JPH083190A - Polypeptide composed of recurring structure of cell-adhesive core sequence - Google Patents

Abstract

PURPOSE:To provide an easily synthesizable new polypeptide having various bioactivities such as cancer metastasis suppressing action and almost free from toxicity. CONSTITUTION:This polypeptide has a recurring structure expressed by the formula (Tyr-Ile-Gly-Ser-Arg)n ((n) is 2-20). The present invention also relates to the pharmacologically permissible salt of the polypeptide and a cancer metastasis suppressing agent containing the polypeptide or salt as an active component.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polypeptide having a repeating structure of an adhesion core / amino acid sequence of a cell adhesion activating protein, and a use of the polypeptide as a cancer metastasis inhibitor and other pharmaceuticals.

[0002]

BACKGROUND OF THE INVENTION Fibronectin, laminin, vitronectin and the like are proteins that are involved in adhesion between cells and interstitial connective tissue and have various physiological activities related to cell functions of animal cells. To be done. For example, fibronectin is a glycoprotein synthesized in the liver and present in human plasma at a concentration of about 0.3 mg / ml.

In fibronectin, a polypeptide A chain having a molecular weight of approximately 250K and a B chain having a molecular weight of approximately 240K are disulfide-bonded near the carboxyl terminal to form a dimer.
The primary structure of fibronectin is described by Corn Bullitt et al. (K
ornblihtt, A .; R. et al: EMBO
Journal, 4 , 1755 (1985)) using molecular cloning techniques. Also,
The primary structure of laminin is described by Sasaki et al. (Sasaki, Me.
t al: Proc. Natl. Acad. Sci.
USA, 84 , 935, 1987; Sasaki,
M. et al: J. Biol. Chem. , 26
2 , 17111 (1987)), and vitronectin is described by Suzuki et al. (Suzuki, S. et al: E.
MBO Journal, 4 , 2519 (1985))
It is decided by each.

[0004] The binding sites involved in cell adhesion activity have also been studied. From the studies on the binding of heparin, collagen, cells and bacteria to the fragments obtained by the limited degradation of fibronectin with proteolytic enzymes, the A chain was obtained. , The binding sites of both B chains have been determined (Yamada,
K. M. : Ann. Rev. Biochem. , 5
2 , 761 (1983)). Furthermore, it was elucidated in 1984 that the core sequence of the cell binding part was Arg-Gly-Asp (RGD) (Pierschbach).
er, M .; D. et al: Nature, 309 ,
30 (1984)). It has been revealed that this RGD sequence is also present in other adhesive proteins such as vitronectin. It has also been elucidated that the core sequence of the cell adhesion site in the laminin molecule is Tyr-Ile-Gly-Ser-Arg (Graf, J. et a.
1: Cell, 48 , 989 (1987)).

[0005] Fibronectin and laminin are bonded to the receptor of the adherent cell through the above core sequence and transmit the information to the adherent cell, and also have the ability to bind to biopolymers such as heparin, collagen and fibrin. It is considered to be involved in adhesion between cells and interstitial connective tissue, cell differentiation and proliferation (Yamada, KM: A).
nn. Rev. Biochem. , 52 , 761 (19
83)).

[0006] As described above, the cell adhesion activity protein has various biological activities, and its application to medicines and the like has been studied. For example, when the amount of fibronectin in plasma decreases, the function of reticuloendothelial system decreases. Examples of such cases include sepsis due to surgery and trauma, disseminated intravascular blood coagulation, burns, severe infections, and surgical shock. Administration of fibronectin is considered to be effective for the improvement of those symptoms. In addition, since fibronectin stimulates the migration ability of fibroblasts and macrophages, its application to wound healing and regulation of immune function is considered. In particular, local treatment for corneal disorders utilizing the wound healing promoting effect has already been tried (Fuj).
ikawa, L .; S. et al: Lab. Inve
st. , 45 , 120 (1981)).

On the other hand, laminin has a binding ability to collagen type IV, heparin sulfate, proteoglycan and cells. In addition, laminin acts on nerve cells and has an effect of promoting neurite outgrowth, and its action in vivo has attracted attention.

[0008] Further, the cell adhesion activating protein has attracted attention as a substance related to 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 a cell adhesion molecule such as fibronectin or laminin is present, the cells form a multicellular mass, which facilitates the growth and survival of cancer cells. In fact, it has been found that when laminin is mixed with cancer cells and administered to animals, cancer metastasis is enhanced.

On the other hand, it has been reported that a protease-degrading fragment derived from laminin has a cancer metastasis inhibiting activity (Barsky, SH et al:
J. Clin. Invest. , 74 , 843 (19
84). Similarly, the tripeptide Arg-Gly-Asp (Humphrie), which is the adhesion core of fibronectin.
S.M. J. et al: Science, 233 ,
467 (1986)) and the pentapeptide Tyr-Ile-Gly-Ser-Arg, which is an adhesive core of laminin.
(Iwamoto, Y. et al: Science
e, 238 , 1132 (1987)) has also been confirmed to suppress cancer metastasis.

[0010]

As described above, cell-adhesive proteins such as fibronectin and laminin have various biological activities, and it is desired to develop a technique for applying the related substances as a medicine. It was In particular, the cancer metastasis-suppressing action of adhesive core sequences such as fibronectin and laminin is considered to have high application value as a drug, but since the cell-adhesive activity of the core sequences is not sufficient, their cancer metastasis-suppressing action is actually It is not sufficiently satisfactory to be applied to the medical treatment, and in this respect, development of a substance having higher activity has been desired. However, since the cell adhesive protein is a natural substance, its supply is limited, and since it is a glycoprotein, it is very difficult to produce it efficiently by a synthetic method or genetic engineering.

[0011] Therefore, the present inventors have conducted diligent research in search of a novel compound that sufficiently retains various biological activities possessed by cell adhesive proteins, is easy to synthesize, and does not show serious side effects on the living body. As a result, a polypeptide compound having an extremely large cancer metastasis inhibitory activity as compared with the above core sequence was found, and the present invention was completed. The novel compound of the present invention was found to have an immunomodulatory activity, a wound healing effect, a platelet aggregation inhibitory effect, and a neurological disease healing effect, in addition to the cancer metastasis inhibitory activity.

Therefore, one of the objects of the present invention is to provide a relatively low molecular weight novel polypeptide compound having various cell-adhesive protein-like activities which can be produced by simpler means.

Another object of the present invention is to provide a novel polypeptide compound having a high cancer metastasis inhibitory activity.

The present invention further aims at providing a pharmaceutical composition containing the above novel polypeptide compound.

[0015]

The polypeptide of the present invention is characterized by comprising a repeating structure of an adhesion core / amino acid sequence of a cell adhesion active protein. A preferred polypeptide compound of the present invention is represented by the following formula: (Tyr-Ile-Gly-Ser-Arg) n (where n represents a number of 2 to 20).
Among them, the molecular weight is about 5,000 to about 15,000,
Particularly, about 10000 is preferable because the biological activity is sufficiently large and the solubility in an aqueous solvent is large.

Production Method The compound of the present invention is a corresponding short-chain peptide synthesized by a conventional method, for example, Tyr-Ile-Gly-Ser-A.
rg to diphenylphosphoryl azide (DPP
A) continuous polymerization method (Nishi, N. et a.
1: Int. J. Biol. Macromol. ，
2 , 53 (1980); Nishi, N .; et a
1: Int. J. Peptide Protein
Res. , 30 , 275, 1987).

Furthermore, it may be possible to produce the compounds of the present invention by genetic engineering techniques.

The polypeptide compound of the present invention includes both L-type amino acids and D-type amino acids. Further, the compound of the present invention, for use as a pharmaceutical, is a pharmaceutically acceptable salt, for example, a salt with an inorganic acid such as a hydrochloride or a sulfate, an acetate, a trifluoroacetate, a lactate or a tartrate. A salt with an organic acid such as the above may be used, and the conversion into such a salt can be carried out by a conventional means.

Action The polypeptide compound of the present invention has a core sequence of a cell adhesive protein repeatedly, and adheres to cells through the core sequence by a mechanism similar to that of the cell adhesive protein. Therefore, they exhibit various biological activities as agonists or antagonists of cell adhesive proteins. In particular, it has a cancer metastasis inhibitory action which is 6 to 10 times stronger than the core sequence of the cell adhesive protein. In addition, a wide range of biological activities such as an immunoregulatory effect, a wound healing effect, an inhibitory effect on platelet aggregation by cancer cells occurring in capillaries, and a healing effect on neurological diseases have been observed. Further, when the polypeptide compound of the present invention was examined using mice, no toxicity was observed.

Accordingly, the polypeptide compound of the present invention comprises
At least one of them can be administered to a patient as a cancer metastasis inhibitor, a wound healing agent, an immunomodulator, a platelet aggregation inhibitor or a neurological disease healing agent, optionally together with a conventional carrier or pharmaceutical auxiliary agent. The dose is 0.2
It is determined in the range of μg / kg to 400 mg / kg based on symptoms, age, body weight and the like.

The polypeptide of the present invention is preferably administered by an administration method generally used for peptide drugs, that is, a parenteral administration method such as intravenous administration, intramuscular administration, subcutaneous administration and the like. In the case of producing such an injectable preparation, the polypeptide of the present invention may be dissolved in PBS or physiological saline as shown in the Examples below to prepare an injectable preparation, or about 0.1N acetic acid. It may be dissolved in water or the like and then used as a freeze-dried preparation. A conventional stabilizer such as glycine or albumin may be added to such a preparation, and collagen or liposome may be used as a carrier for the purpose of extending the half-life in blood.

Furthermore, the polypeptide of the present invention can be used in the form of microcapsules encapsulated in liposomes, for example:
It can be used as an orally administered drug, or can be absorbed through mucous membranes other than the digestive tract in the form of suppositories, sublingual tablets, nasal sprays, and the like.

Biological Activity Test Next, the biological activity of the compound of the present invention will be described based on the results of pharmacological tests and the like.

(1) Adhesion ability to target cells Method of Saiki et al. (Saiki, I. et al; Can
cer Immunol. , Immunother. ，
22, 125 (1986)), the ability of the compound of the present invention to adhere to target cells was examined. That is, the polypeptide of the present invention having a structure of (Arg-Gly-Asp) n having a molecular weight of about 5000, which was synthesized in Synthesis Example 1 described later, and random amino acids of Arg, Gly, and Asp synthesized in Synthesis Example 3 were randomly synthesized. Using a comparative polypeptide (Arg, Gly, Asp) n having a sequence and an average molecular weight of about 5000, the adhesion ability of these compounds to target cells was examined in mouse B16-BL6 melanoma cells.

First, 20 μg / polypeptide to be tested
ml or 5 μg / ml of mouse fibronectin (purchased from Seikagaku Kogyo Co., Ltd.) as a positive control, and microculture wells were pre-coated, followed by B16-BL6 melanoma (2) labeled with radioactive iodinated compound [ ¹²⁵ I] IUdR. × 10 ⁴ ) was added to 0.05 ml per well and incubated at 37 ° C. for 20 minutes, and the number of cells adsorbed in the culture well was determined by measuring radioactivity.
As a negative control, 1% bovine serum albumin (BSA)
Was used. In addition, the label of B16-BL6 melanoma is
0.3 μCi / ml of [ ¹²⁵ I] IUdR (200 mCi
/ Mmol, New England Nuclear, B
Oston, Mass. , USA) 5% FB
It was carried out by culturing cells in a logarithmic growth phase for 24 hours in MEM medium containing S.

The labeled cells were washed twice with physiological saline, suspended in 0.02% EDTA for 1 minute, and then collected. The cells were then suspended in MEM medium without serum and used as a single cell suspension in the above experiments. After removing cells that do not adsorb to the culture well by washing 4 times with PBS,
The cells adsorbed in the culture wells were supplemented with 0.1 N NaOH to 0.1.
It was dissolved by adding 1 ml. The number of cells adsorbed in the culture well was measured by measuring the radioactivity of the cell lysate.

The results are shown in Table 1. As is clear from this result, the Poly (Arg-Gly-As) of the present invention was used.
p) and fibronectin promoted the adsorption of B16-BL6 melanoma cells, but the random sequence Poly (Ar
(g, Gly, Asp) and BSA hardly adsorbed cells. This result indicates that the polypeptide of the present invention has a cell adhesion activity equivalent to that of fibronectin.

[0028]

[Table 1] Table 1 also shows the results of examining the specificity of cell adhesion between Poly (Arg-Gly-Asp) and fibronectin. That is, 2 × 10 ⁴ B16-BL6 cells were placed in a culture well prepared in the same manner as in the above experiment, and the cells were mixed with 100 or 500 μg / ml of Arg-G synthetic peptide.
ly-Asp, 100 or 500 μg / ml Poly
(Arg-Gly-Asp) or H of 500 μg / ml
Incubated at 37 ° C. for 20 minutes in the presence of is-Gly-Gly. As a result, Arg-Gly-Asp or Pol
When cells were cultured in the presence of y (Arg-Gly-Asp), the ability of fibronectin to adhere to cells was inhibited, but in the presence of His-Gly-Gly, such inhibition was confirmed from the results in Table 1. Was not recognized.

The cell adhesion ability of fibronectin is
It was inhibited by the synthetic tripeptide Arg-Gly-Asp in a dose-dependent manner, and also by 5 mM EDTA. These results show that Poly (Arg-Gly-A
It has been shown that the adhesion of sp) to cells is due to a specific mechanism dependent on the presence of divalent metal ions such as calcium and magnesium and the Arg-Gly-Asp sequence. That is, it is suggested that Poly (Arg-Gly-Asp) adheres to cells through the same receptor as fibronectin.

From the above experiment, it was shown that the activity of the polypeptide compound of the present invention as an agonist or antagonist of fibronectin could be applied as a medicine.

(2) Cancer metastasis inhibitory action a) Next, the cancer metastasis inhibitory action of the compound of the present invention having a repeating structure of the core sequence Arg-Gly-Asp was examined. Pol, a synthetic polypeptide with a molecular weight of about 5,000
y (Arg-Gly-Asp), tripeptide Arg-
Gly-Asp and Poly (A with a molecular weight of about 5000
rg, Gly, Asp) each in an amount of 500 μg and B16-BL6 melanoma cells (5 × 10 ^{4 in} the logarithmic growth phase shown in (1) above) as highly metastatic cancer cells were mixed in PBS. , 0.2 ml of which is C57 of 5 animals per group
BL / 6 male mice were injected intravenously. 14 days after the administration, the number of cancer colonies in the lungs of the mice was counted and compared with the control PBS administration group. The results are shown in Experiment 1 in Table 2-1. According to this result, Poly (Arg-Gly-Asp)
By the administration of the drug, metastasis of lung cancer was significantly suppressed. In contrast, Arg-Gly-Asp and Poly (A
Administration of rg, Gly, Asp) did not inhibit such metastasis. Furthermore, as shown in the table, A
In the case of rg-Gly-Asp, Poly (Arg-Gl
In order to obtain a remarkable metastasis-suppressing effect equivalent to that of y-Asp),
A dose of 3000 μg was required, which corresponds to about 6 times.

B) Next, two kinds of Poly (Arg-Gly-Asp) -1500 having a repeating sequence of Arg-Gly-Asp and having a molecular weight of 1500 and 5000.
And Poly (Arg-Gly-Asp) -5000
The cancer metastasis suppressive activity of was investigated. Each compound 100
When 0 μg was administered to mice in the same manner as in Experiment 1, the effect was examined. According to the results shown in Experiment 2 in Table 2-1, both compounds showed a remarkable metastasis-suppressing effect as compared with the control PBS-administered group.

In addition, Poly (Arg-Gly-As
p) -5000 without mixing with B16-BL6 cells
Even when the mice were intravenously administered 5 minutes after the administration of 16-BL6 cells, a high metastasis-suppressing effect was still obtained. This result indicates that the compound of the present invention can be administered by an appropriate method such as intravenous injection to obtain the effect of suppressing cancer metastasis.

[0034]

[Table 2] c) Next, the core sequence Tyr-I for cell adhesion of laminin
having a repeating structure of le-Gly-Ser-Arg,
Polypeptide compound Poly (T
With respect to yr-Ile-Gly-Ser-Arg) (synthesized in Synthesis Example 4 described later), the cancer metastasis inhibitory action was also examined. That is, 5, 20 or 100 μg of the above compound,
5 × 10 ⁴ B16-BL6 cells or 3 × 1 respectively
Mouse C57BL / 6 in the same manner as described in b) above after mixing with 0. ⁴ Lewis lung cancer cells (3LL) cells.
Was administered to examine the cancer metastasis inhibitory effect. The result is the second
Table 2 shows. As is clear from the table, Poly (T
Administration of yr-Ile-Gly-Ser-Arg) significantly suppressed lung cancer metastasis. In contrast, the pentapeptide Tyr-Ile-Gly-Ser-Arg
Almost 100 μg of the compound showed no metastasis-suppressing effect, and although it is not shown in the table, 200 μg or more was required to obtain a significant effect. from this result,
It was confirmed that the polypeptide of the present invention having a pentapeptide repeating sequence has a cancer metastasis suppressing activity that is about 10 times that of the original pentapeptide.

[0035]

[Table 3] (3) Suppressive Effect of Cancer Cell Retention in Lung By the same experiment as in (2) above, it was confirmed that the compound of the present invention suppresses retention of cancer cell in lung. B16-BL6 cells in log phase were labeled with [ ¹²⁵ I] -IUdR,
This labeled cell (2 × 10 ⁴ / mouse) and Po of molecular weight 5000
A 0.2 ml solution containing ly (Arg-Gly-Asp) (500 μg / mouse) was administered through the tail vein of C57BL / 6 mice. Liver, kidney, spleen, lung and blood were collected from each mouse 30 minutes and 24 hours after administration, and the radioactivity was measured. PBS was used as a control. The results are shown in Table 3. No significant difference was observed between the control group and Poly (Arg-Gly-Asp) in the other organs examined, including blood, but in the lung, Pol
It was confirmed that the y (Arg-Gly-Asp) -administered group had significantly lower radioactivity, and the retention of cancer cells in the lung was significantly suppressed.

[0036]

[Table 4] (4) Effect of Suppressing Cancer Metastasis by Spontaneous Metastasis Model Furthermore, it was confirmed by the natural metastasis model that the compound of the present invention suppresses cancer metastasis. That is, B16-BL6
The cells were transplanted into the foot pads of 5 C57BL / mouse per group,
Within a certain period of time after transplantation, Poly (Ar
100 μg or 50 μg of g-Gly-Asp) was directly administered to the transplanted cancer site once or multiple times. The transplanted cancer was excised on the 21st day, and two weeks later, the mouse was dissected to examine the metastasis of the cancer to the lung. Table 4 shows the results. Growth of transplanted cancer itself was not suppressed by single administration of 100 μg of compound on day 1 or 7 after cancer transplantation or multiple administration of 50 μg on days 7, 10, 13, and 16 However, lung cancer metastasis was significantly suppressed.

On the other hand, 100 μg of Poly (Arg, Gly, Asp) having a disordered amino acid sequence was used.
Even when administered on the day, the activity of suppressing the metastasis of lung cancer was not observed.

[0038]

[Table 5] (5) Effect of suppressing platelet aggregation by cancer cells Furthermore, Po for platelet aggregation induced by cancer cells
The influence of ly (Arg-Gly-Asp) was examined. C
Blood was collected from 57BL / 6 mice and 160 x
Platelet-rich fractionation (PRP) by centrifuging at 15g for 15 minutes
I got As a control, a platelet micro fraction (PPP) obtained by centrifuging blood at 1000 × g for 10 minutes was used. Poly (Arg-Gly-Asp) having a molecular weight of 5000 was added to the PRP or PPP fraction (5 × 10 ⁵ / μl) 250 described above.
The solution pre-incubated for 5 to 7 minutes with μl was added to B16-BL6 cells (about 10 ⁶ / ml) suspended in physiological saline, and the mixture was mixed at 37 ° C. at 1000 rpm while being mixed with dual cells.
Aggregometer (DualAggregometer)
Aggregation was observed with model 440 (Chrono-Log, USA). As a comparative control, Poly (Arg-Gl
Poly (Arg, Gly, A instead of y-Asp)
sp) was used. The results are shown in FIGS. 1 to 3. Each figure shows the case where heparinized PRP was treated with PBS 7 minutes before the time point (arrow in the figure) at which B16-BL6 melanoma cells were added ..... Pol. 1 Pol.
When treated with y (Arg, Gly, Asp) 100 μg / ml ... Fig. 2 Pol
y (Arg-Gly-Asp) When treated with 100 μg / ml ... Represents aggregation in FIG. Compound of the present invention Poly (Arg-Gly-As
p) completely suppressed the aggregation of platelets (Fig. 3), while Poly (Arg, Gly, Asp) as a comparative control showed almost no inhibitory effect (Fig. 2).

(6) Toxicity In the tests (1) to (5) above, Poly (Arg-Gly-Asp) having a molecular weight of 5000 or 1500 and Poly (Tyr-Ile-Gly) having a molecular weight of 10,000 are used.
-Ser-Arg) was confirmed not to have cytotoxicity to erythroid cells of the host mouse, spleen and thymocytes, or to have an undesired aggregation effect on serum proteins.

[0040]

EXAMPLES Synthesis Examples Next, production examples of the compounds of the present invention will be described. Amino acid derivatives were purchased from Peptide Institute. Peptide synthesis was carried out by a liquid phase method, and the purity was estimated and the target product was identified by thin layer chromatography (developing solvent: (A) n-butanol: acetic acid: water = 4: 1: 5 upper layer, (B)). n-butanol: pyridine: acetic acid: water = 15: 10: 3: 12
(C) Ammonia water saturated n-butanol, etc.), elemental analysis, and infrared absorption spectrum. Regular and random sequence polypeptide synthesis can be performed using DPPA (Diphe
Nylphophoryl azide) was used to protect the side chain functional groups (Mts and Bz).
The 1-group) was removed with methanesulfonic acid-anisole or trifluoromethanesulfonic acid-thioanisole, and finally the guanide group of the arginine side chain was converted into a hydrochloride with an ion exchange resin (Amberlite IRA-400). The removal of the protecting group was confirmed by infrared absorption spectrum, and the rough molecular weight was estimated by polyacrylamide gel electrophoresis (gel concentration 13%) in the presence of 0.1% SDS (sodium dodecyl sulfate).

Synthesis Example 1 (Polypeptide having a molecular weight of about 5000) (1) t-butoxycarbonylglycyl-β-benzyl-
L-aspartic acid (Boc-Gly-Asp (OBz
l) -OH (I) t-butoxycarbonylglycine (5.3 g, 30 mmo
l) was dissolved in purified THF (100 ml) and triethylamine (4.6 ml, 33 mmol) and isobutyl chloroformate (4.33 ml, 33 mmol) were added at -15 ° C,
Stir at that temperature for 10 minutes. To this mixed acid anhydride solution, β-benzyl-L-aspartic acid (8.0 g, 3
6 mmol) and triethylamine (5.02 ml, 36 mm)
ol) was dissolved in water and cooled to 0 ° C, and the solution was added.
Stir for 15 hours and at room temperature for 15 hours. T by vacuum concentration
HF is distilled off and cold 10% citric acid is added to form a precipitate. The precipitate was extracted twice with ethyl acetate, the extracts were combined, and the ethyl acetate extract was mixed with 1/5 volume of 5%.
Wash 5 times with citric acid and 10 times with 1/10 volume of water. The solution is dried over anhydrous sodium sulfate and concentrated to dryness. The operation of dissolving in ether and precipitating with n-hexane is performed three times and dried. This gives the title compound 6.9.
g was obtained.

(2) Glycyl-β-benzyl-L-aspara
Latate hydrochloride (HCl · H-Gly-Asp (OBz
l) -OH) (II) The compound (I) (3.2 g) obtained in (1 ) above was dissolved in 80 ml of purified dioxane, and 4N HCl / dioxane 8 was added.
Add 0 ml and stir at room temperature for 1 hour. After concentrating to dryness,
Dry ether is added for crystallization, and the product is collected by centrifugation, washed with dry ether several times and dried. This resulted in 2.4 g of the title compound.

(3) N ^α -t-butoxycarbonyl-N ^ω
-Mesitylenesulfonyl-L-arginylglycyl-β
-Benzyl-L-asparatate (Boc-Arg (M
ts) -Gly-Asp (OBzl) -OH) (III) N α -t- butoxycarbonyl -N ^omega - dissolved in mesitylenesulfonyl -L- arginine (3.4 g, 7.4 mmol) and purified THF (70 ml) At −15 ° C., triethylamine (1.13 ml, 8.1 mmol) and isobutyl chloroformate (1.07 ml, 8.1 mmol) are added and stirred at that temperature for 10 minutes (mixed anhydride formation). the above
The compound (II) of (2) (2.4 g, about 8.0 mmol) and triethylamine (2.68 ml, 19.2 mmol) were added to TH.
It is dissolved in a mixed solvent of F (70 ml), DMF (50 ml) and water (10 ml) and cooled to 5 ° C. This solvent is added to the mixed acid anhydride solution, and the mixture is stirred at 5 ° C. for 1 hour and at room temperature for 15 hours. THF and water are distilled off by concentration under reduced pressure, and the residue DM
5% citric acid is added to the F solution and the resulting precipitate is separated by decantation. Wash thoroughly with 5% citric acid and water and dry. After washing twice with ether, the residue is reprecipitated with methanol-ether and separated by decantation. When treated with ether, it becomes a powder, which is then centrifuged, washed with ether and dried. This resulted in 3.9 g of the title compound.

(4) N ^ω -mesitylenesulfonyl-L-a
Luginylglycyl-β-benzyl-L-aspartate
Hydrochloride (HCl · H-Arg (Mts) -Gly-As
p (OBzl) -OH) (IV) Compound (III) (1.5 g, 2.1 mmol) is dissolved in purified dioxane (30 ml), 4N HCl / dioxane (30 ml) is added, and the mixture is stirred for 1 hour. Concentrate to dryness, crystallize by adding dry ether, centrifuge, wash several times with dry ether and dry. This resulted in 1.3 g of the title compound.

(5) L-arginylglycyl asparagine
Acid / hydrochloride (2HCl · H-Arg-Gly-Asp-
OH) (V) Compound (III) (300 mg) was added to methanesulfonic acid (4 m
l) -anisole (1 ml) mixture, dissolved at room temperature.
Stir for 5 hours. Dry ether is added, the precipitate is separated by decantation, washed well with dry ether and dried. Dissolve this in a small amount of water and use amber light (A
mberlite) IRA-400 (Cl type),
Fractions containing the desired compound are concentrated to dryness and the residue is treated with methanol-ether to crystallize. Centrifuge, wash with ether and dry. This resulted in 117 mg of the title compound.

[0046] (6) poly (N ^omega - mesitylenesulfonyl -
L-arginylglycyl-β-benzyl-L-aspara
Tate) (Poly (Arg (Mts) -Gly-As
p (OBzl)) (VI) Compound (IV) (400 mg, 0.58 mmol) purified DMS
It was dissolved in O (1.2 ml) and DPPA (0.19 ml, 0.
87 mmol) and triethylamine (0.285 ml,
2.03 mmol) is added and stirred at 5-8 ° C. for 1 hour and at room temperature for 2 days. The same amounts of DPPA and triethylamine are added and the polymerization reaction is allowed to proceed for another 2 days. The polymer is precipitated with water, centrifuged, washed well with water and methanol and dried. This gave 295 mg of the title compound.
Obtained.

(7) Poly (L-arginylglycyl-L-
Asparagine hydrochloride) (Poly (Arg-Gly-
Asp) HCl) (VII) Compound (VI) (100 mg) was added to methanesulfonic acid (2 ml)
-Treat with anisole (0.4 ml) mixture to remove side chain protecting groups and convert to hydrochloride with ion exchange resin. The operation method is the same as the method for synthesizing compound (V). 60 mg of the title compound was obtained. The molecular weight of this compound was about 5000.

[0048] Synthesis Example 2 (Comparative Compound of random sequence) (1) copoly (N ^omega --L-mesitylenesulfonyl alginate
Nin, glycine, β-benzyl-L-aspartate)
(Copoly (Arg (Mts), Gly, Asp
(OBzl)) (VIII) N ω - mesitylenesulfonyl -L- arginine (356
mg, 1.0 mmol), glycine (75 mg, 1.0 mmol) and β-benzyl-L-aspartate (223 mg,
1.0 mmol) in purified DMSO (2.0 ml),
Polymerize with DPPA (0.97 ml, 4.5 mmol) and triethylamine (1.05 ml, 7.5 mmol). The operating method is the same as in the synthesis of compound (VI).

(2) Copoly (L-arginine hydrochloride, green
Syn, L-aspartic acid) (Copoly (Arg,
Gly, Asp) HCl) (IX) Compound (VIII) (100 mg) was added to methanesulfonic acid (2 m
l) -Anisole (0.4 ml) was treated to remove side chain protecting groups and converted to the hydrochloride salt with an ion exchange resin. The operation method is the same as the method for synthesizing compound (V). This resulted in 50 mg of the title compound. The molecular weight of this compound was about 5000.

Synthesis Example 3 (Polypeptide having a molecular weight of about 1500) Poly (L-arginyl-glycyl-L-asparagine
Acid) hydrochloride (Oligo (Arg-Gly-Asp).
HCI) (X) The polymerization reaction of the compound (VI) synthesis was stopped for 90 minutes to obtain a side chain-protected oligopeptide, and then the target compound was obtained by the same operation as the compound (VII) synthesis. The average molecular weight of this compound was about 1500.

Synthesis Example 4 (Polypeptide having a molecular weight of about 10,000) (1) N ^ω -mesitylenesulfonyl-L-arginine-O
-Methyl ester hydrochloride (HCl-Arg (Mts)-
OMe) (XI) N ^ω -t- butoxycarbonyl -N ^omega - mesitylenesulfonyl -L- arginine (Boc-Arg (Mts) -
2.10 g of OH) is dissolved in dry dioxane (31 ml), 4N HCl-dioxane (31 ml) is added, and the mixture is stirred at room temperature for 1 hour. After this solution was concentrated under reduced pressure to dryness, dry ether was added to crystallize, the desired product was collected by centrifugation and washed with dry ether several more times.
Dry in dessicator. As a result, the HCl.H-Arg (Mts) -OH with the Boc group removed was 1.1
g was obtained. SOCl ₂ (2.9 ml) was gradually added to MeOH (10.6 ml), and the mixture was adjusted to 1 at -10 ° C.
The mixture is stirred for 0 minutes, added to the adjusted solution, stirred for 15 hours, concentrated to dryness, crystallized with dry ether, centrifuged, washed several times with dry ether and dried. As a result, HCl.H-Arg (Mts) -OMe was 0.94.
g was obtained.

(2) N ^ω -t-butoxycarbonyl-β-
Benzyl -L- seryl -N ^omega - mesitylenesulfonyl -
L-arginine methyl ester (Boc-Ser (Bz
l) -Arg (Mts) -OMe) (XII) Boc-Ser (Bzl) -OH (0.69 g, 2.3)
mmol) and HCl-Arg (Mts) -OMe (0.
94 g, 2.3 mmol) was dissolved in rectified DMF (16.3 ml) -dioxane (16.3 ml) and stirred at 0 ° C. for 10 minutes, after which DPPA (0.76 ml, 2.76 mmol) and TEA (0.7. 74 ml, 7.36 mmol) was added, and 2 at room temperature.
Stir for 4 hours. This solution is concentrated under reduced pressure to distill off dioxane, and a precipitate formed by adding brine to the residue is separated by decantation, washed with water, and the residue is extracted with ethyl acetate. The extract was washed well with 5% citric acid, water and sodium bicarbonate, dehydrated with Glauber's salt (Na ₂ SO ₄ ),
Concentrate to dryness and dry.

(3) Boc.Gly-Ser (Bzl)-
Arg (Mts) -OMe (XIII) product (XII) (1.03 g) was added to dry dioxane (2
It was dissolved in 5.3 ml) and 4N-HCl / dioxane (2
5.3 ml) is added and the mixture is stirred at room temperature for 1 hour. The solution is concentrated to dryness, crystallized by adding dry ether, and after centrifugation, washed several times with dry ether and dried. As a result, HCl.H-Ser (Bzl) -Arg (Mt
0.68 g (1.2 mmol) of s) -OMe was obtained. This is Boc-Gly-OH (0.26g, 1.5mmol)
Together with rectified DMF (9.4 ml) / dioxane (9.
4 ml) and stirred at 0 ° C. for 10 minutes, then DPPA
(0.5 ml, 1.8 mmol) and triethylamine (0.49 ml, 4.8 mmol) are added, and the mixture is stirred at room temperature for 24 hours. The obtained solution was concentrated under reduced pressure in the same manner as in the case of the compound (XII), added with a saline solution, washed with the formed precipitate and dried to remove an acidic alkaline substance. Arg (M
0.48 g of ts) -OMe was obtained.

(4) Boc-Ile-Gly-Ser (B
zl) -Arg (Mts) -OMe (XIV) product (XIII) in the same manner as compound (XI)
The group is removed and HCl.H-Gly-Ser (Bzl)
-Arg (Mts) -OMe 0.40g (0.63mm
ol) got. This was added to equimolar Boc-Ile-OH (0.
14 g) and dissolved in rectified DMF (5.4 ml) / dioxane (5.4 ml) and stirred at 0 ° C. for 10 minutes, then DP
PA (0.21 ml) and triethylamine (0.20
ml) and stirred at room temperature for 24 hours. The obtained solution is dried in the same manner as in the compounds (XII) and (XIII) to remove acidic and alkaline substances. Thus, B
oc-Ile-Gly-Ser (Bzl) -Arg (M
0.28 g of ts) -OMe was obtained.

(5) Boc-Tyr (Bzl) -Ile-
Gly-Ser (Bzl) -Arg (Mts) -OMe
In the (XV) product (XIV), the Boc group was removed by the same method as in (1). However, in consideration of the elongation of the peptide chain and the steric hindrance of the Ile side chain, the amount of the solvent and the reaction time are set to the usual 2
It increased three times. That is, 0.28 g of the product (XIV),
Dry dioxane 6.0 ml, 4N HCl / dioxane 6.0 ml, and stirring for 2 hours were used. The obtained peptide (HCl.H-Ile-Gly-Ser (Bzl)-
Arg (Mts) -OMe: Yield 0.14 g, 0.19
mmol) is 0.14 g of Boc-Tyr (Bzl) -OH.
(0.19 mmol) together with rectified DMF (2.8 ml) / dioxane (2.8 ml) and stirred at 0 ° C. for 10 minutes, DPPA (0.12 ml) and triethylamine (0.72 ml) were added, Stir at room temperature for 24 hours. The obtained solution is washed and dried in the same manner as for the compounds (XII) and (XIII). Thereby, Boc-Tyr (Bz
l) -Ile-Gly-Ser (Bzl) -Arg (M
0.12 g of ts) -OMe was obtained.

(6) 0.14 g (0.13 mm) of the product (XV)
ol) was dissolved in dry dioxane (1.95 ml) and 1N
Add NaOH (0.65 ml) and stir at room temperature for 3 hours. After removing dioxane by concentration under reduced pressure, about 20 ml
Water is added and some insoluble matter is removed by filtration. The filtrate is cooled and 10% citric acid (0 ° C) is added to form a precipitate.
The precipitate is filtered, washed twice with a small amount of water and dried to give a Bo
c-Tyr (Bzl) -Ile-Gly-Ser (Bz
0.10 g of 1) -Arg (Mts) -OH was obtained.
Further, this was dried with dioxane (6.0 ml), 4 NH
Cl / dioxane (6.0 ml), B under stirring for 3 hours
The oc group was removed to give HCl.H-Tyr (Bzl)
-Ile-Gly-Ser (Bzl) -Arg (Mt
0.08 g of s) -OH was obtained.

(7) Poly (Tyr-Ile-Gly-
Ser-Arg) HCl (XVII ) HCl.H-Tyr (Bzl) -Ile-Gly-Se
r (Bzl) -Arg (Mts) -OH 60 mg (0.0
61 mmol) was dissolved in rectified DMSO (0.2 ml) to give DP
PA (0.07 ml) and triethylamine (0.06
ml), and the mixture is stirred at 0 ° C. for 1 hour and further at room temperature for 48 hours. After 48 hours the same amounts of DPPA and triethylamine are added again and stirred for 1 hour at 0 ° C. and 48 hours at room temperature. Water was added to the obtained solution, and the mixture was centrifuged several times to obtain D
Remove PPA, etc. This is further added with a small amount of MeOH, centrifuged four times, and dissolved in MeOH to remove low molecular weight peptides. Centrifuge once again with ether,
dry. For removal of the side chain protecting group, Poly (Tyr
(Bzl) -Ile-Gly-Ser (Bzl) -Ar
40 mg of g (Mts)) was added to a scavenger thioanisole (1 ml) together with a mixed solvent of trifluoroacetic acid (2 ml) and trifluoromethanesulfonic acid (0.4 ml), and the mixture was stirred at 0 ° C. for 2 hours. The solution is concentrated to dryness, a precipitate is formed by adding dry ether, and the precipitate is washed four times with ether and dried. Furthermore, in order to convert this into a hydrochloride, it is dissolved in a small amount of water, passed through an anion exchange resin column (Amberlite IRA-400: Cl type), and the fractions containing the target substance in the obtained fractions are concentrated to dryness. Solidify, precipitate the residue with a small amount of MeOH, wash several times with ether, then dry, Poly (Tyr-Il).
22 mg of e-Gly-Ser-Arg) HCl was obtained. The molecular weight of this polypeptide was about 10,000.

[0058]

INDUSTRIAL APPLICABILITY As described above, the novel polypeptide compound having a repeating structure of the present invention has greater cell adhesiveness than the core sequence of cell adhesive protein and has various biological activities such as cancer metastasis inhibitory action. And has almost no toxicity problem. In addition, since its structure is relatively simple, it is easy to synthesize and is highly valuable as a medicine.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of PBS on cancer-induced platelet aggregation.

FIG. 2 is a graph showing the effect of Poly (Arg, Gly, Asp) on cancer-induced platelet aggregation.

FIG. 3 is a graph showing the effect of Poly (Arg-Gly-Asp) on cancer-induced platelet aggregation.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Seiichi Tokura 4-1-1-13 Hachiken 5 Nishi Nishi, Nishi-ku, Sapporo-shi, Hokkaido

Claims (3)

[Claims] 1. The polypeptide according to claim 1, which is represented by the formula: (Tyr-Ile-Gly-Ser-Arg) n (wherein n represents a number of 2 to 20) and its pharmaceutically. Acceptable salt. 2. The polypeptide according to claim 1, which has a molecular weight of about 10,000. 3. The polypeptide according to claim 1, which is represented by the formula: (Tyr-Ile-Gly-Ser-Arg) n (wherein n represents a number of 2 to 20) or a pharmaceutically acceptable form thereof. A cancer metastasis inhibitor comprising an acceptable salt as an active ingredient.