JPH04264097A - Peptide derivative and it's use - Google Patents

Abstract

PURPOSE:To obtain a synthetic cyclic peptide derivative having cell adhesion inhibiting activity, and a preparation containing the same derivative as an active ingredient and capable of inhibiting adhesion of animal cells. CONSTITUTION:A peptide derivative consisting of a synthetic cyclic peptide having an amino acid sequence part expressed by the formula Arg-MeGly-Asp (MeGly is N-methylglycine residue) or salt thereof, and a preparation containing the same derivative as an active ingredient and capable of inhibiting adhesion of animal cells. The peptide derivative is stable to enzymic hydrolysis and the preparation of the present invention is effective in inhibiting the adhesion of animal cells to a substrate, aggregation of platelets and metastasis of cancer cells, etc.

Description

[Detailed description of the invention]

0001

FIELD OF INDUSTRIAL APPLICATION The present invention relates to a novel synthetic cyclic peptide or a peptide derivative consisting of a salt thereof, and an adhesion inhibitor for animal cells such as cancer cells and platelet cells, which contains the same as an active ingredient.

0002

BACKGROUND OF THE INVENTION Fibronectin and vitronectin are known as factors involved in the adhesion of animal cells to extracellular matrices. These cell adhesion factors are -Arg-
It has an adhesive site called Gly-Asp-. Therefore, a compound having the same tripeptide residue as this adhesion site inhibits adhesion by cell adhesion factors. That is, this cell adhesion inhibitor inhibits binding of cell adhesion factors. Examples of such cell adhesion inhibitory factors include Gly-
Arg-Gly-Asp-Ser-Pro is known. Cell adhesion inhibitors are used as reagents for research on the adhesive properties of animal cells, and are also expected to be used as drugs for suppressing cancer cell metastasis (preventing adhesion and immobilization at metastatic sites).

[0003] Since conventionally known cell adhesion inhibitors are linear peptides, it is difficult for them to have a specific tertiary structure stably in solution, making it difficult in some cases to fully exhibit their effects. Furthermore, because of the presence of an amino terminus and a carboxy terminus, they are susceptible to hydrolysis by enzymes such as aminopeptidase and carboxypeptidase, and their stability in solutions containing these enzymes is insufficient.

0004

[Problems to be Solved by the Invention] In order to improve the structural stability of cell adhesion factors, a compound is known in which a polypeptide having the above tripeptide residue is synthesized and cyclized with a disulfide bond (M.D. .Pierschbacher
et al., J. Biol. Chem. ,262,17294
-17298, (1987)). However, cell adhesion inhibitors having disulfide bonds have not yet fully solved the above problems. Furthermore, the synthetic cyclic peptide having the amino acid sequence portion represented by -Arg-Gly-Asp- according to the present inventors' invention (see JP-A-2-174797) does not necessarily have sufficient cell adhesion inhibiting activity. There was a case. Therefore, a cell adhesion factor with higher activity and higher structural stability has been desired.

[0005]

[Means for Solving the Problems] The present invention relates to a novel peptide derivative that solves the above-mentioned problems, and a drug containing the same as an active ingredient that has cell adhesion inhibiting activity.

[0006] In the following, "cyclo[...
The expression "...]" indicates that both ends of the linear peptide in [ ] are connected by a peptide bond to form a cyclic peptide. Furthermore, as amino acids (residues) other than normal amino acids (residues), "MeGly" represents N-methylglycine (residue), and "Phg" represents phenylglycine (residue).

A peptide derivative comprising a synthetic cyclic peptide having an amino acid sequence represented by the following formula (1), or a salt thereof. -Arg-MeGly-Asp-...(1)
A drug that inhibits animal cell adhesion and contains the above peptide derivative as an active ingredient.

[0008] As a synthetic cyclic peptide having an amino acid sequence portion represented by formula (1), a synthetic cyclic peptide having at least one amino acid residue is preferable to a cyclic peptide consisting of only three amino acid residues. is preferred. Such a synthetic cyclic peptide having three or four or more amino acid residues is represented by the following formula (2). However, m represents 0 or 1, and m=0 is a cyclic peptide consisting of only 3 amino acid residues. cyclo[-Arg-MeGly-Asp-(R1)m-
] ...(2)

In formula (2), R1 represents an amino acid residue or an oligopeptide to polypeptide residue, and the number of amino acid residues in the oligopeptide to polypeptide residue is not particularly limited. However, the upper limit of the number of amino acid residues is suitably 16 or less, preferably 8 or less. In particular, 4 or less is preferable. The type of amino acid is not particularly limited, and of course it may be a normal amino acid or other amino acids as described below.

Particularly preferred R1 in the present invention is a phenylglycine residue, or an oligopeptide whose amino terminal side is a phenylglycine residue.
A polypeptide residue. Therefore, the present invention also provides a peptide derivative comprising a synthetic cyclic peptide having an amino acid sequence portion represented by the following formula (3), or a salt thereof. -Arg-MeGly-Asp-Phg-...
・(3)

[0011] If expressed similarly to the above formula (2),
The one represented by the following formula (4) is preferable. However, n
indicates 0 or 1, and n=0 is a cyclic peptide consisting of only 4 amino acid residues. Furthermore, R2 represents a residue excluding one amino acid residue on the amino terminal side of R1. Cyclo[-Arg-MeGly-Asp-Phg-(R
2)n-]...(4)

[0012] In the present invention, amino acids mean not only α-amino acids but also other amino acids (β-amino acids, γ-amino acids, etc.). As amino acids other than α-amino acids, amino acids represented by H2N(CH2)kCOOH (k is an integer of 2 or more) are suitable; for example, 3
-aminopropionic acid, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminocaproic acid, 7-aminopentanoic acid, 8-aminocaprylic acid, and the like. Preferably, k is an integer of 8 or less.

[0013] Furthermore, the α-amino acid may be not only an L-amino acid but also a D-amino acid or a D,L-amino acid. Preferred amino acids in the present invention are α-amino acids, especially L-amino acids (hereinafter, amino acids refer to L-amino acids unless otherwise specified). An amino acid residue refers to a residue obtained by removing one hydrogen atom from an amino group and a hydroxyl group from a carboxyl group.

[0014] In the present invention, the oligo-polypeptide in R1 and R2 refers to a compound in which two or more of the above-mentioned amino acids are linked by a peptide bond. α as an amino acid
- Preferably an amino acid. However, some of the amino acid residues may be residues other than α-amino acids such as H2N(CH2)kCOOH and β-amino acids, or D-amino acid residues. The term "oligo-polypeptide residue" refers to a residue obtained by removing one hydrogen atom from the amino group on the amino terminal side and the hydroxyl group from the carboxyl group on the carboxyl terminal side.

In order for the synthetic cyclic peptide of the present invention to exhibit the cell adhesion inhibiting effect described below, -Arg-Me
A peptide block of Gly-Asp- is required. However, a cyclic peptide consisting only of this peptide block does not necessarily exhibit sufficient effects, and as mentioned above, the presence of at least one amino acid residue is highly necessary.

The cyclic peptides expressed by formulas (2) and (4) are those in which the carboxyl terminus of R1 or R2 and the amino terminus of arginine are linked by a peptide bond. However, the cyclic peptides represented by formulas (1) to (4) do not indicate their synthetic routes. For example, this cyclic peptide can be synthesized not only by synthesizing Arg-MeGly-Asp-R1 and then cyclizing it, but also by cyclizing it by forming a peptide bonding moiety at any other position. . That is, Arg-MeGly, MeGly
-Asp, or any peptide bond within R1 can be formed and cyclized, as well as the peptide bond between Asp and R1.

The cyclic peptides of the present invention are specifically illustrated below, but the present invention is not limited thereto. Incidentally, Abu represents 2-aminobutyric acid.

1. cyclo[-Arg-MeGly-As
p-Phg-]2. cyclo[-Arg-MeGly-
Asp-Ser-]3. cyclo[-Arg-MeGl
y-Asp-Trp-]4. cyclo[-Arg-Me
Gly-Asp-Asp-]5. cyclo[-Arg-
MeGly-Asp-Ala-]6. cyclo[-Ar
g-MeGly-Asp-Phe-]7. Cyclo[-
Arg-MeGly-Asp-Leu-]8. Cyclo[-Arg-MeGly-Asp-Thr-]9. cyclo[-Arg-MeGly-Asp-Abu-]1
0. Cyclo[-Arg-MeGly-Asp-Ser-
Pro-Ala- ]

[0019] Examples of the salts of the peptides include acetic acid, tartaric acid, citric acid, trifluoroacetic acid,
Salts with organic or inorganic acids such as methanesulfonic acid, hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; metal salts such as alkali metal salts such as sodium salts and potassium salts; and alkaline earth metal salts such as calcium salts; It means salts with inorganic bases or organic bases, such as salts with organic amines such as ammonium, ethanolamine, triethylamine, and dicyclohexylamine.

The cyclic peptide of the present invention can be synthesized by conventional peptide synthesis methods. As mentioned above, cyclization is performed in a cyclization reaction after linear peptide synthesis, but the cyclization portion can be performed by forming any peptide bond between adjacent amino acid residues.

The above synthetic cyclic peptide of the present invention is effective as a drug for inhibiting animal cell adhesion. The animal cells are preferably mammalian cells, and examples of the mammalian cells include not only normal somatic cells and reproductive cells but also cancer cells. It is also effective in inhibiting adhesion of nonnucleated cells such as platelets. Particularly targeted animal cells are various cancer cells and platelet cells. Cancer metastasis involves the adhesion of cancer cells to other cells and extracellular matrix. Therefore, inhibiting adhesion of cancer cells is considered to be effective in preventing cancer metastasis. Furthermore, it is thought that if the adhesion of platelets to the inner wall of blood vessels can be inhibited, it will be possible to prevent the occurrence of thrombi and the like.

As shown in the Examples below, the synthetic cyclic peptide of the present invention not only has an excellent effect of inhibiting animal cell adhesion, but is also resistant to hydrolysis by enzymes and has excellent in-vivo stability. Furthermore, even if hydrolyzed, as long as the essential peptide block portion or its vicinity is not hydrolyzed, the linear peptide produced by hydrolysis also has a certain degree of cell adhesion inhibiting effect.

[0023] The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples.

[0024]

[Example] In the following examples, amino acids, protecting groups, active groups, etc. will be explained using IUPAC-IUBCommis.
sion on Biological Nomenc
It may be indicated by abbreviations based on lature and abbreviations commonly used in the field, examples of which are as follows.

Asp: Aspartic acid Arg: Arginine Gly: Glycine MeGly: N-methylglycine Phg: Phenylglycine Boc: t-butoxycarbonyl OBzl: Benzyl ester HOBt: p-hydroxybenzotriazole ONS
u: N-hydroxysuccinimide ester WSC・
HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride TFA: Trifluoroacetic acid OcHex: cyclohexyl ester Tos: p-toluenesulfonyl group

[Example 1] Cyclo[-Arg-MeGl
Synthesis of y-Asp-Phg-]

(1) Boc-Arg(Tos)-Me
Synthesis of Gly-OBzl Boc-Arg(Tos)
3.2g (7.5 mmol) and MeGly-OB
zl 1.3g (7.5 mmol) in DMF 20c
HOBt 1.0g, WSC・
Add 1.6g of HCl and adjust the pH to 6 with N-methylmorpholine.
and stirred overnight. After distilling off DMF under reduced pressure,
Add 100cm3 of ethyl acetate, 100cm3 of water, 1N
HCl 100cm3 (twice), water 100cm3,
100 cm3 of 5% sodium hydrogen carbonate aqueous solution (twice),
The ethyl acetate layer was washed successively with 100 cm3 of water and dried by adding anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain 3.4 g (5.6 mmol, yield 7) of white powder.
6%).

(2) Boc-PhgArg(Tos)M
Synthesis of eGly-OBzl Boc-Arg(Tos)
-MeGly-OBzl 1.2g (2.0mmol)
into a 300cm3 eggplant flask, and add 20cm of TFA.
3 and stirred at room temperature for 30 minutes, the mixture was concentrated under reduced pressure. When 200 cm3 of ether was added thereto, white crystals precipitated, which were filtered, thoroughly washed with ether, and then the ether was removed under reduced pressure. Add 30cm3 of DMF and cool on ice.
-Adjusted to pH 6 with methylmorpholine.

Next, 0.5g of Boc-Phg (2m
mol), HOBt 0.25g, WSC/HCl
0.42 g of the mixture was added thereto, the pH was again adjusted to 6 with N-methylmorpholine, and the mixture was stirred overnight. After distilling off DMF under reduced pressure, add 100 cm3 of ethyl acetate, and add 100 cm3 of water.
3, 1N HCl 100cm3 (twice), water 100cm3
cm3, 5% sodium hydrogen carbonate 100cm3 (2
The ethyl acetate layer was washed with 100 cm3 of water and dried by adding anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain 1.29 g (1.56 mm) of white powder.
ol, yield 78%) was obtained.

(3) Boc-Asp(OcHex)Ph
Synthesis of gArg(Tos)MeGly-OBzl Boc-PhgArg(Tos)MeGly-OBzl
1.2g (1.4mmol) of TFA 30cm3
After stirring at room temperature for 30 minutes, TFA was distilled off under reduced pressure. When 200 cm3 of ether was added thereto, white crystals were generated, which were filtered, washed with ether, and the ether was removed under reduced pressure. This was dissolved in 30 cm3 of DMF, and the pH was adjusted to 6 with N-methylmorpholine under ice cooling. BocAsp (OcHeX) 0.46g, HOB
t and 0.28 g of WSC/HCl were added in this order, and the mixture was adjusted to pH 6 with N-methylmorpholine and stirred overnight.

After distilling off DMF under reduced pressure, ethyl acetate 1
Add 00cm3 of water to dissolve, and dissolve the ethyl acetate layer in 100cm3 of water.
cm3, 1N HCl 100cm3 (2 times), water 1
00cm3, 5% sodium bicarbonate 100cm3
The ethyl acetate layer was washed with 100 cm3 of water (twice) and dried by adding anhydrous sodium sulfate. After distilling off ethyl acetate under reduced pressure, 1.0 g (1.0 mmol) of white powder was obtained.
, yield 70%).

(4) Boc-Asp(OcHex)Ph
Synthesis of gArg(Tos)MeGly-OHBoc-Asp(OcHex)PhgArg(Tos)
Dissolve 1.0 g (1.0 mmol) of MeGly-OBzl in 50 cm3 of methanol, add 1 cm3 of acetic acid,
% Pd-C was added and hydrogen was bubbled through. After filtration, the filtrate was evaporated under reduced pressure, and ether was added to the residue to solidify it, which was filtered and dried. Yield 0.79g (0.85
mmol, yield 85%).

(5) Boc-Asp(OcHex)Ph
Synthesis of gArg(Tos)MeGly-ONSu Boc-Asp(OcHex)PhgArg(Tos)
MeGly-OH 0.79g (0.85mmol)
was dissolved in DMF 15cm3, and HOSu 0.1g
, 0.17 g of WSC·HCl was added, and the mixture was stirred overnight. After distilling off DMF under reduced pressure, the residue was diluted with ethyl acetate.
The ethyl acetate layer was dissolved in 100 cm3 of water,
It was washed with 100 cm3 of 1N HCl and 100 cm3 of water in that order, and dried by adding anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain 0.71 g (0.6 g) of white powder.
8 mmol, yield 80%).

(6) Cyclo[-Asp(OcHex)P
Synthesis of hgArg(Tos)MeGly-]Boc-Asp(OcHex)PhgArg(Tos)
MeGly-ONSu0.71g (0.68mmol)
was dissolved in 10 cm3 of TFA and stirred at room temperature for 20 minutes. After TFA was distilled off under reduced pressure, the residue was solidified with ether and collected by filtration. After drying, dissolve in 10 cm3 of DMF,
The mixture was added dropwise over 30 minutes to 1500 cm3 of pyridine heated to 0.degree. C., and stirred at 55.degree. C. for 8 hours and at room temperature overnight. After distilling off the pyridine under reduced pressure, wash thoroughly with water and dry to obtain the desired product.
．． 33 g (0.41 mmol, yield 60%) was obtained.

(7) Cyclo[-Asp-Phg-Arg
-MeGly-] Synthesis of cyclo[-Asp(OcHex)PhgArg(Tos
)MeGly-] To 0.33 g (0.41 mmol) were added 1 cm3 of anisole and 0.3 cm3 of tricresol, and then 20 cm3 of HF was added and reacted at 0°C for 1 hour. After HF was distilled off under reduced pressure, ether was added to solidify, and the precipitate was washed with ether. This was dissolved in acetic acid and lyophilized to obtain 0.16 g of crude peptide. This was subjected to high performance liquid chromatography (HPLC) using an ODS column to obtain 0.05 g of the target product.

[Example 2] Enzymatic Decomposition Experiment of Cyclic Peptide In order to confirm that the cyclic peptide synthesized in Example 1 is not susceptible to enzymatic decomposition, it was decomposed with trypsin.

[0037] 10mg/cm3 of peptide in distilled water
Buffer (Tr
is-HCl 100mM pH8.5) 0.18cm
3 was added and diluted. Add trypsin solution (CaCl
Trypsin in 5M HCl containing 2 10mM
0.005 cm3 (containing mg/cm3) was added and kept at 37°C for 24 hours. The reaction was terminated by adding 0.01 cm3 of 2N HCl. Of this, 0.05 cm3 was subjected to HPLC for analysis.

Analysis conditions HPLC: Waters Model 510 Column: Waters μ Bonder Sphere Column Detection: A214 Elution: Solution A 0.1% TFA aqueous solution Solution B
0.1% TFA-acetonitrile solution 0%B → 60
%B linear concentration gradient (0 min) (30 min)

The results of the above test are shown in FIG. Figure 1 shows the HPL of the cyclic peptide synthesized in Example 1 (control solid line) and that treated with trypsin for 24 hours (dashed line).
It is a graph showing a C analysis result. As shown in FIG. 1, it was confirmed that the cyclic peptide synthesized in Example 1 was extremely difficult to be degraded by trypsin.

[Example 3] Platelet aggregation inhibitory activity test The in vitro platelet aggregation inhibitory activity of the test drug was assayed using human platelet-rich plasma. 1/ to the collected human blood
Add 9 volumes of 3.8% sodium citrate and centrifuge (1
000 rpm for 10 minutes), and the upper layer was collected as platelet-rich plasma (PRP). Add 0.025 cm3 of test drug to 0.2 cm3 of PRP, incubate at 37°C for 3 minutes, and then add 0.02 to 0.05M ADP (adenosine 5'-diphosphate) solution or epinephrine solution, or 0.05 to 0.025 cm3 of 0.2 μg/cm3 collagen solution or PFA (platelet activating factor) solution was added, and the state of aggregation was measured using an aggregometer. The aggregation inhibition rate is calculated using the following formula,
The results are shown in Table 1. Peptide C in Table 1 represents the cyclic peptide synthesized in Example 1.

Aggregation inhibition rate: (1-T/T0)×10
0 %T0: Transmittance when no test drug is added T: Transmittance when test drug is added

Furthermore, as a comparative example, Peptide A (linear peptide) and Peptide B (cyclic peptide) shown below were synthesized and similar tests were conducted, and the results are also shown in Table 1. Peptide A: Gly-Arg-Gly-Asp-Ser
-Pro-Ala Peptide B: cyclo[-Arg-G
ly-Asp-Phg-] Peptide C: cyclo[-A
rg-MeGly-Asp-Phg-]

[0043]

[Table 1]

[0044]

Effects of the Invention As shown in Example 2, the cyclic peptide of the present invention is resistant to decomposition by hydrolytic enzymes and is thought to be able to maintain its activity in vivo for a long period of time. Moreover, as shown in Example 3, the cyclic peptide of the present invention has extremely high activity, and is more than 10 times more active than the comparative cyclic peptide.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the analysis results of Example 2.

Claims (10)

[Claims] 1. A peptide derivative comprising a synthetic cyclic peptide having an amino acid sequence portion represented by the following formula (1), or a salt thereof. -Arg-MeGly-Asp-...(1)
However, -MeGly- represents an N-methylglycine residue. 2. A peptide derivative comprising a synthetic cyclic peptide represented by the following formula (2) or a salt thereof. cyclo[-Arg-MeGly-Asp-(R1)m-
] ...(2) However, cyclo[-Arg-
MeGly-Asp-(R1)m-] indicates that both ends of the linear peptide in [] are connected by a peptide bond to form a cyclic peptide, and -MeGly- is N-
methylglycine residue, R1 is an amino acid residue or oligopeptide to polypeptide residue, m is 0 or 1
shows. 3. The peptide derivative according to claim 2, wherein R1 is an oligopeptide to polypeptide residue having 16 or less amino acid residues. 4. A peptide derivative comprising a synthetic cyclic peptide having an amino acid sequence portion represented by the following formula (3), or a salt thereof. -Arg-MeGly-Asp-Phg-...
-(3) However, -MeGly- represents an N-methylglycine residue, and -Phg- represents a phenylglycine residue. 5. A peptide derivative comprising a synthetic cyclic peptide represented by the following formula (4) or a salt thereof. Cyclo[-Arg-MeGly-Asp-Phg-(R
2) n- ] ... (4) However, cyclo[-Arg-MeGly-Asp-Ph
g-(R2)n-] indicates that both ends of the linear peptide in [] are connected by a peptide bond to form a cyclic peptide, and -MeGly- represents an N-methylglycine residue, - Phg- represents a phenylglycine residue, R2 represents an amino acid residue or an oligopeptide to polypeptide residue, and n represents 0 or 1. 6. The peptide derivative according to claim 5, wherein R2 is an oligopeptide to polypeptide residue having 15 or less amino acid residues. 7. A drug for inhibiting adhesion of animal cells, which contains the peptide derivative of claim 1, 2, 4, or 5 as an active ingredient. 8. The drug according to claim 7, wherein the animal cell is a mammalian somatic cell. 9. The drug according to claim 7, wherein the animal cell is a cancer cell. 10. A drug that inhibits platelet aggregation, which contains the peptide derivative of claim 1, 2, 4, or 5 as an active ingredient.