JPH06116287A - Propenamide derivative, its polymer and its use - Google Patents

Abstract

PURPOSE:To obtain the derivative free from toxicity useful as a raw material for an inhibitor against cancerous metastasis, having an active site sequence peptide of cell adhesion protein laminin at the side chain, having high cell adhesiveness and various biological activities such as inhibitory action on cancer ous metastasis. CONSTITUTION:A pentapeptide of the formula Tyr-IIe-Gly-Ser-Arg which is a core sequence of a cell adhesion site of laminin is synthesized by successively binding a terminal C amino acid containing a protected carboxyl group and a protected side-chain functional group to an amino acid containing a protected alpha-amino group and a protected side-chain functional group in the presence of a condensation agent in a solvent, eliminating a terminal N amino protecting group of the peptide, reacting the resultant substance with carboxyethylmethacrylamide and then removing the protecting group to give the objective propenamide derivative of formula I [R<1> and R<2> are H or carboxyl; R<3> is H, methyl, etc.; A is group of formula II (X is Glu, Asp, etc.; Y is amino, OH, etc.; R<4> is H, 1-11C alkylene, etc.; (n) is 1-3)].

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a propene amide derivative having an active site sequence peptide of laminin, which is a cell adhesive protein, in its side chain, a polymer thereof 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 called cell adhesive proteins. For example, fibronectin is a glycoprotein that is biosynthesized in the liver and is present in human plasma at a concentration of about 0.3 mg / ml.

The primary structure of fibronectin has been determined using molecular cloning (Koarnblihtt, A.
R. et al., EMBO Journal, 4, 2519 (1985)), A chain which is a polypeptide having a molecular weight of about 250K, and B having a molecular weight of about 240K.
It has been shown that the chain is a disulfide-linked protein with a disulfide bond near the C-terminus. As for laminin, Sasaki et al. (Sasaki, M. et al., Proc. Natl. Aca
d. Sci. USA., 81, 935 (1987), Sasaki, M. et al., J.
Biol. Chem., 262, 17111 (1987)) has determined its primary structure. 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-S.
It has also been elucidated to be a pentapeptide represented by er-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 via 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 adhesive protein has various biological activities, vigorous studies have been conducted using its active site sequence peptide. 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 (JP-A-1-309682, JP-A-1-309682) 1-305960, WO 90/0503
6A 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 90/11297 A patent) Method for Inhibiting Platelet Aggregation Using Various Cyclic and Chain Oligopeptides Having RGD Sequences or Analogues Thereof
Vol., 3149 (1989), JP-A-2-74797, JP-A-3-118.
No. 330, JP-A-3-118331, JP-A-3-118398, JP-A-3-118397, JP-A-3-118333,
WO 91/01331 patent, WO 91/07429 patent), a method of using a peptide having an RGD sequence as a cell migration control agent (Japanese Patent Application Laid-Open No. 2-4716), and a membrane having a peptide having an RGD sequence immobilized thereon is a cell adhesion film. (Polymer Society of Japan Proceedings, Vol. 37, 705 (1988)), a method of using a polypeptide having an RGDS sequence as a platelet protective agent for extracorporeal blood (JP-A-64-6217), intra-polypeptide molecule A method of using a peptide having cell adhesion activity as an artificial functional polypeptide by adding the peptide (Japanese Patent Laid-Open No. 3-34996)
Gazette) is disclosed.

Furthermore, in recent years, cell adhesive 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)).

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, 23 (1989), ibid., 11, 226 (1989), Jpn. J.
Cancer Res., 60, 722, (1989), Japanese Patent Application Laid-Open No. 2-174798), or a method for preventing tumor recurrence (Japanese Patent Application Laid-Open No. 2-2400).
No. 20) has also 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 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-A-3-2196, Iwamoto, I.
Science 238, 1132 (1987), Graf, J. Biochemistry 2
6, 6896 (1987), Mayumi, T. et al., Biochem. Biophy.
s. Res. Commun., 174, 1159 (1991), Mayumi, T. et.
al., Peptide chemistry, 145 (1991), Tanaka, NG et
al., Cancer Res., 51, 903 (1991)). It is believed that they exhibit activity by inhibiting cell adhesion to laminin.

[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 not sufficient as compared with the natural cell adhesive protein, and in this respect, the development of a more effective substance has been required. Therefore, various methods as described in the section of the prior art have been reported, but many of them are still insufficient in biological activity or difficult to synthesize. Further, a method of linking the core sequence with a polymer such as polyethylene glycol (PEG) by a polymer reaction and a method of repeating the core sequence to form a polypeptide are also known. However, radically polymerizable monomers having the core sequence in the side chain have not been known, and neither change of charge balance nor control of hydrophilicity / hydrophobicity by copolymerization with other monomers has been realized. Accordingly, an object of the present invention is to provide a radical-polymerizable monomer having a cell-adhesive core sequence peptide in its side chain, which has sufficient cell-adhesive protein-like activity and can be synthesized by a simpler means, and the monomer. To provide a polymer containing The present invention further aims to provide a peptide derivative having a high cancer metastasis inhibitory activity,
Further, it is also an object to provide a drug composition containing the above peptide derivative.

[0011]

Means for Solving the Problems The above problems have been achieved by finding peptide derivatives represented by the following general formula (I) and salts thereof. In the general formula ^{(I) R 1 R 2 C} = C (R 3) CO- [NH] -A formula, R ^1, R ² represents a hydrogen atom or a carboxyl group, R ³ is a hydrogen atom, a methyl group, ethyl Represents one of a group, a halogen atom and a carboxymethyl group. A represents an adhesive peptide group represented by the following general formula (II). Formula ^{(II) - [R 4]} - [CO] - ([X] -Tyr-Ile-Gly-Ser-A
rg) n- [Y]-In the formula, X represents a Glu or Asp residue. Y is -NR
⁵ represents R ⁶ or —OH. Here, R ⁵ and R ⁶ represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ⁵ and R ⁶ may be the same or different, but these two are not hydrogen atoms at the same time. However, when the carbon number is 3 or 4, it may have a branched structure or a cyclic structure. Preferred alkyl groups include a methyl group, an ethyl group and an isopropyl group, with R being particularly preferred.
Is a hydrogen atom or a methyl group. R ⁴ is a hydrogen atom, a linear or branched alkylene group having 1 to 11 carbon atoms, or an arylene group having 6 to 11 carbon atoms, and may have a substituent. n represents an integer of 1 to 3. In the general formulas (I) and (II), [] represents the presence or absence of the presence. The group A represented by the general formula (II) is bonded to R ¹ R ² C═C (R ³ ) CO— [NH] — from either the left end side or the right end side of the general formula (II). However, when R ⁴ is present and is a hydrogen atom, it is not bonded from the left end side, and when Y is present, it is not bonded from the right end side. Each amino acid may be in the D-form, L-form or racemic form, but is preferably the L-form. Examples of the monomer bonded to the N-terminus of the cell adhesive peptide fragment of the present invention include N-methacryloylglycine, N-methacryloyl-β-alanine, N-methacryloylalanine, N-methacryloyl-β-aminopropionic acid, N-methacryloyl-γ-aminobutyric acid, N-methacryloyl valine,
Examples include propene amide derivatives such as N-methacryloyl leucine, N-methacryloyl isoleucine, N-methacryloyl norvaline and N-methacryloyl norleucine. C of the cell adhesive peptide fragment of the present invention
Examples of the monomer bonded to the terminal include monomethacrylamides of diaminoalkyl derivatives and diaminoaryl derivatives, preferably ethylenediamine monomethacrylamide, 1,3-diaminopropane monomethacrylamide, and 1,4-diamino. Butane monomethacrylamide and 1,6-diaminohexane monomethacrylamide. The present invention also provides homopolymers, copolymers and crosslinked polymers of the compound of the above formula (I) and salts thereof (hereinafter, these are referred to as "the peptide derivative of the present invention"). The comonomer used in the copolymer of the present invention is not particularly limited as long as it is an ordinary polymerizable vinyl monomer, and anionic, cationic and nonionic monomers can be used. Examples of the anionic monomer include N-methacryloyl derivatives of glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminocaproic acid, 12-aminolauric acid, 4-aminobenzoic acid, leucine and glutamic acid. And N-acryloyl derivatives, acrylic acid, methacrylic acid, itaconic acid, maleic anhydride and the like. Examples of the cationic monomer, chlorotrimethylammonioethylmethacrylamide, chlorotrimethylammoniopropylmethacrylamide, chlorotrimethylammonioethylmethacrylate, chlorotrimethylammoniopropylmethacrylate, dimethylaminoethylmethacrylamide, dimethylaminopropylmethacrylamide, Methacrylic acid derivatives such as dimethylaminoethyl methacrylate, dimethylaminopropyl methacrylate, and acrylic acid derivatives having similar side chains are included. Examples of nonionic monomers are N-vinylpyrrolidone, N, N-dimethylmethacrylamide, N, N-dimethylacrylamide, 2-hydroxypropylmethacrylamide, ethoxytriethyleneglycolmethacrylate, 3-hydroxypropylmethacrylamide, 2- Hydroxypropyl acrylamide, hydroxyethyl methacrylamide, hydroxyethyl acrylamide, N-methacryloyl glycine amide, N-acryloyl glycine amide, N-methacryloyl serine amide, N-acryloyl serine amide, N-methacryloyl glutamine amide, N-acryloyl glutamine amide, etc. Can be mentioned. The polymer of the compound of formula (I) of the present invention preferably has a molecular weight of about 3,000 to 200,000. For the binding between the propenoic acid derivative and the cell adhesive peptide, the active ester method, mixed acid anhydride method, azide method, acid chloride method, symmetrical acid anhydride method, DCC method, DCC-additive method, carbonyldiimidazole method An amide bond synthesizing method utilizing the above can be used. Further, the polymer and crosslinked polymer of the propene amide derivative can be obtained by a general radical polymerization method or ionic polymerization method. The water-soluble polymer can be subjected to specific molecular weight fractionation by a gel filtration method, a dialysis method or the like. By changing the composition of the polyfunctional monomer, the crosslinked polymer can be made into a hydrogel having different physical properties such as water content and gel strength.

The ionic group present in the peptide derivative of the present invention may form a salt with a suitable counterion. The biological activity of the peptide is sufficiently maintained even in the salt state.
However, the salt must be physiologically and pharmacologically acceptable. Specifically, salts with inorganic acids such as hydrochlorides, sulfates, nitrates, phosphates, acetates, lactates,
Examples thereof include salts with organic acids such as tartrate salts, sodium salts, potassium salts and the like. Among them, hydrochlorides and acetates are particularly preferable. Conversion to such salts can be carried out by conventional means and methods.

Specific examples of the compound of the present invention are shown below.
The present invention is not limited to these. Compound 1 (monomer 1) H ₂ C = C (CH ₃ ) CO-NH- (CH ₂ ) _2- CO-Tyr-Ile-Gly-Ser-Arg-OH

The method for synthesizing the compound of the present invention will be described below. The method for synthesizing the peptide is not particularly limited, and examples thereof include a solid phase method, a synthetic method using an automatic peptide synthesizer utilizing the solid phase method, and a liquid phase method. For the solid phase method and the method of synthesizing by the peptide automatic synthesizer using the solid phase method, see Biochemistry Laboratory, Protein Chemistry IV p.207.
(Biochemistry Society of Japan, Tokyo Kagaku Doujin), Sequel Biochemistry Laboratory, Protein Chemistry (below) p.641 (Japanese Biochemical Society, Tokyo Kagaku Doujin), etc.

The peptide derivative of the present invention can also be synthesized by a liquid phase method. That is, it is a method of starting from a protected arginine or an amide derivative of the protected arginine which becomes the C-terminal, removing the N-terminal protecting group, and successively condensing the protected amino acid. In addition, a method of performing fragment condensation between the Tyr-Ile-Gly residue and the Ser-Arg residue 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, the DCC-Additive method using 1-hydroxybenzotriazole and DCC 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, trifluoromethanesulfonic acid / thioanisole / m-cresol mixed system treatment and the like are available, but needless to say, various other methods are possible depending on the type of protective group. The target 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. Since the peptide derivative of the present invention has a YIGSR sequence in the side chain of the polymer, it is less susceptible to degradation by proteases and the like in vivo, and its life span in blood is prolonged, so that it exhibits a remarkable cancer metastasis inhibitory activity. The peptide derivative of the present invention acts on the laminin receptor on malignant cells at multiple points, and inhibits the binding to laminin to prevent malignant cell adhesion, colonization, and destructive erosion. The peptide derivative of the present invention is useful for breast cancer, epidermal cancer, muscle melanoma (muscle).
line melanoma), epidermal neuroblastoma x glyoma (epide
rmal line neuroblastoma x glioma), chondrocytes, and fibrosarcoma, which are effective in inhibiting adhesion and metastasis of various cells.

Further, the peptide derivative of the present invention was found 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 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. This drug composition is based on Remington's Pharmaceutical Sciences.
ces, Merck, 16, (1980)) and may be prepared 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 dose of the peptide derivative of the present invention is usually in the range of about 0.2 μg / kg to 200 mg / kg per day, and is determined depending on the age, body weight, symptom and administration method of the patient. Further, since the propene amide derivative of the present invention and its polymer have a high binding property to the laminin receptor on the cell surface, they can be suitably used as a culture substrate for animal cell culture. When the propene amide derivative of the present invention and its polymerized product are used as a cell culture substrate in culturing animal cells, they can be used in the same manner as a conventional cell culture substrate. Not limited. For example, a method in which an adhesive peptide is suspended in a bead culture solution that has been covalently bound and agitated at a low speed to adhere animal cells to the surface of a microcarrier and culture, a petri dish roller that is covalently treated with an adhesive peptide. A method of culturing animal cells on a bottle or the like, a method of refluxing a culture solution to a hollow fiber covalently treated with an adhesive peptide to adhere animal cells to the inner surface of the hollow fiber and culturing, a microbe covalently treated with an adhesive peptide Examples thereof include a method of using a column packed with a carrier, a method of treating with a polymer solution supporting an adhesive peptide to coat the surface of a base material, and culturing animal cells on the surface.

The cell culture substrate of the present invention can be used for culturing various cells, and the type of cells is not particularly limited, and examples thereof include living cells, hybridomas and the like.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The synthetic route of peptide 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 Mts: mesitylenesulfonyl HOBt: 1-hydroxybenzotriazole DCC: dicyclohexylcarbodiimide iPr ₂ NEt: diisopropylethylamine DMF: dimethylformamide CDI: carbonyldiimidazole acetic acid TFA. TFMSA: trifluoromethanesulfonic acid

[0025]

【Example】

Example 1 Synthesis of Monomer 1 1) Synthesis of BocArg (Mts) OBn BocArg (Mts) (21.4 g, 47 mmol), benzyl bromide (8.0 g, 47 m
mol), triethylamine (8.0 g, 47 mmol) in ethyl acetate 20
It was dissolved in 0 ml and refluxed for 8 hours. The generated salt is filtered off,
The filtrate was concentrated and then n-hexane was added for crystallization to obtain 23.1 g (90%) of the desired BocArg (Mts) OBn as a colorless powder.

2) Synthesis of BocSer (Bn) Arg (Mts) OBn BocArg (Mts) OBn (20 g, 37 mmol) in methylene chloride (50
ml) solution was added trifluoroacetic acid (50 ml) 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 19.5 g of trifluoroacetic acid salt. Meanwhile, BocSer (Bn) (5.84g, 21 mmol)
Dissolve it in DMF (20 ml) and add CDI (3.
A solution of 41 g, 21 mmol) in DMF (20 ml) was added. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (11.6 g, 21 mmol) and diisopropylethylamine (2.84 g, 22 mmol) DMF obtained by the above operation were added.
(30 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 is diluted with an appropriate amount of ethyl acetate, water, 1
Wash with M citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure.
14.0 g (94%) of c-Ser (Bn) Arg (Mts) OBn was obtained as a colorless powder. FAB-MS: (M + H) + 713

3) Synthesis of BocGlySer (Bn) Arg (Mts) OBn Trifluoroacetic acid (50 ml) was added to a solution of BocSer (Bn) Arg (Mts) OBn (13.3 g, 18.7 mmol) in methylene chloride (50 ml). 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 12.3 g of trifluoroacetic acid salt. Meanwhile, BocGlyOH (3.20g, 1
8.3 mmol) was dissolved in DMF (20 ml), and a DMF (20 ml) solution of CDI (2.97 g, 18.3 mmol) was added thereto while cooling with ice. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (12.3
g, 16.9 mmol) and diisopropylethylamine (2.45
g, 19 mmol) in DMF (30 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 obtain the desired BocGlySer (Bn) Arg (Mts ) OBn was obtained as a colorless powder, 12.4 g (95.3%). FAB-MS: (M + H) + 770

4) Synthesis of BocIleGlySer (Bn) Arg (Mts) OBn Trifluoroacetic acid (50 ml) was added to a solution of BocGlySer (Bn) Arg (Mts) OBn (12.8 g, 16.6 mmol) in methylene chloride (50 ml). 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 12.8 g of trifluoroacetic acid salt. Meanwhile, BocIleOH (3.79 g,
16.4 mmol) was dissolved in DMF (20 ml), and a DMF (20 ml) solution of CDI (2.66 g, 16.4 mmol) was added thereto while cooling with ice. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (12.7
g, 16.2mmol) and diisopropylethylamine (2.33
g, 18.0 mmol) in DMF (30 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 obtain the desired BocIleGlySer (Bn) Arg (Mts ) OBn was obtained as a colorless powder in 13.8 g (96.5%). FAB-MS: (M + H) + 883

5) BocTyr (Bn) IleGlySer (Bn) Arg (Mts) OBn
Synthesis of BocIleGlySer (Bn) Arg (Mts) OBn (13.7 g, 15.5 mmol) in methylene chloride (40 ml) in trifluoroacetic acid (40 ml)
Was added 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 13.4 g of trifluoroacetic acid salt. On the other hand, BocTyr (Bn) (5.94
g, 16.0 mmol) was dissolved in DMF (15 ml), and a DMF (20 ml) solution of CDI (2.60 g, 16.0 mmol) was added thereto while cooling with ice. The reaction mixture was stirred for 1 hour while cooling with ice, and then the trifluoroacetic acid salt (1
3.4 g, 14.9 mmol) and diisopropylethylamine (2.2
A solution of 0 g, 17.0 mmol) in DMF (30 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 the desired Boc-Tyr (Bn) IleGlySer (Bn) Arg.
11.1 g (65.6%) of (Mts) OBn was obtained as colorless crystals. FAB-MS: (M + H) + 1136

6) CEMA-Tyr (Bn) IleGlySer (Bn) Arg (Mts) O
Synthesis of Bn BocTyr (Bn) IleGlySer (Bn) Arg (Mts) OBn (1.42 g, 1.25 m
mol) in methylene chloride (10 ml) in trifluoroacetic acid
(40 ml) was added 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 1.44 g (quantitative) of trifluoroacetic acid salt. Carboxyethyl methacrylamide (CEMA) (0.22g, 1.4mmol)
Was dissolved in 5 ml of DMF and contained CDI (0.23 g, 1.4 mmol) at 0 ° C.
5 ml of MF was added and reacted for 2 hours. Add the above trifluoroacetate salt and diisopropylethylamine (0.18g, 1.4mm
10 ml of DMF containing ol) was added and reacted at 0 ° C. for 30 minutes and at room temperature for 24 hours. The solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and 1N aqueous sodium hydrogen carbonate solution and saturated saline solution were added to each 200
It was washed with ml and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure and recrystallized from ethyl acetate, and CEMA-Tyr (Bn) IleGlySer (Bn) Arg (Mts) OBn
Was obtained as a colorless powder (1.31 g) (89%). FAB-MS: (M + H) + 1175

7) Deprotection CEMA-Tyr (Bn) IleGlySer (Bn) Arg (Mts) OBn (1.14 g, 0.97
(3 mmol) in trifluoroacetic acid (12 ml) solution, trifluoromethanesulfonic acid (12 g), thioanisole (10 ml)
, M-cresol (8.7 ml), trifluoroacetic acid (45 ml)
Was added while cooling with ice, and the reaction mixture was stirred under cooling with ice for 1 hour. The reaction solution was added dropwise to ether (800 ml) and stirred slowly for 30 minutes. The precipitated crude peptide was dissolved in a small amount of water, purified by ion exchange chromatography (Amberlite IRA-400), and lyophilized to obtain the desired monomer 1 as a colorless powder (610 mg, yield).
85.6%) got. FAB-MS: (M + H) + 733

Example 2 Synthesis of Homopolymer of Monomer 1 A radical polymer of Monomer 1 was synthesized. Of monomer 1
550 mg was dissolved in 8 ml DMF, and polymerization was carried out at 65 ° C. for 6 hours in a nitrogen stream using V-65 as an initiator. V-65 7.5 mg was added at the start of polymerization and after 3 hours. After completion of the reaction, the DMF solution was poured into n-hexane / acetone (400 ml / 100 ml) to precipitate a polymer. The precipitate was recovered, dissolved in water, dialyzed against ion-exchanged water using Spectra 7 (molecular fraction 3500), and then freeze-dried. Yield 170 mg (31%) (Polymer 1). The molecular weight is TSKgel G3000 manufactured by Tosoh Corporation.
Using PW _XL and G4000PW _XL column, mobile phase was a 0.2M phosphate buffer (pH 7.4), was determined at a flow rate of 1.0 ml / min. The polyethylene glycol (PEG) equivalent molecular weight was about 22000.

Example 3 Synthesis of Copolymer of Monomer 1 and Carboxyethylmethacrylamide (CEMA) A radical copolymer of Monomer 1 and CEMA was synthesized. DM of monomer 1500mg (0.65mmol) and CEMA 410mg (2.6mmol)
Dissolve in F20 ml and use V-65 as an initiator under nitrogen stream 6
Polymerization was carried out at 5 ° C for 6 hours. The initiator was added 9 mg each after 0 and 3 hours. The polymer was reprecipitated in acetone and then purified in the same manner as in Example 2. Yield 300 mg (33%), molecular weight 30700 (Polymer 2).

Amino acid analysis revealed that the copolymer contained 25% of the unit of monomer 1 (β-Al.
Calculated from the values of a and Gly). Amino acid analysis value (nmol / 50μl): β-Ala (4.9241), Tyr
(1.0346), Ile (1.1708), Gly (1.2251), Ser (0.9947), Arg
(1.1379)

Comparative Example 1 Carboxyethylmethacrylamide (CEMA) homopolymer CEMA 500 mg (3.18 mmol) was dissolved in DMF 8 ml, and V-65 5m
l was added and the mixture was polymerized at 65 ° C. for 6 hours in a nitrogen stream. Note that V
-65 added an additional 5 mg after 3 hours. Hereinafter, Example 3
Purified in the same manner as. Yield 380 mg (76%), molecular weight 26500
(Polymer 3).

Example 4 Cancer Metastasis Inhibitory Action The cancer metastasis inhibitory action of the peptide derivative of the present invention was examined by an experimental lung metastasis model system. Polymers 1 and 2 of the present invention, polycarboxyethyl methacrylamide (Polymer 3) as a comparative example and commercially available Tyr-Ile-Gly-Ser-
Arg and Tyr-Ile-Gly-Ser-ArgNH _{2 were} used. B16-, which is a highly metastatic cancer cell with 1000 μg of each of these polymers
BL6 melanoma cells (5 x 10 ^{4 in} logarithmic growth phase) each
The mixture was mixed in PBS, and 0.2 ml of the mixture was intravenously injected into 5 C57BL / 6 female mice per group by tail vein injection. 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 in Table 1 below.

Table 1 Experimental lung metastases ────────────────────────────── Administered compound Lung metastases Mean ± SD ( Range) ────────────────────────────── PBS (untreated) 320 ± 119 (157-498) Polymer 1 148 ± 52 (93-216) ^** Polymer 2 87 ± 38 (52-156) ^** Polymer 3 208 ± 62 (130-304) Tyr-Ile-Gly-Ser-Arg 217 ± 42 (159-278) Tyr-Ile- Gly-Ser-ArgNH ₂ 306 ± 37 (250-352) ────────────────────────────── ^{* *} Not tested by t-test P <0.05 ^** P <0.01 compared to treated control

According to these results, cancer metastasis to the lung was significantly suppressed by the administration of the polymers 1 and 2 of the present invention.
On the other hand, a simple core sequence Tyr-Ile-Gly-Ser-Ar
It can be seen that g, Tyr-Ile-Gly-Ser-ArgNH ₂ and polymer 3 having no peptide moiety have almost no such inhibitory effect on transfer. Among them, the polymer 2 of the present invention
Is particularly effective. Therefore, the cancer metastasis inhibitory effect of the propene amide derivative of the present invention and its usefulness 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.

[0040]

INDUSTRIAL APPLICABILITY As described above, the propene amide derivative of the present invention has a large cell adhesiveness as compared with the core sequence of laminin, which is a cell adhesive protein, and has sufficient biological activity such as cancer metastasis inhibitory action. It retains 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 (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical display location C07K 99:00 (72) Inventor Ikuo Suiki 3-5 West 12 Atsetsukita, Atsubetsu-ku, Sapporo-shi, Hokkaido -6 (72) Inventor Ichiro Higashi 5-3 Makomanaikamimachi, Minami-ku, Sapporo-shi, Hokkaido

Claims (8)

[Claims] 1. A propenamide derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof. In the general formula ^{(I) R 1 R 2 C} = C (R 3) CO- [NH] -A formula, R ^1, R ² represents a hydrogen atom or a carboxyl group, R ³ is a hydrogen atom, a methyl group, ethyl Represents one of a group, a halogen atom and a carboxymethyl group. A represents an adhesive peptide group represented by the following general formula (II). Formula ^{(II) - [R 4]} - [CO] - ([X] -Tyr-Ile-Gly-Ser-A
rg) n- [Y]-In the formula, X represents a Glu or Asp residue. Y is -NR
⁵ represents R ⁶ or —OH. Here, R ⁵ and R ⁶ represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ⁵ and R ⁶ may be the same or different, but these two are not hydrogen atoms at the same time. R ⁴ is a hydrogen atom, a linear or branched alkylene group having 1 to 11 carbon atoms, or an arylene group having 6 to 11 carbon atoms, and may have a substituent. n represents an integer of 1 to 3. In the general formulas (I) and (II), [] represents the presence or absence of the presence. 2. A homopolymer, copolymer or salt of the propene amide derivative according to claim 1. 3. A molecular weight of about 3,000 to 200,000.
The polymerized product of the propene amide derivative according to claim 2 or a salt thereof, which is within the range of. 4. A crosslinked polymer of the propene amide derivative according to claim 1 or a salt thereof. 5. An animal cell adhesion inhibitor comprising the propene amide derivative according to claim 1, 2 or 3 as a polymer or a salt thereof as an active ingredient. 6. An animal cell culture substrate containing the crosslinked polymer of the propene amide derivative according to claim 4 or a salt thereof. 7. An animal cell culture substrate containing the immobilized propene amide derivative polymer according to claim 3 or a salt thereof. 8. A cancer metastasis inhibitor comprising the propene amide derivative according to claim 1, 2 or 3 or a polymer thereof or a salt thereof as an active ingredient.