JPH05222092A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To provide a peptide derivative containing Arg-Gly-Asp as a core sequence, having remarkably improved cancer metastasis suppressing activity in spite of simple modification of the peptide chain terminal and exhibiting broad carcinostatic spectrum. CONSTITUTION:The peptide derivative of the formula R<1>-X-Arg-Gly-Asp-Ser-R<2> or its physiologically permissible salt (Arg, Gly, Asp and Ser are residues of arginine, glycine, aspartic acid and serine, respectively; X is amino acid or peptide residue; R<1> is H or (substituted) acyl group; R<2> is group of the formula OR<3> or NR<4>R<5>; R<3>, R<4> and R<5> are H or <=3C alkyl; X may be absent). A cancer metastasis suppressing agent can be produced by using the compound as an active component.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide derivative having a core sequence (Arg-Gly-Asp) of a cell adhesion peptide, and a cancer metastasis inhibitor containing the peptide derivative.

[0002]

BACKGROUND OF THE INVENTION Fibronectin is a protein involved in cell-extracellular matrix adhesion and is also considered to be involved in platelet aggregation and cancer metastasis. The cell-extracellular matrix interaction is mediated by a series of cell surface receptors,
Although fibronectin is a macromolecule with a molecular weight of about 250,000, these receptors have Arg-
It was revealed that the Gly-Asp sequence (core sequence) was specifically recognized, and it was reported that the core sequence is important for the interaction with the receptor (Nature, vol. 309). , P. 30, 1984). This Arg-Gly-A
The sp sequence is also present in other adhesive proteins such as vitronectin, and these adhesive proteins bond with the receptor of the adherent cell via the above core sequence and transmit the information to the adherent cell. In addition, this Arg-Gly-Asp sequence also has the ability to bind to biopolymers such as heparin, collagen, and fibrin, and is considered to be involved in adhesion between cells and interstitial connective tissue, cell differentiation, and proliferation. Has been. As described above, the cell adhesion-activating protein has various biological activities, so that
Application to medical materials is being studied.

For example, a method of inhibiting platelet aggregation using various chain and cyclic oligopeptides having Arg-Gly-Asp sequence (Polymer Preprints,
Japan), vol. 38, p. 3149, 1989; JP-A-2-174797.
No.), a method of using a peptide having an Arg-Gly-Asp sequence as a cell migration inhibitor (JP-A No. 2-4716), Arg-Gly-As.
Method of using pMA-immobilized PMMA membrane as cell adhesion membrane (Polymer Preprints, Japan), Volume 37, 7
(P. 05, 1988) is reported. Also, for polymers
A method in which a peptide having Arg-Gly-Asp as an essential constituent unit is covalently bonded and used as a substrate for animal cell culture, a substrate for a biocomposite artificial organ (JP-A-1-309682, JP-A-1-305960),
Method for using a polypeptide having an Arg-Gly-Asp-Ser sequence as a platelet protective agent for extracorporeal blood (JP-A-64-6217)
Etc. are also disclosed.

Furthermore, in recent years, cell adhesion-activating proteins have been attracting attention as substances 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 is present, the cells form a multicellular mass, which facilitates growth and survival of cancer cells. However, at this time,
Tripeptide Arg-Gl, the adhesion core of fibronectin
It has been reported that, when y-Asp coexists, it competitively binds to a receptor on cancer cells and thereby exhibits cancer metastasis inhibitory activity (Science, 238, 467, 1986).

As described above, the cell adhesion-activating protein such as fibronectin or its peptide fragment has various biological activities, and it has been desired to develop a technique for applying the related substance as a drug. In particular, the cancer metastasis-suppressing action of the adhesive core sequence is considered to have high application value as a drug. However, since the suppression effect is not so great with only the core sequence, for the purpose of enhancing the effect, a method for suppressing cancer metastasis using an oligopeptide having this sequence, a cyclic oligopeptide, or a polypeptide having a repeating structure thereof is also available. Disclosed (Int. J. Bio
l. Macromol., Vol. 11, p. 23, 1989; ibid., Vol. 11,
226, 1989; Jpn. J. Cancer Res., 60, 722.
Page, 1989; JP-A-2-174797). However, for example, the synthesis of cyclic peptides is more difficult and the yield is lower than that of linear ones,
In addition, there are problems such as difficulty in specifying the structure of a polypeptide, and a method for significantly improving the cancer metastasis suppressing ability by a simpler modification of the core sequence has been desired.

[0006]

Therefore, an object of the present invention is to improve the ability to suppress cancer metastasis, while the modification of the end of the core sequence (Arg-Gly-Asp) is simple, and It is to provide a peptide derivative having a broad anticancer spectrum. Another object of the present invention is to provide a cancer metastasis inhibitor containing this peptide derivative as an active ingredient.

[0007]

An object of the present invention is to provide a peptide derivative represented by the following general formula (I), a pharmacologically acceptable salt thereof, and a cancer metastasis inhibitor containing them as an active ingredient. Achieved by In the general formula ^{(I) R 1 -X-Arg} -Gly-Asp-Ser-R 2 formula represents Arg, Gly, Asp and Ser, respectively arginine, glycine, aspartic acid and serine residues. X represents an amino acid or peptide residue. R
¹ represents a hydrogen atom or an optionally substituted acyl group. R ² represents -OR ³ or -NR ⁴ R ^5. R ³ , R ⁴ ,
R ⁵ each represents a hydrogen atom or an alkyl group having 3 or less carbon atoms. X may or may not be present.

Examples of the salt of the peptide derivative of the present invention include salts with inorganic acids such as hydrochlorides, sulfates, nitrates, phosphates and borates, and acetates, trifluoroacetates and trifluoromethanesulfones. Examples thereof include salts with organic acids such as acid salts, lactate salts and tartrate salts, and conversion to such salts can be carried out by a conventional means.

The amino acid constituting the peptide derivative of the present invention may be L-form or D-form, but the L-form is preferred. However, the arginine residue has the same effect in the D-form as in the L-form. X represents an amino acid or peptide residue, which may or may not be present, but when present, particularly preferred examples include a glycine residue, an aspartic acid residue, and a glutamic acid residue. In this case, the L-form and the D-form are likewise preferred.

R ¹ represents a hydrogen atom or an optionally substituted acyl group. At this time, the carbon number of the acyl group is preferably 12 or less, and more preferably 4 or less, excluding carbonyl carbon and carboxyl carbon. Preferred R
Examples of the acyl group of ¹ include an acetyl group, a propionyl group, a butyryl group, a valeryl group, and an isobutyryl group. When R ¹ is an acyl group, it may be substituted with an appropriate substituent. Examples of preferable substituents include anionic substituents such as a carboxyl group and a sulfo group.
Preferred examples of the acyl group substituted with the carboxyl group of R ¹ include a succinyl group, a glutaryl group and an adipoyl group. R ² represents -OR ³ or -NR ⁴ R ^5. R ³ , R ⁴ and R ⁵ are each a hydrogen atom or have 3 carbon atoms.
The following alkyl groups are represented. As a particularly preferable example,-
_{OH, -OCH 3, -OC 2 H} 5, -NH 2, include -NHCH _3.

Examples of the compounds of the present invention are shown below, but the present invention is not limited thereto. When R ¹ is a hydrogen atom and R ² is —OH, the description is omitted according to the convention of the notation in the field. Amino acids are represented by the three-letter notation and represent the L-form unless otherwise specified. Also Su
c-, Glt-, and Adp-represent a succinyl group, a glutaryl group, and an adipoyl group, respectively.

Compound 1 Suc-Arg-Gly-A
sp-Ser

Compound 2 Suc-Gly-Arg-G
ly-Asp-Ser

Compound 3 Suc-D-Arg-Gly-
Asp-Ser

Compound 4 Suc-Arg-Gly-A
sp-Ser-OCH ₃

Compound 5 Suc-Arg-Gly-A
sp-Ser-NH ₂

Compound 6 Glt-Arg-Gly-A
sp-Ser

Compound 7 Glt-Arg-Gly-A
sp-Ser-OCH ₃

Compound 8 Suc-D-Arg-Gly-
Asp-Ser-OCH ₃

Compound 9 Adp-Arg-Gly-A
sp-Ser

Compound 10 Asp-Arg-Gly-A
sp-Ser

Compound 11 CH ₃ C (O) -Asp-A
rg-Gly-Asp-Ser

Compound 12 CH ₃ C (O) -Glu-A
rg-Gly-Asp-Ser

Compound 13 CH ₃ C (O) -D-Asp-A
rg-Gly-Asp-Ser

Compound 14 CH ₃ C (O) -Asp-D-A
rg-Gly-Asp-Ser

Compound 15 C ₂ H ₅ C (O) -Asp-A
rg-Gly-Asp-Ser

Compound 16 C ₄ H ₉ C (O) -Asp-A
rg-Gly-Asp-Ser

Compound 17 CH ₃ C (O) -Asp-Ar
g-Gly-Asp-Ser-OCH ₃

Compound 18 CH ₃ C (O) -Asp-Ar
g-Gly-Asp-Ser-NH ₂

Compound 19 Suc-Asp-Arg-G
ly-Asp-Ser

The method for synthesizing the peptide is not particularly limited, and examples thereof include a liquid phase method, a solid phase method, and a synthetic method using an automatic synthesizer. For more information on these synthetic methods,
Biochemistry Experiment Course "Protein Chemistry IV" p207-49
5 (Biochemical Society of Japan, Tokyo Kagaku Doujin), "Continuing Biochemistry Experiment Course, Protein Chemistry (2)" (Japanese Biochemical Society, Tokyo Kagaku Doujin), Izumiya et al. "Basics and Experiments of Peptide Synthesis"
(Maruzen). It is also possible to use a commercially available synthetic peptide.

The peptide derivative of the present invention or a salt thereof is
It has a core sequence Arg-Gly-Asp of a cell adhesion protein, and adheres to cells through the core sequence by a mechanism similar to that of the cell adhesion protein. Therefore, it shows various physiological activities as an agonist or antagonist of cell adhesive protein, and has a wide range of biological activities such as immunomodulatory action, wound healing action, platelet aggregation inhibitory action by cancer cells occurring in capillaries, and neurological disease healing action. Show. Therefore, the peptide derivative or a salt thereof of the present invention, at least one of them is used as a cancer metastasis inhibitor, a wound healing agent, an immunomodulator, a platelet aggregation adhesion inhibitor, together with a conventional carrier or a pharmaceutical aid, in patients. It is possible to administer. The dose is
It is determined in the range of 0.2 μg / kg to 400 mg / kg per dose based on symptoms, age, body weight and the like.

The peptide derivative of the present invention or a salt thereof is
It is preferable to administer by a generally used administration method 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 peptide derivative of the present invention or a salt thereof may be dissolved in PBS or physiological saline as shown in Examples below to give an injectable preparation, or 0.1N. It may be dissolved in a moderate amount of acetic acid water or the like and then made into a freeze-dried preparation. A conventional stabilizer such as glycine or albumin may be added to such a preparation. Furthermore, the peptide derivative of the present invention or a salt thereof can be orally administered, for example, in the form of microcapsules encapsulated in liposomes, microspheres, or hydrogels, such as suppositories, sublingual tablets, and dots. If it is in the form of a nasal spray, it can be absorbed through mucous membranes other than the digestive tract.

[0034]

The present invention will be further described below with reference to synthetic examples of the compounds of the present invention and test examples of their activity, but the present invention is not limited to these. In the following examples, the following abbreviations are used in addition to those used above.

T-Boc: t-butoxycarbonyl Bzl: benzyl Z: benzyloxycarbonyl DCC: dicyclohexylcarbodiimide HOBt: hydroxybenzotriazole TFA: trifluoroacetic acid DIEA: diisopropylethyl ether DMF: dimethylformamide

Synthesis Example 1 Synthesis of Compound 1 Compound 1 was synthesized according to the route shown below.

[0037]

[Chemical 1]

[0038]

[Chemical 2]

Synthesis of (1a) 14.8 g (50 mmol) t-Boc-Ser (Bzl), NaHCO ₃
g (0.1 mol) in DMF solution (150 ml)
To the mixture, 8.55 g (50 mmol) of benzyl bromide was added dropwise at room temperature, and the mixture was stirred at room temperature for 24 hours. 4% NaHCO _{3 in the} reaction solution
Aqueous solution and ethyl acetate were added for extraction and liquid separation. The ethyl acetate layer was washed with water, then brine, and then dried over sodium sulfate. The ethyl acetate was distilled off under reduced pressure to obtain 17.0 g (4.4 mmol) of oil (1a). Yield 8
8%.

Synthesis of (1b) 17.0 g (44 mmol) of (1a) was added to 30 m of methylene chloride.
After dissolving in 1 l, 30 ml of trifluoroacetic acid (TFA) was added and stirred at room temperature for 3 hours. Methylene chloride and TF
After A was distilled off with a rotary evaporator, ethyl acetate and a 4% sodium carbonate aqueous solution were added to the residue for extraction and separation. The ethyl acetate layer was washed with water and then with brine, and then dried over magnesium sulfate. After distilling off ethyl acetate under reduced pressure, the residue was diluted with methylene chloride (60 ml) and DM.
Dissolved in a mixed solvent of F (40 ml) and t-Boc-Asp (OBzl)
15 g (46 mmol) and hydroxybenzotriazole monohydrate (HOBt.H ₂ O) 6.7 g (44 mmo)
1) was added and the mixture was stirred with ice cooling. DCC1 was added to this reaction solution.
0.3 g was added, and the mixture was stirred with ice cooling for 2 hours and at room temperature overnight.
The precipitated urea derivative was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate and 4% aqueous sodium carbonate solution were added to the residue for extraction and separation. The ethyl acetate layer was washed with water and then with brine, the newly precipitated urea derivative was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2/1) to give 15.8 g (26.7 mmo) of white solid (1b).
l) was obtained. Yield 61%.

Synthesis of (1c) 15.5 g (26.2 mmol) of (1b) was added to 30 ml of methylene chloride.
It was dissolved in ml, 30 ml of TFA was added, and the mixture was stirred at room temperature for 30 minutes. Methylene chloride and TFA were distilled off by a rotary evaporator, a mixed solvent of n-hexane and ether (5: 1) was added to the residue, and the white solid (1c) precipitated was collected by filtration. 15.0 g (24.8 mmol). Yield 95%.

Synthesis of (1d) (1c) 9 g (14.9 mmol), t-Boc-Gly 3 g (17.
1 mmol), HOBt · H ₂ O 2.3 g (2.5 mmo
l), diisopropyl ethyl ether 2.9 ml (16.
6 mmol) containing methylene chloride (60 ml) and DMF
The reaction solution of (30 ml) was stirred with ice cooling.

DCC (3.45 g) was added to the reaction solution, and the mixture was stirred with ice cooling for 2 hours and at room temperature overnight. The precipitated urea derivative was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was washed with 10% citric acid aqueous solution, 4% sodium carbonate aqueous solution, water, and brine in this order. The newly precipitated urea derivative was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / ethyl acetate = 3/1) to give a white solid (1d).
Obtained 8.64 g (13.3 mmol). Yield 90%.

Synthesis of (1e) 3.5 g (5.4 mmol) of (1d) was added to 20 ml of methylene chloride.
Was dissolved in, 20 ml of trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 1 hour. Methylene chloride and TFA
After distilling off with a rotary evaporator, ethyl acetate and a 4% aqueous sodium carbonate solution were added to the residue for extraction and liquid separation. The ethyl acetate layer was washed with water and then with brine, and then dried over magnesium sulfate. After distilling off ethyl acetate under reduced pressure, the residue was diluted with methylene chloride (40 ml) and DM.
Dissolve in a mixed solvent of F (20 ml) and t-Boc-Arg (Z) ₂ 2.
86 g (5.27 mmol) and 830 mg of hydroxybenzotriazole monohydrate (HOBt.H ₂ O) (5.
3 mmol) was added and the mixture was stirred with ice cooling. D to this reaction solution
CC 1.24 g was added, and the mixture was stirred for 2 hours with ice cooling and overnight at room temperature. The precipitated urea derivative was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate and 4% aqueous sodium carbonate solution were added to the residue for extraction and separation. The ethyl acetate layer was washed with water and then with brine, and the newly precipitated urea derivative was filtered off and dried over magnesium sulfate. Ethyl acetate was concentrated under reduced pressure, and the residue was recrystallized from a mixed solvent of ethyl acetate and n-hexane to obtain 5.0 g (4.66 mmol) of white solid (1e). Yield 88%.

Synthesis of (1f) 1.07 g (1 mmol) of (1e) was dissolved in 7 ml of methylene chloride, 7 ml of trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 1 hour. After methylene chloride and TFA were distilled off by a rotary evaporator, ethyl acetate and a 4% sodium carbonate aqueous solution were added to the residue for extraction and separation. After washing the ethyl acetate layer with water and then with saline,
Dry over magnesium sulfate. The ethyl acetate was distilled off under reduced pressure to obtain a de-t-Boc derivative of (1e).

This was dissolved in methylene chloride (15 ml), 120 mg of succinic anhydride was added, and the mixture was stirred at room temperature for 2 hours. The solvent was concentrated under reduced pressure, and the residue was recrystallized from acetonitrile to obtain 1.00 g (0.93 mmol) of (1f).
(Yield 93%)

Synthesis of Compound 1 720 mg (0.67 mmol) of (1f) was dissolved in 15 ml of acetic acid, 80 mg of 10% palladium carbon was added, and hydrogenolysis was carried out as follows. First, the reaction temperature is set to 55 ° C, the temperature is increased to 35 ° C in 1 hour, and the temperature is increased to 2 ° C in 1 hour.
When the temperature was lowered to 5 ° C, precipitation was observed. Water 7m
When 1 was added, the precipitate redissolved, so hydrogenolysis was continued at room temperature for 5 hours.

After the catalyst was filtered off and the solvent was distilled off under reduced pressure, ether was added to the residue and the precipitate was collected by filtration. This crude peptide was dissolved in pure water, activated carbon was added, and the mixture was stirred at room temperature for 15 minutes.
Activated carbon was filtered off, water was distilled off under reduced pressure, and then freeze-dried.
30 mg of compound 1 was obtained as a white amorphous product. In proton NMR, a peak of the methyl group of acetic acid was observed at 2.05 ppm, and it was found from the integrated intensity that Compound 1 was in the form of monoacetate. Yield 83%.

FAB Mass 534 (M + H) ⁺ , 556
(M + Na) ⁺

Synthesis Example 2 Synthesis of Compound 6 In the synthesis of the compound (1f) of Synthesis Example 1, the same reaction was carried out by using glutaric anhydride instead of succinic anhydride to obtain a fully protected compound 6. It was This was subjected to deprotection by hydrogenolysis similar to the synthesis of compound 1 and subsequent purification to obtain compound 6.

FAB Mass 548 (M + H) ⁺ , 568
(M + Na) ⁺

Synthesis Example 3 Synthesis of Compound 4 t-Boc-Ser (Bzl) 8.86 g (30 mmol), N, N-dimethylaminopyridine 360 mg, methanol 1.4 ml
A methylene chloride solution (60 ml) containing the above was stirred with ice cooling, and D
CC 6.2 g was added. After stirring at room temperature overnight, methylene chloride was distilled off under reduced pressure, and ethyl acetate was added to the residue. The urea derivative was filtered off, and the ethyl acetate filtrate was washed with an aqueous citric acid solution, an aqueous sodium hydrogencarbonate solution and brine in this order, and dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain a colorless oily crude product of t-Boc-Ser (Bzl) -CH ₃ 9.3.
g was obtained.

Using this instead of (1a), the above Synthesis Example 1
A fully protected form of compound 4 (4f) was obtained by the same route as described above. The yield at each step is described below. Note that (4b) to (4f) correspond to compounds in which the benzyl ester of the serine residue of (1b) to (1f) is changed to a methyl ester, respectively. (4b) 79% (4c) 86% (4d) 88% (4e) 70% (4f) 84%

850 mg (0.85 mmol) of (4f) was dissolved in 15 ml of acetic acid, and 80 mg of 10% palladium carbon was dissolved.
In addition, hydrogenolysis was carried out at 40 ° C. for 1 hour and then at room temperature overnight. After completion of the reaction, the mixture was heated again to 40 ° C. to dissolve the precipitate, the catalyst was filtered off, the solvent was distilled off under reduced pressure, ether was added to the residue, and the precipitate was collected by filtration. This crude peptide was dissolved in pure water, activated carbon was added, and the mixture was stirred at room temperature for 15 minutes. Activated carbon was filtered off, water was distilled off under reduced pressure, and then freeze-dried to give 330
Obtained mg of compound 4 as a white amorphous. Proton N
In the MR, a peak of the methyl group of acetic acid was observed at 2.07 ppm, and it was found from the integrated intensity that Compound 4 was in the form of monoacetate. Yield 77%.

FAB Mass 548 (M + H) ⁺

[0056] In the synthesis of Compound 1 of Synthesis Example 1 Synthesis Example 4 Compound 3, the t-Boc-Arg (Z) the same operation using the t-Boc-D-Arg ( Z) 2 in the _second alternative Done,
Compound 3 was obtained in the form of the monoacetate salt. FAB Mass 534 (M + H) ⁺ , 556
(M + Na) ⁺

Synthesis Example 5 Synthesis of Compound 5 Compound 5 was synthesized by the same route as in Synthesis Example 1 using t-Boc-Ser (Bzl) -NH ₂ instead of (1a). FAB Mass 533 (M + H) ⁺

Synthesis Example 6 Synthesis of Compound 10 Compound 10 was synthesized according to the route shown below. The compound (1e) was synthesized in the same manner as in Synthesis Example 1.

[0059]

[Chemical 3]

Synthesis of (10f) (1e) 4.0 g (3.73 m) synthesized in the same manner as in Synthesis Example 1
(mol) was dissolved in 20 ml of methylene chloride, 20 ml of trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 1 hour. After methylene chloride and TFA were distilled off by a rotary evaporator, ethyl acetate and a 4% sodium carbonate aqueous solution were added to the residue for extraction and separation. The ethyl acetate layer was washed with water and then with brine, and then dried over magnesium sulfate. After distilling off ethyl acetate under reduced pressure, the residue was dissolved in a mixed solvent of methylene chloride (30 ml) and DMF (30 ml), and 1.3 g (4 mmol) of t-Boc-Asp (OBzl) and hydroxybenzotriazole / 1 water were added. Japanese products (HOBt
H ₂ O) (610 mg, 4 mmol) was added, and the mixture was stirred with ice cooling. To this reaction solution, 0.85 g of DCC was added, which was cooled with ice to 2
The mixture was stirred at room temperature overnight. The precipitated urea derivative was filtered off, and the filtrate was concentrated under reduced pressure. Ethyl acetate and 4% aqueous sodium carbonate solution were added to the residue for extraction and separation. The ethyl acetate layer was washed with water and then with brine, and the newly precipitated urea derivative was filtered off and dried over magnesium sulfate.
Ethyl acetate was concentrated under reduced pressure, and the residue was recrystallized from a mixed solvent of ethyl acetate and n-hexane to give a white solid (10f) 3.4.
g (2.66 mmol) was obtained. Yield 71%.

Synthesis of (10 g) 3.3 g (2.58 mmol) of (10f) was added to methylene chloride 15
Dissolve in 15 ml of trifluoroacetic acid (TFA)
Was added and the mixture was stirred at room temperature for 1 hour. Methylene chloride and T
After FA was distilled off by a rotary evaporator, ethyl acetate and a 4% sodium carbonate aqueous solution were added to the residue for extraction and separation. The ethyl acetate layer was washed with water and then with brine, and then dried over magnesium sulfate. The ethyl acetate was distilled off under reduced pressure, the residue was recrystallized from ethyl acetate, and (10 g)
2.7 g (2.29 mmol) was obtained. Yield 89%.

Synthesis of Compound 10 (10 g) 910 mg (0.77 mmol) was added to acetic acid 15 ml.
Was dissolved in 100 mg of 10% palladium carbon and hydrogenolysis was carried out at room temperature for 10 hours. After the catalyst was filtered off and the solvent was distilled off under reduced pressure, ether was added to the residue and the precipitate was collected by filtration. This crude peptide was dissolved in pure water, activated carbon was added, and the mixture was stirred at room temperature for 15 minutes. Activated carbon was filtered off, water was distilled off under reduced pressure, and then freeze-dried to obtain 280 mg of Compound 1 as a white amorphous substance. 2.05 pp in proton NMR
A peak of the methyl group of acetic acid was observed at m, and it was found from the integrated intensity that Compound 1 was in the form of 1/2 acetate. Yield 63%. FAB Mass 549 (M + H) ⁺

Synthesis Example 7 Synthesis of Compound 11 1.1 g (0.94 mmol) of (10 g) synthesized in Synthesis Example 6
Was dissolved in 15 ml of methylene chloride and 0.1 ml of acetic anhydride was added with stirring at room temperature. Immediately, precipitation was observed. After continuing stirring at room temperature for 30 minutes, methylene chloride was distilled off under reduced pressure, and the residue was recrystallized from acetonitrile to obtain 910 mg (0.746 mmol) of a protected compound 11 of compound 11. 850 mg (0.697 mmol) of this protected compound was subjected to deprotection by hydrogenolysis similar to the synthesis of compound 10 and subsequent purification to obtain 380 mg of compound.
In the same manner as in Synthesis Example 6, the compound
Was found to be in the form of 1/2 acetate. Yield 88%. FAB Mass 591 (M + H) ⁺ , 613
(M + Na) ⁺

Synthesis Example 8 Synthesis of Compound 12 In the synthesis of (10f) of Synthesis Example 6, t-Boc-Glu (OBzl) was used in place of t-Boc-Asp (OBzl), and the same procedure as in Synthesis Example 7 was followed. The acetate salt of compound 12 was obtained through the steps of de-t-Boc, acetylation and hydrogenolysis. FAB Mass 605 (M + H) ⁺

Synthesis Example 9 Synthesis of Compound 13 In the synthesis of (10f) of Synthesis Example 6, t-Boc-D-Asp (OBzl) was used in place of t-Boc-Asp (OBzl). Acetate of compound 13 was obtained through the same steps of det-Boc, acetylation and hydrogenolysis. FAB Mass 591 (M + H) ⁺

[0066] Using the Synthesis Example 10 Compound 14 t-Boc-Arg (Z ) 2 in place of t-Boc-D-Arg ( Z) 2 in the synthesis of the (1e) Synthesis Example 1, hereinafter Synthesis Example The acetate salt of compound 14 was obtained through the same steps as 6 and 7. FAB Mass 591 (M + H) ⁺

Synthesis Example 11 Synthesis of Compound 17 t-Boc-Ser (Bzl) 8.86 g (30 mmol), N, N-dimethylaminopyridine 360 mg, methanol 1.4 ml
A methylene chloride solution (60 ml) containing the above was stirred with ice cooling, and D
CC 6.2 g was added. After stirring at room temperature overnight, methylene chloride was distilled off under reduced pressure, and ethyl acetate was added to the residue. The urea derivative was filtered off, and the ethyl acetate filtrate was washed with an aqueous citric acid solution, an aqueous sodium hydrogencarbonate solution and brine in this order, and dried over magnesium sulfate. Ethyl acetate was distilled off under reduced pressure to give a colorless oily crude product of t-Boc-Ser (Bzl) -OCH ₃ 9.3.
g was obtained. Using this instead of (1a), the acetate salt of compound 17 was obtained through the same steps as in Synthesis Examples 1, 6 and 7. FAB Mass 605 (M + H) ⁺

Synthesis Example 12 Synthesis of Compound 19 (10 g) synthesized in Synthesis Example 6 (1.1 g, 0.94 mmol)
Was dissolved in 15 ml of methylene chloride and 120 mg of succinic anhydride was added with stirring at room temperature. Immediately, precipitation was observed. After continuously stirring at room temperature for 30 minutes, methylene chloride was distilled off under reduced pressure, and the residue was recrystallized from acetonitrile to obtain 910 mg of a protected compound 2 (0.712 mmo).
l) was obtained. This protector 850mg (0.697mmo
1) was subjected to deprotection by hydrogenolysis similar to the synthesis of Compound 1 and subsequent purification to obtain 150 mg of Compound 19.
Obtained. Yield 33%. FAB Mass 649 (M + H) ⁺ , 671
(M + Na) ⁺

Synthesis Example 12 Compounds 2, 7, 8, 9, 15, 16 and 18 were synthesized in the same manner as above. Compound 2 FAB Mass 591 (M + H) ⁺ Compound 7 FAB Mass 562 (M + H) ⁺ Compound 8 FAB Mass 548 (M + H) ⁺ Compound 9 FAB Mass 562 (M + H) ⁺ Compound 15 FAB Mass 605 (M + H) ⁺ Compound 16 FAB (M + H) ⁺ Compound 18 FAB Mass 590 (M + H) ⁺

Test Example (Experimental Lung Metastasis) The compound of the present invention and the RGD compound having no aspartic acid or glutamic acid residue or an acyl group substituted with a carboxyl group at the N-terminal of arginine were compared for their cancer metastasis inhibiting effects. .. Compounds 1, 3, 4, 5,
6, 9, 10, 11, 12, 13, 14, 19 and Arg-Gly-Asp-Ser (RGD for comparison).
S), Gly-Arg-Gly-Asp-Ser (GR
GDS), Ac-Arg-Gly-Asp-Ser (A
c-RGDS) and Arg-Gly-Asp-Ser-
OCH ₃ (RGDS-OCH ₃ ) 1000 μg each, and B16-BL6 melanoma cells 5 as highly metastatic cancer cells
After mixing 10 ⁴ × 10 ⁴ each in PBS 0.2 ml,
Each of the C57BL / 6 male mice was injected intravenously. Administration 1
Four days later, the number of lung melanoma cell colonies of the mice was counted and compared with the control PBS-administered group. The results are shown in Table 1.

Compound 1 and Arg for comparison
-Gly-Asp-Ser (RGDS) 3000μ each
g and L5178Y ML25 T-lymphoma ⁴ × 10 ⁴ each in PBS
After mixing in 0.2 ml, 1 group of 5 (BALB / C x DBA ₂ ) F ₁ · CDF ₁
Female mice were injected intravenously. Fourteen days after the administration, the liver and spleen of the mice were weighed and compared with the control PBS-administered group and the untreated group. The results are shown in Table 2.

Further, compound 12 and G for comparison
ly-Arg-Gly-Asp-Ser (GRGDS)
1000 μg of each and L5178Y ML25 T-lymphoma ⁴ × 10 ⁴ were mixed in 0.2 ml of PBS, and then 5 C57BL / groups were prepared per group.
Six male mice were injected intravenously. 14 days after administration
Lungs and spleens were weighed and compared to control PBS-treated and untreated groups. The results are shown in Table 3.

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[Table 1] Table 1 Effect of peptides on experimental lung metastasis of cancer induced by injection of B16-BL6 melanoma cells ────────────────────── ───────────── Dose Compound Dose Lung metastases (μg) Mean ± SD (range) ──────────────────── ──────────────── PBS (control group) −91 ± 33 (53-135) Compound 1 1000 19 ± 10 (4-32) ^** 3 1000 38 ± 17 (18) -66) ^* 4 1000 33 ± 12 (18-47) ^* 5 1000 25 ± 14 (6-46) ^** 6 1000 36 ± 19 (13-70) ^* 9 1000 31 ± 16 (10-54) ^* 10 1000 96 ± 16 (78-116) ^* 11 1000 25 ± 9 (10-33) ^** 12 1000 28 ± 9 (18-43) ^{* *} 13 1000 55 ± 8 (45-67) ^** 14 1000 27 ± 15 (7-46) ^** 19 1000 30 ± 10 (20-45) ^** RGDS 1000 114 ± 35 (53-135) GRGDS 1000 108 ± 17 (85-121) ^* Ac-RGDS 1000 95 ± 23 (57-121) RGDS-OCH ₃ 1000 127 ± 43 (68-165) ^* ────────────────── ─────────────────── ^{* *} t-test compared to control group p <0.01 ^** t-test compared to control group p <0.001

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[Table 2] Table 2 Effect of peptide on experimental liver and spleen transition of cancer induced by injection of L5178Y ML25 T-lymphoma cells ─────────────────── ───────────────── Dose Compound Dose Weight Average ± SD (range) (g) (μg) Liver Spleen ────────────── ────────────────────── PBS (control group) −4.56 ± 0.47 0.28 ± 0.04 RGDS 3000 4.42 ± 0. 75 0.25 ± 0.06 Compound 1 3000 1.08 ± 0.08 ^* 0.14 ± 0.04 ^{* No} cell administration − 1.06 ± 0.00 0.11 ± 0.01 ───── ─────────────────────────────── ^* p <0.001 compared to the control group by t-test

[0075]

[Table 3] Table 3 Effect of peptides on experimental liver and spleen transition of cancer induced by injection of L5178Y ML25 T-lymphoma cells ─────────────────── ───────────────── Dose Compound Dose Weight Average ± SD (range) (g) (μg) Liver Spleen ────────────── ────────────────────── PBS (control group) −3.83 ± 0.60 0.23 ± 0.01 GRGDS 1000 2.82 ± 0. 62 0.19 ± 0.04 Compound 12 1000 1.71 ± 0.35 ^* 0.15 ± 0.03 ^{* No} cell administration-1.16 ± 0.02 0.08 ± 0.01 ───── ─────────────────────────────── ^* p <0.001 compared to untreated controls by t-test

From the above results, it was found that the cancer metastasis-inhibiting ability is improved by modifying the amino terminal of the RGDS sequence with an acyl group substituted with a carboxyl group or an aspartic acid or glutamic acid residue.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ichiro Higashi 5-3-2 Makomanaikamimachi, Minami-ku, Sapporo-shi, Hokkaido

Claims (4)

[Claims] 1. A peptide derivative represented by the following general formula (I):
Conductor, or a physiologically acceptable salt thereof. General formula (I) R¹-X-Arg-Gly-Asp-Ser-R² (Wherein Arg, Gly, Asp and Ser are respectively
Arginine, glycine, aspartic acid and serine
Represents a residue. X represents an amino acid or peptide residue.
R¹Is a hydrogen atom or an optionally substituted acyl group
Represent R²Is -OR ³Or -NR^FourR^FiveRepresents R³,
R^Four, R^FiveIs a hydrogen atom or an alkane having 3 or less carbon atoms, respectively.
Represents a kill group. X may or may not be present. ) 2. The compound according to claim 1, wherein X is absent or represents a glycine residue, and R ¹ represents an acyl group substituted with a carboxyl group. 3. The compound according to claim ¹ , wherein X represents an aspartic acid or glutamic acid residue, and R ¹ represents a hydrogen atom or an acyl group. 4. A cancer metastasis inhibitor comprising the compound according to any one of claims 1 to 3 as an active ingredient.