cyclohexane ring or a benzene ring. As a substituent, a hydroxyl group, a halogen atom, a carbonyl group, a methyl group, a lower alkyl group such as an ethyl group, a lower acyloxy group represented by an acetoxy group, a methoxy group, a lower alkoxy group such as an ethoxy group, a nitro group, a cyano group, Examples thereof include a sulfo group and the like, and preferred substituents are a carbonyl group, a methyl group, a hydroxyl group and an acetoxy group. When W is an arylene group, it preferably has 6 to 14 carbon atoms and may have a substituent. Substituents that may be present include a hydroxyl group, a halogen atom, a lower alkyl group such as a methyl group and an ethyl group, a lower acyloxy group represented by an acetoxy group, a lower alkoxy group such as a methoxy group and an ethoxy group, a nitro group, and a cyano group. Examples of the substituent include a methyl group, a hydroxyl group, and an acetoxy group. These may be substituted at any position on the ring.

X, which may or may not be present, represents the Gly residue when present. Y may or may not be present, but if present, Ser (serine), T
hr (threonine), Asp (aspartic acid), Va
1 represents an amino acid residue or a peptide residue selected from the group consisting of 1 (valine) and Ser-Pro (serine-proline). Among them, Y, Ser, Thr, Se
The r-Pro residue is preferred.

R represents either --OH or --NR ¹ R ² . Here, R ¹ and R ² represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. These R ¹ and R ² may be the same or different and may be linked to each other to have a cyclic structure. Further, when R ¹ and R ² have 3 or more carbon atoms, they may have a branched structure or a cyclic structure. Examples of preferable R include —OH, —NH ₂ , methylamino group, ethylamino group, isopropylamino group, dimethylamino group, diethylamino group and cyclohexylamino group.

Q represents an acidic sugar residue (excluding sialic acid residue) bound to W via a glycosidic bond. In this case, the glycoside bond may be either O-glycoside or S-glycoside. N-glycosides are not suitable for the present invention because they are unstable. The acidic sugar residue in the present invention means a sugar residue having at least one carboxyl group, sulfate group or phosphate group on the sugar chain.
As a sugar residue having at least one carboxyl group on the sugar chain, an oligosaccharide composed of uronic acid and uronic acid, composed of 2-acetamido-2-deoxyuronic acid and 2-acetamido-2-deoxyuronic acid Oligosaccharides,
6-O-carboxymethyl-D-glucose, 6′-O-carboxymethyl-D-maltose, oligosaccharide composed of glucuronic acid and N-acetylglucosamine, oligosaccharide composed of glucuronic acid and glucosamine, glucuronic acid And oligosaccharides composed of N-acetylgalactosamine, oligosaccharides composed of glucuronic acid and galactosamine, oligosaccharides composed of glucuronic acid and glucose, oligosaccharides composed of glucuronic acid and galactose, glucuronic acid and mannose Oligosaccharides composed of galacturonic acid and N-acetylglucosamine, oligosaccharides composed of galacturonic acid and glucosamine, oligosaccharides composed of galacturonic acid and N-acetylgalactosamine, galacturonic acid and galactosamine Composed At least one selected from the group consisting of oligosaccharides composed of galacturonic acid and glucose, oligosaccharides composed of galacturonic acid and galactose, and oligosaccharides composed of galacturonic acid and mannose.

Among the above sugar residues, uronic acid, oligosaccharide composed of uronic acid, 6-O-carboxymethyl-D-
Glucose, oligosaccharide composed of glucuronic acid and galactose, oligosaccharide composed of glucuronic acid and N-acetylglucosamine, oligosaccharide composed of glucuronic acid and glucosamine, oligo composed of glucuronic acid and N-acetylgalactosamine Particularly preferred are sugars, oligosaccharides composed of glucuronic acid and galactosamine.

Examples of uronic acid include iduronic acid, galacturonic acid, glucuronic acid and mannuronic acid. Examples of oligosaccharides composed of uronic acid include β-glucuronosyl glucuronic acid and β-galacturonosyl galacturonic acid. Examples of oligosaccharides composed of glucuronic acid and galactosamine include chondrosin. Examples of oligosaccharides composed of glucuronic acid and glucosamine include hyalobiouronic acid and heparosin. Needless to say, these sugars may be naturally occurring sugars or chemically synthesized sugars.
Also preferred are oligosaccharides of non-naturally occurring linkage type that are obtained by chemical synthesis. For example, 6-O-carboxymethyl-D-glucose is 1,2,3,4-tetra-O-
Alkylation of benzyl-β-D-glucose with methyl bromoacetate in the presence of sodium hydride can be synthesized as a key step.

As the sugar residue having at least one sulfate group or phosphate group on the sugar chain, glucose-6-
Sulfuric acid, galactose-6-sulfuric acid, N-acetylglucosamine
-6-sulfate, N-acetylgalactosamine-6-sulfate, N-acetylgalactosamine-4-sulfate, mannose-6-phosphate, glucose-6-phosphate, or oligosaccharides containing these sugars as constituent sugars be able to. In particular, sugars whose hydroxyl groups are sulfated are constituent components of mucopolysaccharides that are widely distributed in the living body, and through the interaction with sugar chains present on the cell surface, the compound of the present invention and the cancer cell receptor are further It is thought that the bond is strengthened.

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. Specifically, hydrochloride, sulfate, nitrate,
Examples thereof include salts with inorganic acids such as phosphates, acetates, lactates, salts with organic acids such as tartrates, and ammonium salts, sodium salts, potassium salts, magnesium salts and the like. , Acetate, and sodium salt are particularly 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.

[0021]

[Chemical 1]

[0022]

[Chemical 2]

Next, a method for synthesizing the compound of the present invention will be described. The compound of the present invention can be synthesized by various methods. However, since it has a peptide part and a sugar residue as essential constituents, the peptide part and the sugar part must be synthesized separately after the synthesis. A method of synthesizing both components by linking and further deprotection is advantageous. First, examples of the method for synthesizing the peptide part include a solid phase method, a synthetic method using an automatic peptide synthesizer utilizing the solid phase method, and a liquid phase method. Regarding the solid phase method and the synthetic method using the automatic peptide synthesizer using the solid phase method, Biochemistry Experiment Course / Protein Chemistry IV p.207 (edited by the Biochemical Society of Japan, Tokyo Kagaku Dojin), Zokusei Chemistry Experiment Course / Protein Chemistry (below) p.6
41 (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin) and the like.

It is also possible to synthesize the peptide portion by a liquid phase method. That is, it is a method of starting from a protected C-terminal component, removing the N-terminal protecting group, and then successively condensing protected amino acids. A method of performing fragment condensation is also effective. The method for condensing the protected amino acid or the protected peptide is not particularly limited, and all known methods can be basically used. Peptide synthesis is described in detail in "Basics and Experiments of Peptide Synthesis" (Maruzen) edited by Nobuo Izumiya et al., 1-hydroxybenzotriazole and DC.
The DCC-Additive method using C or the condensation method using carbonyldiimidazole gave the best results.

The synthesis of the sugar moiety can be performed using the glycosylation reaction as a key step. Various methods are known for the glycosylation reaction, and the reaction conditions can be appropriately set in consideration of the type of sugar, availability, deprotection conditions, and the like. The reaction is basically carried out by condensing an alcohol serving as a sugar donor and a sugar acceptor in the presence of a promoter. For example, when sugar chloride or bromide is used as the sugar donor, silver oxide, silver carbonate, silver perchlorate, silver trifluoromethanesulfonate, mercuric cyanide, mercury bromide or the like is used as the promoter. When a fluorinated sugar is used as a sugar donor, stannous chloride / silver perchlorate, zirconocene dichloride / silver perchlorate or the like can be used as a promoter. When an alkylthio compound is used as the sugar donor, dimethyl (methylthio) sulfonium triflate, trifluoromethanesulfonic acid methyl ester, phenylselenyl triflate, etc. are used as the promoter. When imidate is used as the sugar donor, the reaction is carried out using a Lewis acid (eg boron trifluoride diethyl ether complex). In carrying out the above-mentioned glycosylation reaction, a dehydrating and drying agent such as molecular sieve or drylite can be used. When the reducing end of the sugar residue is a 2-acetamido-2-deoxy sugar such as N-acetylglucosamine, glycosylation by the oxazoline method is effective.

When the sugar residue is an aryl glycoside, the reaction can be carried out by heating the polyacetylated sugar and phenol in a nonpolar solvent (benzene, toluene, etc.) as a key step. In particular, p-nitrophenyl glycosides can be purchased.

Further, S-glycoside is reacted with thiol using sugar chloride and bromide as sugar donors Koenig-Knorr
Can be synthesized by the method. The reaction proceeds easily because thiol has a stronger nucleophilic reactivity than alcohol.

The sugar moiety and the peptide moiety are linked by the following general formula:
It can be carried out by condensing both components represented by (II) and (III). In the general formulas (II) and (III), Q,
W, [X], [Y] and R have the same meanings as in formula (I). Q-W-COOH (II) H- [X] -Arg-Gly-Asp- [Y] -R (III)

At this time, the reactive functional groups other than the carboxyl group and the amino group at the N-terminal of the peptide involved in the reaction are
It is desirable to protect with a suitable protecting group. The condensation method is not particularly limited, and basically the same method as in the case of peptide bond formation can be used. Specific examples thereof include a condensation method using carbonyldiimidazole.

The conditions for removing the protecting group depend on the type of protecting group used. Commonly used methods are hydrogenolysis,
Needless to say, various methods such as alkali saponification can be used depending on the type of the protecting group. The desired peptide derivative can be deprotected and then purified by a known method such as ion exchange chromatography or gel filtration chromatography.

Next, the action and use of the peptide derivative of the present invention will be described. The peptide derivative of the present invention is Arg-Gly-As, which is the smallest unit involved in cell adhesion.
Since it has a tripeptide sequence p and an acidic sugar residue as essential constituent units, it is a compound that can be expected to enhance the ability to bind to the receptor by multipoint binding. That is, the peptide derivative of the present invention acts on the fibronectin receptor on malignant cells and inhibits the binding of malignant cells to fibronectin, thereby preventing adhesion of malignant cells to connective tissue, colonization, and destructive erosion. .. Furthermore, it is considered that the presence of the sugar residue further enhances the binding between the peptide derivative of the present invention and the cell through the interaction with the sugar chain present on the cell surface. It is considered that this peptide derivative also contributes to the extension of blood life in blood. The peptide derivative of the present invention includes breast cancer, epidermal cancer,
Muscle line melanoma, epidermal line neuroblastoma x
glioma), chondrocytes, and various cells including fibrosarcoma, which are effective in inhibiting adhesion and metastasis.

Further, the peptide derivative of the present invention is recognized to have a wide range of biological activities such as a wound healing action and an inhibitory action on platelet aggregation by cancer cells occurring in capillaries. In addition, when the peptide derivative of the present invention was subjected to a toxicity test using mice, no toxicity was observed at all.

The peptide derivative of the present invention or a salt thereof is
It can be used by an administration method generally used for peptide drugs, and is usually administered as a drug composition containing an excipient. This drug composition is, for example, a product of Remington's Pharmaceutical Sciences, Merm.
ck, 16, (1980)), and may be produced by any known method. Distilled water as an excipient,
Physiological saline, buffer solution containing buffer salts such as phosphate or acetate, antioxidants such as sodium chloride and sucrose as osmotic pressure adjusting agents, or ascorbic acid, or pharmaceutically acceptable these There is a combination of.

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 used as a freeze-drying agent after being dissolved in an acetic acid buffer solution of about 0.1 N. It is also possible to use it in the form of microcapsules or microspheres encapsulated in liposomes.

The dose of the peptide derivative of the present invention is usually
The range is 0.2 μg / kg to 200 mg / kg, which is determined by the patient's age, body weight, symptoms, and administration method.

The present invention will be described in more detail with reference to the following examples. The method for synthesizing Exemplified Compound 1 will be described in detail, but other Exemplified Compounds were basically synthesized in the same manner. The synthetic route of Exemplified Compound 1 is shown below. The following abbreviations are used in the notation of commonly used solvents, reagents, and protecting groups.

Boc: t-butoxycarbonyl Bn: benzyl Ac: acetyl Me: methyl HOBt: 1-hydroxybenzotriazole DCC: dicyclohexylcarbodiimide iPr ₂ NEt: diisopropylethylamine AcOH: acetic acid AcOEt: ethyl acetate DMF: dimethylbenzylformamide Z Carbonyl CDI: Carbonyldiimidazole TFA: Trifluoroacetic acid

[0038]

[Chemical 3]

[0039]

[Chemical 4]

[0040]

【Example】

Example 1 Synthesis of Exemplified Compound 1 1) Synthesis of Intermediate 1 Boc-Ser (Bn) -OH (20.4 g, 69 mmol), benzyl bromide
(13 g, 76 mmol), diisopropylethylamine (9.8
g, 76 mmol) was dissolved in ethyl acetate (100 ml), and the reaction mixture was heated under reflux for 5 hours. After allowing to cool to room temperature, the generated salt was filtered off, and the filtrate was 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 an intermediate product. Body 1 was obtained as a colorless oil. This was used in the next reaction without purification.

2) Synthesis of Intermediate 2 Methylene chloride (80%) of Intermediate 1 obtained by the method described in the preceding paragraph.
ml) solution was added trifluoroacetic acid (80 ml) and the reaction mixture was stirred at room temperature for 40 minutes. After completion of the reaction, the solution was concentrated under reduced pressure to remove most of the solvent, and the residue was diluted with ethyl acetate.
The extract was washed with saturated aqueous sodium hydrogen carbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the amine compound as a colorless oil.

The obtained amine compound and Boc-Asp (Bn) -OH (22.6
g, 70 mmol), 1-hydroxybenzotriazole (10.7
g, 70 mmol) in DMF (80 ml) and methylene chloride (80 ml)
Dissolved in a mixed solvent of DCC (14.4 g, 70 mm
ol) was added. The reaction mixture was stirred under ice-cooling for 2 hours 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, 5% sodium carbonate solution, 1 M citric acid solution,
The extract was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give an oil. Purify by silica gel column chromatography (eluent: hexane / ethyl acetate = 4/1) to obtain the target intermediate 2 as colorless crystals.
35.8 g (87% yield in 3 steps) were obtained.

3) Synthesis of Intermediate 3 Intermediate 2 (35.7 g, 60.5 mmol) of methylene chloride (100 m
l) Trifluoroacetic acid (100 ml) was added to the solution and the reaction mixture was stirred at room temperature for 40 minutes. After completion of the reaction, the solvent was distilled off and the residue was crystallized from ether to obtain 32.5 g of trifluoroacetic acid salt.

The resulting trifluoroacetic acid salt (18.9 g, 3
1.3 mmol), Boc-Gly-OH (5.75 g, 33 mmol), 1-hydroxybenzotriazole (5.04 g, 33 mmol), diisopropylethylamine (4.45 g, 34.4 mmol) in DMF (20 m
l) and methylene chloride (50 ml) were dissolved in a mixed solvent, and DCC (6.8 g, 33 mmol) was added while cooling with ice. The reaction mixture was stirred under ice-cooling for 2 hours and further stirred while warming to room temperature overnight, and then filtered through Celite to remove the formed precipitate. The filtrate was diluted with an appropriate amount of ethyl acetate, washed with water, 5% sodium carbonate solution, 1 M citric acid solution, and saturated saline, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a colorless solid. .. This was recrystallized from hexane / ethyl acetate (2/1) to obtain Intermediate 3 (17.9 g, yield 87%).

4) Synthesis of Intermediate 4 Intermediate 3 (17.9 g, 27 mmol) in methylene chloride (60 ml)
Trifluoroacetic acid (60 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 trifluoroacetate 15.9
got g. On the other hand, Boc-Arg (Z) 2-OH (5.0 g, 9.2 mmol)
Dissolve it in DMF (20 ml) and add CDI (1.51
g, 9.3 mmol) in DMF (10 ml) was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (6.22 g, 9.2 mmol) obtained by the above operation and diisopropylethylamine (1.32 g, 9.5 mmol) DMF (2
0 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 obtained solid was recrystallized from ether to obtain 10.8 g (quantitative) of the target Intermediate 4 as colorless crystals.

FAB-MS: (M + H) ⁺ 1072.

5) Synthesis of Intermediate 5 Intermediate 4 (11.5 g, 11 mmol) in methylene chloride (40 ml)
Trifluoroacetic acid (40 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 dissolved in ethyl acetate. The ethyl acetate layer is saturated aqueous sodium hydrogen carbonate,
The extract was washed with saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was recrystallized from ether to obtain Intermediate 5 (11.5 g, quantitative).

FAB-MS: (M + H) ⁺ 972, (M + Na) ⁺ 994.

6) Synthesis of Intermediate 6 Includes 2- (2-N-benzyloxycarbonylaminoethoxy) ethanol (7.2 g, 30 mmol), silver carbonate (12 g), drylite (40 g), dried alcohol A mixture of unexisting chloroform (100 ml) was stirred for 1 hour under shaded conditions to remove water in the system. After adding iodine (4 g), (2,3,4-tri-O-acetyl-α-D-glucopyranosyl bromide) -uronate methyl ester [8 g, 20 mmo
l, Reference (YA Hassan, J. Carbohydrates. Nucreoside
S. Nucreotides, Vol. 4, p. 77 (1977)] in chloroform (50 ml) was added over 30 minutes, and the reaction mixture was stirred at room temperature for 30 hours. Insoluble components were removed by filtration through Celite, and the Celite layer was washed with chloroform. The filtrate and washings were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give an oil. Purification by silica gel column chromatography (eluent: chloroform) gave 8.9 g (yield 80%) of the target intermediate 6 as a colorless oil.

FAB-MS: (M + H) ⁺ 556, (M + Na) ⁺ 578.

7) Synthesis of Intermediate 7 Intermediate 6 (5.4 g, 9.7 mmol) in methanol (100 ml)
10% Pd-C (300 mg) was added to the solution, and the reaction mixture was stirred under a hydrogen atmosphere at room temperature for 13 hours. After completion of the reaction, the catalyst was removed by filtration through Celite, and the Celite layer was washed with methanol. The filtrate and washings are combined and concentrated under reduced pressure.
g was obtained as a colorless oil.

The obtained amine compound was treated with methylene chloride (50 ml).
Dissolved in water, triethylamine (1.02 g, 10 mmol) was added, and succinic anhydride (980 mg, 9.8 mm was added while cooling with ice.
ol) was added. After stirring the reaction mixture at room temperature for 1 hour,
The reaction was stopped by adding an appropriate amount of water. After separating the organic layer, the aqueous layer was extracted with chloroform. The chloroform layers were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 5.0 g (quantitative) of the target Intermediate 7 as an oily substance.

FAB-MS: (M + H) ⁺ 552.

8) Synthesis of Intermediate 8 Intermediate 7 (1.86 g, 3.6 mmol) was dissolved in dry DMF (15 ml), and CDI (620 mg, 3.8 mmol) in dry DMF (10 ml) was added thereto while cooling with ice. ml) solution was added. The reaction mixture was stirred for 1 hour with ice cooling, then Intermediate 5 (3.5 g, 3.6 mmol)
Of DMF (20 ml) was added. The reaction mixture was stirred under ice-cooling for 2 hours, further stirred for 20 hours while warming to room temperature, and then the solvent was evaporated under reduced pressure. The residue was recrystallized from ether to give the desired intermediate 8 as colorless powder 3.7 g (yield 70%)
Obtained.

FAB-MS: (M + H) ⁺ 1475.

9) Synthesis of Intermediate 9 10% of Intermediate 8 (1.6 g, 1 mmol) in acetic acid (40 ml) was added.
Pd-C (200 mg) was added, and the reaction mixture was stirred under a hydrogen atmosphere at room temperature for 36 hours. The catalyst is filtered off with Celite,
The Celite layer was washed with acetic acid. The filtrate and washings were combined and concentrated under reduced pressure. The residue is dissolved in a small amount of water and freeze-dried,
900 mg (yield 88%) of Intermediate 9 was obtained as a colorless powder.

FAB-MS: (M + H) ⁺ 937.

10) Synthesis of Exemplified Compound 1 Powdered sodium methoxide (180 mg, 3.3 mmol) was added to an aqueous solution (20 ml) of Intermediate 9 (650 mg, 0.7 mmol),
The reaction mixture was stirred at room temperature for 4 hours. After completion of the reaction, dilute acetic acid was added to neutralize the reaction solution and concentrated under reduced pressure. The residue was dissolved in a small amount of water, subjected to ion exchange chromatography (Amberlite IRA-93ZU), and then subjected to gel filtration chromatography using Sephadex G-10 for purification and lyophilization to give the desired compound 1 as a colorless amorphous solid. 470
mg (yield 84%) was obtained.

FAB-MS: (M + H) ⁺ 797.

Example 2 Synthesis of Exemplified Compound 2 Exemplified Compound 2 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 5 and Intermediate 10 represented by the following formula as a key step.

[0061]

[Chemical 5]

Yield 1.05 g, FAB-MS: (M + H) ⁺ 696.

Example 3 Synthesis of Exemplified Compound 3 Exemplified Compound 3 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 5 and Intermediate 11 represented by the following formula as a key step.

[0064]

[Chemical 6]

Yield 230 mg, FAB-MS: (M + H) ⁺ 801.

Example 4 Synthesis of Exemplified Compound 4 Exemplified Compound 4 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 5 and Intermediate 12 represented by the following formula as a key step.

[0067]

[Chemical 7]

Yield 370 mg, FAB-MS: (M + H) ⁺ 899, (M +
Na) ⁺ 21.

Example 5 Synthesis of Exemplified Compound 5 Exemplified Compound 5 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 5 and Intermediate 13 represented by the following formula as a key step.

[0070]

[Chemical 8]

Yield 110 mg, FAB-MS: (M + H) ⁺ 858.

Example 6 Synthesis of Exemplified Compound 6 Exemplified Compound 6 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 7 and Intermediate 14 represented by the following formula as a key step.

[0073]

[Chemical 9]

Yield 510 mg, FAB-MS: (M + H) ⁺ 854.

Example 7 Synthesis of Exemplified Compound 7 Exemplified Compound 7 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 7 and Intermediate 15 represented by the following formula as a key step.

[0076]

[Chemical 10]

Yield 490 mg, FAB-MS: (M + H) ⁺ 752.

Example 8 Synthesis of Exemplified Compound 8 Exemplified Compound 8 was synthesized by a method similar to that described in Example 1 with the condensation of Intermediate 12 and Intermediate 16 represented by the following formula as a key step.

[0079]

[Chemical 11]

Yield 210 mg, FAB-MS: (M + H) ⁺ 998.

Example 9 Cancer Metastasis Inhibitory Effect The cancer metastasis inhibitory effect of the compound of the present invention was examined by an experimental lung metastasis model system. Exemplary compound 1 of the present invention,
2, 4, 5, 7, 8 and Arg-Gly-Asp-Ser as comparative examples
And Gly-Arg-Gly-Asp-Ser were used. B16-BL, which is a highly metastatic cancer cell with 1000 μg of each of these peptides
6 melanoma cells (5 x 10 ⁴ cells in logarithmic growth phase) each
Mix in 0.2 ml of BS and use 0.2 ml of C57BL / 6 per group.
Female mice were injected with 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.

────────────────────────────────── Number of metastases to the lung Mean dose SD ± range (range) ────────────────────────────────── PBS (untreated) 139 ± 58 (62-219) Exemplified Compound 1 54 ± 11 (34-64) * Exemplified Compound 2 51 ± 27 (23-92) * Exemplified Compound 4 20 ± 6 (10-28) ** Exemplified Compound 5 6 ± 5 (0-15) ** Exemplified Compound 7 34 ± 12 (21-48) ** Exemplified Compound 8 25 ± 10 (12-43) ** Arg-Gly-Asp-Ser 177 ± 68 (78-246) Gly-Arg-Gly-Asp-Ser 162 ± 28 (128-193) ────────────────────────────────── * t-test compared to untreated control P <0.01 ** P <0.001

According to these results, cancer metastasis to the lung was significantly suppressed by the administration of the exemplified compounds 1, 2, 4, 5, 7, 8 of the present invention. On the other hand, Ar which is just a core array
It can be seen that g-Gly-Asp-Ser and Gly-Arg-Gly-Asp-Ser have no such inhibitory effect on metastasis. Therefore, the above results clearly show the cancer metastasis inhibitory effect of the peptide derivative of the present invention.

Although the present invention has been described with reference to particular examples by way of examples, it should not be construed as limiting. Obviously, various changes and modifications can be made without departing from the essence and scope of the present invention, and such an invention is included in the present invention.

[0085]

INDUSTRIAL APPLICABILITY As described above, the peptide derivative of the present invention is the smallest unit involved in cell adhesion, Arg-G.
Since it has a tripeptide sequence ly-Asp and an acidic sugar residue as essential structural units, it has greater cell adhesion than a simple fibronectin core sequence fragment, and sufficiently retains various biological activities such as cancer metastasis suppressive action. However, there is almost no toxicity problem. Therefore, the peptide derivative of the present invention is highly valuable as a medicine.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ikuo Suiki 5-3, Atsetsu Kita 3-jo Nishi, Atsubetsu-ku, Sapporo-shi, Hokkaido 12-6 (72) Inventor Ichiro Higashi 5-3, Makomanaikamicho, Minami-ku, Sapporo-shi, Hokkaido

Claims (5)

[Claims] 1. A peptide derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof. General formula (I) Q-W-CO- [X] -Arg-Gly-Asp- [Y] -R (I) In formula, W is a C1-C15 linear or substituted alkylene group or arylene. Represents a group, -O-, -N
It may be via H-, -S-, ester bond, amide bond, urethane bond, or urea bond. X may or may not be present, but when present represents a Gly residue.
Y may or may not be present, but if present S
represents an amino acid residue or peptide residue selected from the group consisting of er, Thr, Asp, Val and Ser-Pro. R represents either -OH or -NR ¹ R ^2. Here, R ¹ and R ² are hydrogen atoms or have 1 to 6 carbon atoms.
Represents an alkyl group, and R ¹ and R ² may be connected to each other to form a cyclic structure. Q represents an acidic sugar residue (excluding sialic acid residue) bound to W via a glycosidic bond. 2. The peptide derivative according to claim 1, wherein Q is a sugar residue having at least one of a carboxyl group, a sulfate group and a phosphate group on the sugar chain, or a pharmaceutically acceptable derivative thereof. salt. 3. Q is an oligosaccharide composed of uronic acid and uronic acid, 2-acetamido-2-deoxyuronic acid, 2-
Acetamide-2-deoxyuronic acid-containing oligosaccharide, 6-O-carboxymethyl-D-glucose, 6'-O
-Carboxymethyl-D-maltose, glucuronic acid and N-
From oligosaccharides composed of acetylglucosamine, oligosaccharides composed of glucuronic acid and glucosamine, oligosaccharides composed of glucuronic acid and N-acetylgalactosamine, oligosaccharides composed of glucuronic acid and galactosamine, and glucuronic acid and glucose Constituted oligosaccharides, oligosaccharides composed of glucuronic acid and galactose, oligosaccharides composed of glucuronic acid and mannose, oligosaccharides composed of galacturonic acid and N-acetylglucosamine, composed of galacturonic acid and glucosamine Oligosaccharides, oligosaccharides composed of galacturonic acid and N-acetylgalactosamine, oligosaccharides composed of galacturonic acid and galactosamine, oligosaccharides composed of galacturonic acid and glucose, galacturonic acid and galactose Et constituted oligosaccharide, sugar residue selected from the group consisting of consisting oligosaccharide galacturonic acid and mannose, peptide derivative or a pharmaceutically acceptable salt thereof according to claim 1. 4. Q is composed of uronic acid, oligosaccharide composed of uronic acid, 6-O-carboxymethyl-D-glucose, oligosaccharide composed of glucuronic acid and galactose, and glucuronic acid and N-acetylglucosamine. Sugar residue selected from the group consisting of oligosaccharides composed of glucuronic acid and glucosamine, oligosaccharides composed of glucuronic acid and N-acetylgalactosamine, oligosaccharides composed of glucuronic acid and galactosamine The peptide derivative according to claim 1, which is a group, or a pharmaceutically acceptable salt thereof. 5. A cancer metastasis inhibitor comprising a peptide derivative represented by the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.