JPH0625290A - Cell adhesion active peptide - Google Patents

Abstract

PURPOSE:To obtain a new cell adhesion active peptide, composed of a cyclic peptide having a specific amino acid sequence, having biological activity such as suppressing actions on cancer metastasis and blood platelet agglutination, etc., and useful as a cancer metastatic suppressor, a blood platelet agglutination suppressor, etc. CONSTITUTION:This cell adhesion active peptide expressed by formula IV [(n) is 2-10] is obtained by dissolving a protected tetrapeptide expressed by formula I (Boc is benzyloxycarbonyl; cHex is cyclohexyl; Bzl is benzyl; Tos is p-toluenesulfonyl; Pac is phenacyl) in acetonitrile, adding methanesulfonic acid thereto under cooling the resultant solution with ice, stirring the prepared solution at ambient temperature, further adding DMF and triethylamine thereto under cooling the mixture solution, subsequently adding a C-terminal-free protected tetrapeptide of formula II together with a condensing agent, carrying out the reaction, providing a protected oligopeptide of formula III, then removing the protecting groups at both terminals of the resultant compound, activating the obtained compound with N-hydroxysuccinimide, subsequently adding a condensing agent thereto, carrying out the cyclizing reaction and further removing the whole protecting groups.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide having cell adhesion activity. Specifically, it relates to a novel cyclic peptide having a repeating structure of a peptide or a pharmaceutically acceptable salt thereof. Further, the present invention relates to a cancer metastasis inhibitor or a platelet aggregation inhibitor containing these cyclic peptides or a pharmaceutically acceptable salt thereof as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, proteins which are involved in the adhesion between cells and interstitial connective tissue and have various physiological activities related to the cell function of animal cells are called cell adhesion activating proteins. Known proteins include, for example, fibronectin, laminin, vitronectin and the like. Regarding these cell adhesive activity proteins, studies have been conducted on the binding sites involved in cell adhesive activity. For example, a fragment obtained by limited degradation of fibronectin with a proteolytic enzyme and binding of heparin, collagen, cells and bacteria Studies have determined the binding sites for both the A and B chains (Yamada, KM: Ann. Rev. B).
iochem. , 52 , 761 (1983)). Furthermore, the core sequence of the cell binding site is arginine-glycine-aspartic acid [Arg-Gly-Asp (hereinafter,
RGD sequence))] 1984
Clarified in the year (Pierschbachr, MD.
Et al., Nature, 309 , 30 (1984)). It has been revealed that this RGD sequence is also present in the sequences of other cell adhesion activating proteins such as vitronectin (Suzuki, S. et al .: J. Biol. Che.
m. , 259 , 15307 (1984)).

[0003] Fibronectin bonds with the receptor of adherent cells via the above RGD sequence and transmits the information to the adherent cells, and is involved in adhesion between cells and interstitial connective tissue, differentiation and proliferation of cells. (Ya
mada, K .; M. : Ann. Rev. Bioche
m. , 52 , 961 (1983)). As described above, the cell adhesion-activating 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 is decreased, the function of the reticuloendothelial system is decreased, and, for example, sepsis due to surgery or trauma, disseminated intravascular blood coagulation, burns, serious infection, surgical shock, etc. occur. Administration of fibronectin is considered to be effective for the improvement of those symptoms. Moreover, since fibronectin stimulates the migration ability of fibroblasts and macrophages, its application to the healing of wounds and the regulation of immune function is considered. In particular, local treatment of corneal disorders using the wound healing promoting effect has already been attempted (Fujik).
awa, L .; S. Et al: Lab. Invest. , 45 ,
120 (1981)). Furthermore, the cell adhesion active protein is
It is also attracting 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, when cell adhesion activating proteins such as fibronectin and laminin are present, the cells form a multicellular mass. , Is known to facilitate the growth and survival of cancer cells.

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. Cli).
n. Invest. , 74 , 843 (1984)). Similarly, the tripeptide Arg-Gly-Asp (Humphiries, M .;
J. Et al .: Science, 233 , 467 (198).
6)) and pentapeptide Ty, which is the adhesive core of laminin
r-Ile-Gly-Ser-Arg (Iwamot
o, Y. Et al .: Science, 238 , 1132 (19
It has been confirmed that 87)) also suppresses cancer metastasis.
Furthermore, the polymer peptide consisting of the repeating structure of these adhesive cores exhibits a stronger platelet aggregation inhibitory activity (activity to inhibit the platelet aggregation by cancer cells occurring in capillaries) and cancer metastasis inhibitory activity than its monomer peptide. Is known (Japanese Patent Laid-Open No. 2-174798).
Issue).

However, one of the problems in administering the above peptides into a living body is that the in vivo clearance is very fast. As one of useful means for solving such a problem, a peptide having a cysteine residue in the peptide chain of a peptide having cell adhesion activity (hereinafter, abbreviated as cell adhesion activity peptide) is synthesized to produce disulfide. A compound cyclized by a bond is known (MD Pierschbadhen
J. J. et al. Biol. Chem. , 262 , 17294-
17298 (1987). ). In addition, a cyclic cell-adhesive active peptide has already been known (Japanese Patent Laid-Open No. 2-1.
No. 74797).

[0006]

As described above, cell adhesion activity proteins such as fibronectin and laminin have various biological activities, and it is desired to develop a technique for applying related substances thereof as a medicine. It was In particular, the cancer metastasis suppressive action of adhesive core sequences such as fibronectin and laminin is
Although it is considered to have high application value as a medicine, since the cell adhesion activity of the fragment of the core sequence is not sufficient, it is not sufficiently satisfactory to apply them as a cancer metastasis inhibitor and a platelet aggregation inhibitor. There wasn't. In this respect, it has been desired to develop a substance having higher activity.

Therefore, the object of the present invention is to provide a cell adhesion active peptide having a structure with improved stability in vivo, and to have a biological activity effective as a cancer metastasis inhibitor or a platelet aggregation inhibitor. To provide a peptide.

[0008]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that the peptide represented by the following formula (1) and a pharmaceutically acceptable salt thereof. Have been found to have cell adhesion activity and have completed the present invention. That is, the present invention uses the formula (1)

[0009]

[Chemical 4]

(Wherein n represents an integer of 2 or more and 10 or less) or a pharmaceutically acceptable salt thereof, or a peptide or a pharmaceutically acceptable salt thereof as an active ingredient. And a peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

The peptide of the present invention has the structure represented by the above formula (1), is involved in the adhesion between cells and stromal connective tissue, and has various physiological activities related to the cell function of animal cells, such as platelets. It is a peptide having physiological activities such as aggregation inhibitory activity and cancer metastasis inhibitory activity. In the peptide of the present invention, the positive integer represented by n in the above formula (1) is particularly preferably 2 or more and 5 or less from the viewpoint of having a stable conformation. The peptides of the present invention include both those consisting of L-type amino acids and those consisting of D-type amino acids.
-Type amino acids are preferred.

In the present specification, amino acids, protecting groups,
The active groups, solvents and the like may be indicated by abbreviations based on IUPAC-IUB and abbreviations commonly used in this field, and examples thereof are as follows. Abbreviations for amino acid residues or amino acid derivatives are as follows. Symbol Name (Structure) Asp aspartic acid Gly Glycine Ile Isoleucine Leu Leucine Pro Proline Arg Arginine Ser Serine Tyr Tyrosine Asn asparagine Thr threonine His Histidine Phe Phenylalanine Val Valine Lys Lysine Gln Glutamine Met Methionine Trp tryptophan Glu glutamic acid Ala Alanine Asx aspartic acid or Asparagine

Abbreviations for protecting groups are as follows: Abbreviation Name Boc: benzyloxycarbonyl cHex: cyclohexyl Bzl: benzyl Tos: p-toluenesulfonyl Pac: phenacyl OSu: N-hydroxysuccinimide ester

The peptide of the present invention can be synthesized by a method in which the primary sequence of the intermediate is clear at each stage of the synthesis, and by constructing the primary sequence of the desired product. As such a method, a synthetic method using a chemical synthetic method and a genetic engineering method is known, and the peptide of the present invention can be synthesized by any method. As the chemical synthesis method, a liquid phase method and a solid phase method are known, but any method can be used for the synthesis. In addition, a stepwise extension method in which the desired primary sequence is sequentially constructed from the N-terminus or the C-terminus, and a fragment condensation method in which the desired primary sequence is divided into appropriate fragments and the fragments are condensed to construct the desired product. Are known, but they can be synthesized by any method or a combination thereof. At this time, the functional groups (amino group, carboxyl group, guanidino group, hydroxyl group) in the fragment can be protected if necessary.

Regarding the condensation method, activation method, protecting group, reaction conditions and the like in the above peptide synthesis, "Basics and Experiments of Peptide Synthesis" (Maruzen Co., Ltd., 1985),
The methods, protecting groups, reaction conditions and the like used in ordinary peptide synthesis described in “Biochemistry Experiment Course, Volume 1, Protein Chemistry IV” (Tokyo Kagaku Dojin, 1976) can be used. The peptide of the present invention synthesized as described above can be further purified, if necessary, according to a conventional peptide purification method such as reverse phase HPLC, ion exchange chromatography, gel filtration chromatography and the like. The purity of the peptide of the present invention thus obtained is preferably 90% or more by area percent by HPLC, and more preferably 95% or more.

The peptide represented by the formula (1) synthesized as described above contains 4 residues (Arg-Gly) containing the RGD sequence part which is the core sequence part of the cell binding site of the cell adhesion activity protein. -Asp-Thr) is a cyclic peptide having a repeating structure and retains cell adhesion activity. It is considered that the peptide adheres to cells via the core sequence portion by a mechanism similar to that of the cell adhesion-activating protein. Therefore, various biological activities as agonists or antagonists of the cell adhesion activity protein, for example, biological activity such as the inhibitory action of platelet aggregation is shown, and in particular, the peptide of the present invention has a biological activity as compared with a simple core sequence of the cell adhesion activity protein. It has a cancer metastasis inhibitory action several times stronger.

Furthermore, the peptide of the present invention has a significantly delayed in vivo clearance (that is, prolonged persistence) as compared with the mere core sequence of the cell adhesion activity protein, and exhibits its physiological activity effectively for a long time. Therefore, the peptide of the present invention is extremely effective as a drug or veterinary drug. Further, the peptide of the present invention, for use as a pharmaceutical, a pharmaceutically acceptable salt, for example, hydrochloride, a salt with an inorganic acid such as sulfate,
It may be a salt with an organic acid such as an acetate salt, a trifluoroacetate salt, a lactate salt or a tartrate salt, and conversion to such a salt can be carried out by a conventional means.

Further, the peptide of the present invention and a pharmaceutically acceptable salt thereof are usually prepared from an appropriate pharmaceutical composition using a carrier, a diluent and the like which are known per se (eg, a pharmaceutical composition).
It is orally or parenterally administered to mammals (eg, cows, horses, pigs, sheep, humans) as capsules, injections, etc.). Specifically, when administered to humans as a cancer metastasis inhibitor or a platelet aggregation inhibitor, the cell adhesion-active peptide of the present invention is 0.2 μg / kg to 400 mg / kg.
The dose is determined based on symptoms, age, body weight, etc. so as to be in the range (dose / body weight), and the dose can be administered once to several times a day.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In addition, in the following examples, the amino acid analysis value of peptides means an acid decomposition product of these peptides (6N hydrochloric acid-
Amino acid analysis value in phenol, decomposition product after treatment at 110 ° C. for 24 hours).

Example 1

[0021]

[Chemical 5]

Preparation of a) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gly) ₂ -OPac Boc-Asp (OcHex) Thr (Bzl) Arg (Tos) Gly-OPac 8.46 g
(8.5 mmol) was dissolved in 40 ml of acetonitrile,
6.56 g (0.068mo) of methanesulfonic acid under ice cooling
l) was added dropwise, and the mixture was stirred at room temperature for 1 hour. The mixture was ice-cooled again, DMF (35 ml) was added dropwise, and further triethylamine (6.04 g, 0.060 mol) was added dropwise. Subsequently, Boc-Asp (OcHex) Thr (Bzl) Arg (Tos) Gly-OH 7.83
g (9.0 mmol), 1-hydroxybenzotriazole (hereinafter abbreviated as HOBt) 1.39 g (0.103 m)
ol), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (hereinafter abbreviated as WSC) 1.60 g
(0.103 mol) was added under ice cooling, and the mixture was stirred for 30 minutes and then at room temperature for 3 hours. The reaction solution was 1% saline solution 6
The mixture was poured into 00 ml and stirred under ice cooling for 30 minutes, then the precipitate was filtered, washed with water, washed with ether and dried to give the title compound 1.
4.4 g was obtained.

B) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gl
y) Preparation of _2- OH 14.36 g of the compound prepared according to Example 1 (a)
(8.2 mmol) was dissolved in 250 ml of 90% acetic acid, and 10.7 g (0.164 mol) of zinc powder was added every 3 minutes under ice cooling, and the mixture was returned to room temperature and stirred vigorously.
After 1.5 hours, the reaction solution was filtered through Celite, the filtrate was poured into 600 ml of 1% saline, and the mixture was stirred under ice cooling, and the precipitate was collected by filtration, washed with water, washed with ether and dried to give the title compound. 12.0 g was obtained.

C) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gl
y) Preparation of _2- OSu 6.89 g of the compound prepared according to Example 1 (b)
(4.2 mmol) of methylene chloride 70 ml and DMF10
Dissolve in a mixed solution of ml, and under ice cooling, 0.73 g (6.3 mmol) of N-hydroxysuccinimide, and then 0.5M
Dicyclohexylcarbodiimide methylene chloride solution (hereinafter abbreviated as DCC-CH ₂ Cl ₂ ) 12.7 ml
(6.3 mmol) was added, and the mixture was stirred at room temperature for 23 hours.
The insoluble material was filtered off, and the filtrate was evaporated under reduced pressure. A little D
After dissolving in MF, the mixture was poured into 500 ml of 1% saline and stirred under ice cooling. The precipitate is collected by filtration, washed with water and then with ether,
After drying, 10.1 g of the title compound was obtained.

D)

[0026]

[Chemical 6]

Preparation of 9.86 g of the compound prepared according to Example 1 (c)
(5.7 mmol) was dissolved in 50 ml of trifluoroacetic acid, and the mixture was stirred for 20 minutes under ice cooling. After the solvent was distilled off, 300 ml of ether was added, and the mixture was stirred again under ice cooling for 30 minutes. The precipitate was collected by filtration, washed with ether and dried to obtain 7.72 g of Boc-free body. 7.72 g of this de-Boc body was added to DMF.
It was dissolved in 50 ml and slowly dropped into pyridine heated to 60 to 65 ° C over about 1 hour. 30 at 60-65 ° C
After stirring for a minute, the mixture was allowed to cool and the solvent was distilled off under reduced pressure. The residue was recrystallized from acetone-ether to give the title compound 5.9.
6 g was obtained.

E)

[0029]

[Chemical 7]

Preparation of 5.85 g of the compound prepared according to Example 1 (d) was added at a temperature of -78 ° C. under reduced pressure with 60 ml of anhydrous hydrogen fluoride in the presence of 9 ml of anisole and 2.25 ml of methyl ethyl sulfide.
It was treated at -20 ° C for 1 hour and at 0 ° C for 1 hour. Hydrogen fluoride was removed under reduced pressure at 0 ° C. and the residue was triturated with ether. The obtained solid was dissolved in 5% acetic acid water, the insoluble matter was filtered off, and the filtrate was freeze-dried to obtain 3.07 g of a crude product of the title compound. 3.07 g of this crude product was added to the reverse phase HPL
C (column: YMC-ODS, 50 mmφ x 500 mm, 1
5-30μ; Flow rate: 14 ml / min; Detection wavelength: 220
nm; eluent: (A) water-0.1% trifluoroacetic acid (B) acetonitrile-0.1% trifluoroacetic acid; gradient: (B) 0% → (180 minutes) 3% → (120
Min) 5%-> (120 min) 8%-> (60 min) 10%) to give 1.66 g of the title compound.

MS (FAB) m / e 859 (M
⁺ H ⁺ ) Amino acid analysis value: Asx (1.91), Gly ^* (2.
00), Arg (1.84), Thr (1.77) [G
Calculation based on ly ^* ] Reversed-phase HPLC column: YMC-ODS, 5μ 4.6 mmφ × 250 mm Eluent: gradient A solution: water-0.1% trifluoroacetic acid B solution: acetonitrile-0.1% trifluoro Acetic acid initial solution B concentration: 7% Concentration gradient: 0.5% / min Flow rate: 1 ml / min Detection wavelength: 220 nm Retention time: 6.23 min

Example 2

[0033]

[Chemical 8]

Preparation of a) Preparation of Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gly) ₃ -OPac 10.0 g of the compound prepared according to Example 1 (a)
(5.7 mmol) was dissolved in 60 ml of acetonitrile,
8.25 g of methanesulfonic acid under ice cooling (0.086 mo
l) was added dropwise, and the mixture was stirred at room temperature for 1 hour. The mixture was ice-cooled again, 50 ml of DMF was added dropwise, and further 5.25 g (0.080 mol) of triethylamine was added dropwise. Then, Boc-Asp (OcHex) Thr (Bzl) Arg (Tos) Gly-OH 5.25
g (6.0 mmol), HOBt 0.93 g (6.9 m)
mol) and 1.07 g (6.9 mmol) of WSC were added under ice cooling, and the mixture was stirred for 30 minutes and then at room temperature for 3 hours. The reaction solution was poured into 1.0 liter of 1% saline solution, stirred for 30 minutes under ice cooling, and the precipitate was filtered, washed with water, washed with ether and dried to obtain 12.5 g of the title compound.

B) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gl
y) Preparation of ₃ -OH 7.42 g of the compound prepared according to Example 2 (a)
(2.9 mmol) was dissolved in 90 ml of 90% acetic acid, and 3.85 g (0.059 mol) of zinc powder was added every 3 minutes under ice cooling, and the mixture was returned to room temperature and stirred vigorously.
After 4.5 hours, the reaction solution was filtered through Celite, the filtrate was poured into 600 ml of 1% saline, and the mixture was stirred under ice cooling, and the precipitate was collected by filtration, washed with water, washed with ether and dried to give the title compound. Obtained 6.62 g.

C) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gl
y) Preparation of ₃ -OSu 6.62 g of the compound prepared according to Example 2 (b)
(2.8 mmol) of methylene chloride 40 ml and DMF20
It was dissolved in a mixed solution of 1.5 ml, and under ice cooling, 1.58 g (0.014 mol) of N-hydroxysuccinimide was added.
5M of _{DCC-CH 2 Cl 2 27.5ml (} 0.014
mol) was added and the mixture was stirred at room temperature for 23 hours. The insoluble material was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolved in a small amount of DMF, then poured into 500 ml of 1% saline and stirred under ice cooling.
The precipitate was collected by filtration, washed with water, washed with ether and dried to give the title compound (7.72 g).

D)

[0038]

[Chemical 9]

Preparation of 7.72 g of the compound prepared according to Example 2 (c)
(3.1 mmol) was dissolved in 50 ml of trifluoroacetic acid and stirred under ice cooling for 30 minutes. After the solvent was distilled off, 300 ml of ether was added, and the mixture was stirred again under ice cooling for 30 minutes. The precipitate was collected by filtration, washed with ether, and dried to obtain 6.77 g of Boc-free body. 6.77 g of this de-Boc body was added to DMF.
It was dissolved in 50 ml and slowly dropped into pyridine heated to 60 to 65 ° C over 1.5 hours. 3 at 60-65 ° C
After stirring for 0 minutes, the mixture was allowed to cool and the solvent was distilled off under reduced pressure. The residue was recrystallized from acetone-ether to give the title compound 5.
94 g were obtained.

E)

[0041]

[Chemical 10]

Preparation of 5.10 g of the compound prepared in Example 2 (d) was added in the presence of 9 ml of anisole and 2.25 ml of methyl ethyl sulfide to 60 ml of anhydrous hydrogen fluoride at -78 ° C under reduced pressure, and -20 It was treated at 0 ° C. for 1 hour and at 0 ° C. for 1 hour. Hydrogen fluoride was removed under reduced pressure at 0 ° C. and the residue was triturated with ether. The obtained solid was dissolved in 5% acetic acid water, the insoluble material was filtered off, and the filtrate was freeze-dried to obtain 2.59 g of the crude product of the title compound. 2.02 g of this crude product was added to reverse phase HP
LC (column: YMC-ODS, 50 mmφ × 500 mm,
Flow rate: 14 ml / min; Detection wavelength: 22
0 nm; eluent: (A) water-0.1% trifluoroacetic acid (B) acetonitrile-0.1% trifluoroacetic acid; gradient: (B) 0% → (180 minutes) 8% → (180
Min) 17%) to give 0.96 g of the title compound.
Got

MS (FAB) m / e 1288 (M ⁺ H
⁺ ) Amino acid analysis value: Asx (2.85), Gly ^* (3.
00), Arg (2.88), Thr (2.79) [G
Calculation based on ly ^* ] Reversed phase HPLC analysis conditions were the same as in Example 1. Retention time: 10.36 minutes

Example 3

[0045]

[Chemical 11]

Preparation of a) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gly) ₄ -OPac Boc-Asp (OcHex) Thr (Bzl) Arg (Tos) Gly-OPac 2.50 g
(2.5 mmol) is dissolved in 30 ml of acetonitrile,
Methanesulfonic acid 1.94g (0.020mo under ice cooling
l) was added dropwise, and the mixture was stirred at room temperature for 1 hour. The mixture was ice-cooled again, 30 ml of DMF was added dropwise, and 1.79 g (0.018 mol) of triethylamine was added dropwise. Subsequently, 6.36 g (2.6 mmo) of the compound of Example 2 (b) was used.
l), HOBt 0.41 g (3.0 mmol), WS
0.47 g (3.0 mmol) of C was added under ice cooling and 3
After stirring for 0 minutes, the mixture was stirred at room temperature for 4.5 hours. The reaction mixture was poured into 600 ml of 1% saline and stirred for 30 minutes under ice cooling. The precipitate was filtered, washed with water, washed with ether and dried to obtain 6.37 g of the title compound.

B) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gl
y) Preparation of _4- OH 6.37 g of the compound prepared according to Example 3 (a)
(1.9 mmol) was dissolved in 200 ml of 90% acetic acid, and 5.69 g (0.087 mol) of zinc dust was added every 5 minutes under ice cooling, and the mixture was returned to room temperature and stirred vigorously.
After 2.5 hours, the reaction solution was filtered through Celite, the filtrate was poured into 600 ml of 1% saline, and the mixture was stirred under ice cooling, and the precipitate was collected by filtration, washed with water, washed with ether and dried to give the title compound. Obtained 5.95 g.

C) Boc- (Asp (OcHex) Thr (Bzl) Arg (Tos) Gl
y) Preparation of _4- OSu 5.95 g of the compound prepared according to Example 3 (b)
(1.9 mmol) was dissolved in 60 ml of DMF, and the mixture was cooled with ice.
1.08 g (9.4 m) of N-hydroxysuccinimide
mol) and then 0.5 M DCC-CH ₂ Cl ₂ 1
8.8 ml (9.4 mmol) was added, and the mixture was stirred at room temperature for 21 hours. The insoluble material was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolved in a small amount of DMF, then poured into 1.0 liter of 1% saline and stirred under ice cooling. The precipitate is collected by filtration, washed with water,
The crystals were washed with ether and dried to give the title compound (6.59 g).

D)

[0050]

[Chemical 12]

Preparation of 6.59 g of the compound prepared according to Example 3 (c)
(2.0 mmol) was dissolved in 50 ml of trifluoroacetic acid, and the mixture was stirred for 30 minutes under ice cooling. After the solvent was distilled off, 300 ml of ether was added, and the mixture was stirred again under ice cooling for 30 minutes. The precipitate was collected by filtration, washed with ether and dried to obtain 6.30 g of Boc-free body. 6.30 g of this de-Boc body was added to DMF.
It was dissolved in 50 ml and slowly dropped into pyridine heated to 60 to 65 ° C over 2 hours. After stirring at 60 to 65 ° C for 30 minutes, the mixture was allowed to cool and the solvent was distilled off under reduced pressure. The residue was recrystallized from acetone-ether to give the title compound 4.78.
g was obtained.

E)

[0053]

[Chemical 13]

Preparation of 4.29 g of the compound prepared in Example 3 (d) was added to 7.5 ml of anisole and 1.88 ml of methyl ethyl sulfide.
50 ml of anhydrous hydrogen fluoride was added under reduced pressure at -78 ° C, and the mixture was treated at -20 ° C for 1 hour and at 0 ° C for 1 hour. Hydrogen fluoride was removed under reduced pressure at 0 ° C. and the residue was triturated with ether. The obtained solid was dissolved in 5% acetic acid water, the insoluble matter was filtered off, and the filtrate was freeze-dried to obtain 2.85 g of a crude product of the title compound. 2.85 g of this crude product was added to reverse phase HP
LC (column: YMC-ODS, 50 mmφ × 500 mm,
Flow rate: 14 ml / min; Detection wavelength: 22
0 nm; eluent: (A) water-0.1% trifluoroacetic acid (B) acetonitrile-0.1% trifluoroacetic acid; gradient: (B) 0% → (180 minutes) 8% → (60
Min) 11% → (60 min) 13%) to give 0.40 g of the title compound.

MS (FAB) m / e 1717 (M ⁺ H
⁺ ) Amino acid analysis value: Asx (3.77), Gly ^* (4.
00), Arg (3.69), Thr (3.39) [G
Calculation based on ly ^* ] Reversed phase HPLC analysis conditions were the same as in Example 1. Retention time: 11.73 minutes

[0056]

INDUSTRIAL APPLICABILITY According to the present invention, it has an effective biological activity such as a cancer metastasis inhibitory action and a platelet aggregation inhibitory action.
It has become possible to provide a cell adhesion active peptide which is a cyclic peptide having a Gly-Asp-Thr sequence.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keiichi Ono 3-98 Kasugadeka, Konohana-ku, Osaka Sumitomo Pharmaceutical Co., Ltd.

Claims (4)

[Claims] 1. Formula (1): (In the formula, n represents an integer of 2 or more and 10 or less), or a pharmaceutically acceptable salt thereof. 2. In the formula (1), n is 2, 3 or 4
The peptide according to claim 1 or a pharmaceutically acceptable salt thereof. 3. Formula (1): (In the formula, n represents an integer of 2 or more and 10 or less.) A cancer metastasis inhibitor comprising a peptide represented by the formula or a pharmaceutically acceptable salt thereof as an active ingredient. 4. Formula (1): (In the formula, n represents an integer of 2 or more and 10 or less.) A platelet aggregation inhibitor comprising a peptide represented by the formula or a pharmaceutically acceptable salt thereof as an active ingredient.