JPH04297495A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To provide a peptide derivative containing an octapeptide unit of formula Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr and useful as a adhesion-inhibiting agent for animal cell and platelet coagulation and adhesion inhibitor to suppress the metastasis of cancer. CONSTITUTION:The objective peptide derivative of formula (X and Y are amino acid or none; W is none or CO; Z is O or NH; R<1> and R<2> are H, 8-24C alkyl, etc.) is produced by bonding amino acid having protected alpha-amino group and side-chain functional group according to the amino acid sequence of the objective peptide by a DCC-additive method using l-hydroxybenzotriazole in combination with dicyclohexylcarbodiimide, removing the alpha-amino protecting group, repeating the above steps to synthesize a protected peptide containing the octapeptide unit of formula Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, reacting an alcohol such as 1-hexadecanol to the peptide in the presence of a condensation agent and finally removing all protection groups.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides an octapeptide unit of glutamic acid-isoleucine-leucine-aspartic acid-valine-proline-serine-threonine.
Peptide derivatives or their salts that are optimal for forming molecular aggregates such as liposomes or micelles, and animal cell adhesion inhibitors and platelet aggregation inhibitors containing these as active ingredients.
Relating to adhesion inhibitors.

0002

BACKGROUND OF THE INVENTION Fibronectin is a protein involved in cell-extracellular matrix adhesion, and is also thought to be involved in platelet aggregation and cancer metastasis. These interactions are mediated by a series of cell surface receptors, and although fibronectin is a large molecule with a molecular weight of approximately 250,000, these receptors
(abbreviated as )) has been shown to specifically recognize sequences, and has been reported to be important for interaction with receptors (Nature, 309, 30 (1)
984)). Since then, research has been conducted using oligos or polypeptides having the Arg-Gly-Asp sequence.For example, methods for inhibiting platelet aggregation using various linear and cyclic oligopeptides having the Arg-Gly-Asp sequence. (Proceedings of the Society of Polymer Science and Technology)
er Preprints, Japan), 38,
3149 (1989), Japanese Patent Application Publication No. 2-174797)
, a method using a peptide having an Arg-Gly-Asp sequence as a cell migration inhibitor (Japanese Patent Application Laid-Open No. 2-4716
PM with immobilized Arg-Gly-Asp
Method of using MA membrane as a cell adhesion membrane (Proceedings of the Society of Polymer Science and Technology (Polymer Preprints, Jap
an), 37, 705 (1988)). Furthermore, a method in which a peptide having Arg-Gly-Asp as an essential constituent unit is covalently bonded to a polymer and used as a substrate for animal cell culture and a substrate for biocomposite artificial organs (JP-A-1-309682, JP-A-1-305960)
, a method of using a polypeptide having the Arg-Gly-Asp-Ser sequence as a platelet protecting agent for extracorporeal blood has been disclosed (Japanese Patent Laid-Open No. 64-6217). Furthermore, a method of suppressing cancer metastasis using an oligopeptide having an Arg-Gly-Asp sequence or a polypeptide having a repeating structure thereof is known (Int
．． J. Biol. Macromol. , 11
, 23, (1989), same magazine, 11, 226 (1086
), Jpn. J. Cancer Res. , 6
0, 722 (1989)).

On the other hand, recently, A
It has also been revealed that there are cell adhesion sequences other than the rg-Gly-Asp sequence, one of which is III CS.
(type III homology connection
The CS1 peptide (containing the glutamic acid-isoleucine-leucine-aspartic acid-valine-proline-serine-threonine sequence) present in the ng segment) region has been attracting attention (J. Biol. Chem
．． , 262, 6886 (1987)). This peptide, like the Arg-Gly-Asp peptide, is recognized by the fibronectin receptor and is thought to contribute to the adhesion specificity of fibronectin. It has now been revealed that the minimum unit of adhesive activity is an octapeptide having the sequence glutamic acid-isoleucine-leucine-aspartic acid-valine-proline-serine-threonine (hereinafter abbreviated as EILDVPST) (J. Cell Biol., 107, 2
189 (1988)).

[0004] On the other hand, there are no oligopeptides having the EILDVPST sequence or peptide derivatives having repeating structures thereof, which are optimal for forming molecular assemblies such as liposomes or micelles, and these compounds are difficult to bind to receptors. It is expected that this drug will enhance the drug's ability and stabilize it in the blood.

[0005]

[Problems to be Solved by the Invention] The purpose of the present invention is to
An object of the present invention is to provide a peptide derivative having an octapeptide unit of LDVPST and a method for synthesizing the same. Another object of the present invention is to provide an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing this as an active ingredient.

[0006]

[Means for Solving the Problems] The compound of the present invention is a peptide derivative defined by the following general formula [I], and the ionic group present in the molecule may form a salt with an appropriate ion. . R1-W-([X]-Glu-Ile-Leu-
Asp-Val-Pro-Ser-Thr-[Y]
) n-Z-R2 [I] In the formula, Glu, Ile
, Leu , Asp , Val , Pro , Ser
, Thr are glutamic acid, isoleucine, and
Represents leucine, aspartic acid, valine, proline, serine, and threonine residues. [X] and [Y] represent amino acid residues that are present or absent. When present, [X] and [Y] are preferably amino acid residues selected from serine, glycine, valine, asparagine, proline, cysteine, and threonine residues. In particular, it is preferable that [X] is a glycine residue. Also,
It is also particularly preferable that both [X] and [Y] are absent. n represents an integer from 1 to 5, with integers from 1 to 3 being particularly preferred. W is absent or -CO-
represents a group. Z represents an -O- group or an -NH- group. R1
and R2 represents hydrogen or a linear or branched alkyl group having 8 to 24 carbon atoms, and may have a substituent or an unsaturated group. The preferred number of carbon atoms in the alkyl group is 12 to 18. For example, preferred examples include myristoyl group, stearyl group, and 3,7,11,15-tetramethylhexadecyl group.

In the present invention, the amino acid residues are L-, D-
Although it may be either the - or racemic form, the L-form is preferred. Furthermore, the asymmetric carbon present in the molecule may be either a racemic form or an optically active form. Examples of preferred salts of the compounds of the invention include sodium, potassium, ammonium, magnesium, hydrochloride, sulfate, nitrate, acetate.

Examples of preferred compounds of the present invention are listed below, but the present invention is not limited thereto. Compound Example Compound 1 (SEQ ID NO: 1) H-(Glu-Ile-Leu-Asp-Val-Pr
o-Ser-Thr)-O-C16H33 Compound 2
(SEQ ID NO: 1) H-(Glu-Ile-Leu-Asp-Val-Pr
o-Ser-Thr)-O-C20H41 compound 3
(SEQ ID NO: 1) H-(Glu-Ile-Leu-Asp-Val-Pr
o-Ser-Thr)-O-R3R3=CH2CH2C
H(CH3)(CH2)3CH(CH3)(CH2)3
CH(CH3)(CH2)3CH(CH3)2 Compound 4 (SEQ ID NO: 2) H-(Glu-Ile-Leu-Asp-Val-Pr
o-Ser-Thr)2-O-C14H29 Compound 5
(SEQ ID NO: 1) H-(Glu-Ile-Leu-Asp-Val-Pr
o-Ser-Thr)-NH-C18H37 compound 6
(SEQ ID NO: 1) H-(Glu-Ile-Leu-Asp-Val-Pr
o-Ser-Thr)-NH-C14H29 Compound 7
(SEQ ID NO: 3) C16H33-(Glu-Ile-Leu-Asp-V
al-Pro-Ser-Thr)-OH compound 8 (
SEQ ID NO: 3) C14H29-(Glu-Ile-Leu-Asp-V
al-Pro-Ser-Thr)-OH compound 9 (
SEQ ID NO: 3) R4-CO-(Glu-Ile-Leu-Asp-Va
l-Pro-Ser-Thr)-OHR4=CH2CH
(CH3)(CH2)3CH(CH3)(CH2)3C
H(CH3)(CH2)3CH(CH3)2 Compound 10 (SEQ ID NO: 3) C13H27-CO-(Glu-Ile-Leu-As
p-Val-Pro-Ser-Thr)-OH compound 1
1 (SEQ ID NO: 4) R4-CO-(Glu-Ile-Leu-Asp-Va
l-Pro-Ser-Thr)-O-R3R3=CH2
CH2CH(CH3)(CH2)3CH(CH3)(C
H2)3CH(CH3)(CH2)3CH(CH3)2 R4=CH2CH(CH3)(CH2)3CH(CH3
)(CH2)3CH(CH3)(CH2)3CH(CH
3)2 The compound of the present invention is expected to enhance binding ability with receptors and stabilize in blood, and utilizes the ability of the EILDVPST site to bind to fibronectin receptors present on the surfaces of cancer cells, platelets, lymphocytes, etc. It can be used for the purposes of inhibiting cancer metastasis, inhibiting platelet aggregation, and activating lymphocytes.

Next, the method for synthesizing the compound of the present invention will be explained. The compound of the present invention is synthesized in the following three steps. ■ Sequential extension of protected amino acids ■ Introduction of an alkyl group to the N- or C-terminus of the protected peptide ■ Deprotection and purification Each step will be explained in detail below. ■ The method of sequentially elongating protected amino acids is a known method, i.e., "Basics and Experiments of Peptide Synthesis" by Izumiya et al.
Maruzen) and “PRINCIPLE” by Bodanszky
S OF PEPTIDE SYNTHESIS”,”
THE PRACTICE OF PEPTIDES
YNTHESIS” (Springer Verla
All methods described in J. G., New York) are effective. In the condensation reaction stage, DCC-add
Although any of the itive method, azide method, mixed acid anhydride method, and active ester method may be employed, the DCC-additive method using a combination of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide gives the best results. ■ In general formula [I], in order to introduce R1 into the protected peptide, if W is not present, the amino group at the N-terminus of the protected peptide may be released and reacted with an alkyl halide in the presence of a base; -CO- group, D
The condensation reaction may be carried out by a CC-additive method, a CDI method, or the like. To introduce R2 into the protected peptide,
When Z is -O- group, use a long chain alcohol, and when Z is -NH
In the case of a - group, a long-chain amine may be used to carry out a condensation reaction with a protected peptide whose C-terminus has been released, by a DCC-additive method, a CDI method, or the like. ■ The conditions used to deprotect a protecting group are highly dependent on the type of protecting group used. Commonly used deprotection conditions include hydrolysis,
Examples include trifluoroacetic acid, anhydrous hydrogen fluoride, trifluoromethanesulfonic acid-thioanisole mixed system, trifluoroacetic acid-thioanisole mixed system, etc., but more various means are possible depending on the type of protecting group. In addition, purification of the target product is performed by using silica gel chromatography, ion exchange chromatography, gel filtration, and the like.

[0010] To prepare a preparation by dispersing the compound of the present invention, it may be carried out either by dispersing it alone in an aqueous medium or by using a dispersion aid in combination. Dispersion aids include natural or synthetic lipids such as egg yolk lecithin, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiolipin, or Triton X-100. , polyethylene glycol, benzyl alcohol, gelatin, and other pharmaceutical additives approved as dispersion aids, depending on the compound. Regarding these pharmaceutical excipients, please refer to the Pharmacia Review N published by the Pharmaceutical Society of Japan in 1987.
o. It is described in detail in 22 “Pharmaceutical Excipients”.
It is preferable to select from among these.

The amount of the compound represented by the general formula [I] in the preparation of the molecular assembly according to the present invention is not particularly limited, but is preferably 0.1 to 9.0 (1) to 1 (1) of the dispersion aid. weight ratio). Additionally, additives such as sterols (for example, cholesterol, β-sitosterol, stigmasterol, carbenseterol, etc.) may be mixed.

[0012] As a method for forming a molecular assembly by mixing the compound represented by the general formula [I] and a phospholipid, a conventional liposome formation method, that is, a vortexing method (J.
Mol. Biol. , 13, 238 (1965)
), sonication method (Biochem., 8,
344 (1969)), prevesicle method (Neuros
ci. Res. Prog. Bull. , 9 ,
273 (1971)), ethanol injection method (Bioch
em. Biophys. Acta, 298, 10
15 (1973)), French press method (FEBS.
Lett. , 99, 210 (1973)), cholic acid removal method (J. Biol. Chem., 246,
5477 (1971)), Triton X-100 batch method (Eur. J. Biochem., 85, 25
5 (1978)), Ca2+ fusion method (Biochem.
Biophys. Acta, 394, 483 (
1975)), ether injection method (Biochem. B)
iophys. Acta, 443, 629 (19
76)), annealing method (Biochem. Bio
phys. Acta, 443, 313 (1976)
), freeze-thaw fusion method (J. Biol. Chem.,
252, 7384 (1977)), W/O/W emulsion method (J. Colloid Interfa
ce Sci. , 62, 149 (1977)), reversed-phase evaporation method (Pro. Natl. Acad. Sci.
．． USA, 75, 4194 (1978)), but the present invention may use any of the above preparation methods, and is not limited thereto.

[0013] The peptide derivative used in the present invention can be administered by the administration method generally used for peptide drugs, that is, by parenteral administration, such as intravenous administration, intramuscular administration, subcutaneous administration, etc. preferable. When producing such an injectable preparation, the peptide derivative of the present invention may be mixed with PBS (Na
H2PO4 5mM, NaCl 70mM) or physiological saline to form an injection preparation, or after dispersing in approximately 0.1N acetic acid water or the like, a lyophilized preparation may be prepared. At this time, the above-mentioned dispersion aids may be used. Conventional stabilizers such as glycine and albumin may be added to such preparations, and collagen, liposomes, and the like may be used as carriers for purposes such as prolonging the blood half-life.

Furthermore, the peptide derivative of the present invention can be administered orally if it is made into a microcapsule encapsulated in a liposome, or if it is made into a suppository, sublingual, nasal spray, etc. It is also possible to absorb it through mucous membranes from areas other than the gastrointestinal tract. The peptide derivative of the present invention comprises the core sequence of cell adhesion protein (EILDVP).
ST) and adheres to cells via this core sequence in a similar mechanism to cell adhesion proteins. Therefore, they exhibit various biological activities as agonists or antagonists of cell adhesion proteins. In addition, a wide range of biological activities are recognized, including immunomodulatory effects, wound healing effects, suppressive effects on platelet aggregation by cancer cells that occur in capillaries, and neurological disease healing effects.

[0015] Therefore, the peptide derivative of the present invention
At least one of them can be administered to a patient as a cancer metastasis inhibitor, wound healing agent, immunosuppressant, platelet aggregation inhibitor, or neurological disease therapeutic agent, optionally together with a conventional carrier or pharmaceutical preparation. In particular, use as an animal cell adhesion inhibitor or platelet aggregation/adhesion inhibitor is preferred. The dosage is 0.2 μg/kg to 400 mg/k
The weight range is determined based on symptoms, age, weight, etc.

Synthesis examples of the compounds of the present invention are shown below.
The present invention is not limited to these. Note that amino acids, various protecting groups, and deprotecting reagents are expressed using commonly used abbreviations. Other examples of compounds can also be synthesized by the methods exemplified here.

[0017]

EXAMPLES Example 1 A synthesis example of Compound 1 of the present invention is shown below. Compounds 2 to 6 can also be synthesized by the methods exemplified here. Synthesis of protected peptide 1) BocSer(Bzl)Thr(Bzl)OBz
Synthesis of BocThr (Bzl) (purchased from Kokusan Kagaku Co., Ltd.) (15.5 g, 50 mmol), diisopropylethylamine (6.46 g), p-nitrobenzyl bromide (10.8 g), ethyl acetate (200 g)
ml) was heated under reflux for 3 hours. After the reaction solution was allowed to cool to room temperature, it was washed with 200 ml each of 1N aqueous sodium bicarbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a colorless oil, BocThr(Bzl)OBz.
l(NO2) was obtained quantitatively. Add chloroform (1
00 ml) and trifluoroacetic acid (50 ml) were added, and the mixture was reacted at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, ethyl acetate (250 ml) was added, and the mixture was washed with 200 ml each of a 1N aqueous sodium bicarbonate solution and 2 saturated brine, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to give a colorless oil, Thr(Bzl)OB.
zl(NO2) was obtained quantitatively. BocSer for this
(Bzl) (purchased from Kokusan Kagaku Co., Ltd.) (14.8g,
50 mmol), DCC (11.4 g, 55 mmol)
, HOBt (6.9 g, 45 mmol), DMF (15
0 ml) was added and reacted at 0°C for 30 minutes and at room temperature for 24 hours. After removing DCUrea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and the mixture was washed with 200 ml each of 1N aqueous sodium bicarbonate solution and saturated brine, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and purified by silica gel chromatography (eluent: hexane/ethyl acetate 40:1) to obtain BocSer(Bzl)Thr(Bzl).
OBzl(NO2) was obtained as a colorless oil (28.3g). 2) BocProSer(Bzl)Thr(Bzl)
Synthesis of OBzl(NO2) BocSer(Bzl)Th
r(Bzl)OBzl(NO2) (28.0g, 45m
mol), BocPro (purchased from Kokusan Kagaku Co., Ltd.) (
9.69g, 45mmol), DCC (10.3g, 5
0 mmol), HOBt (6.1 g, 40 mmol),
DMF (150 ml) was added and reacted at 0°C for 30 minutes and at room temperature for 24 hours. After removing DCUrea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and 1N
It was washed with 200 ml each of aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by silica gel chromatography (eluent: chloroform/methanol 99:1).
oSer(Bzl)Thr(Bzl)OBzl(NO2
) was obtained as a colorless oil (29.8 g). 3) BocValProSer(Bzl)Thr(B
zl) Synthesis of OBzl(NO2) BocProSer
(Bzl) Thr (Bzl) OBzl (NO2) (28
．． 8g, 40mmol), BocVal (purchased from Kokusan Kagaku Co., Ltd.) (8.7g, 40mmol), DCC (9
．． 3g, 45mmol), HOBt (5.4g, 35mmol)
mol), add DMF (150 ml), and add BocPro
Ser(Bzl)Thr(Bzl)OBzl(NO2)
Synthesis was carried out in the same manner as for . BocValProSer(
Bzl)Thr(Bzl)OBzl(NO2) was obtained as a colorless oil (29.4g). 4) BocAsp(OBzl)ValProSer(
Bzl) Thr (Bzl) Synthesis of OBzl (NO2) BocValProSer (Bzl) Thr (Bzl)
OBzl(NO2) (28.6g, 35mmol), B
ocAsp (OBzl) (purchased from Kokusan Kagaku Co., Ltd.) (
11.3g, 35mmol), DCC (8.3g, 40
mmol), HOBt (4.6 g, 30 mmol), D
Add MF (150 ml) and add BocProSer (Bz
l) The same procedure as the synthesis of Thr(Bzl)OBzl(NO2) was performed. BocAsp (OBzl) ValProS
er(Bzl)Thr(Bzl)OBzl(NO2)
was obtained as a colorless oil (33.7g). 5) BocLeuAsp(OBzl) ValProS
er(Bzl)Thr(Bzl)OBzl(NO2)
Synthesis of BocAsp (OBzl) ValProSer (Bzl
)Thr(Bzl)OBzl(NO2)(32.7g,
32 mmol), BocLeu (purchased from Kokusan Kagaku Co., Ltd.) (7.4 g, 32 mmol), DCC (7.2 g,
35 mmol), HOBt (4.6 g, 30 mmol)
, DMF (150 ml) was added, and BocProSer (
Synthesis of Bzl)Thr(Bzl)OBzl(NO2) was performed in the same manner. BocLeuAsp(OBzl)Va
lProSer(Bzl)Thr(Bzl)OBzl(
NO2) was obtained as a colorless oil (33.5 g). 6) BocIleLeuAsp(OBzl)ValP
roSer(Bzl)Thr(Bzl)OBzl(NO
2) Synthesis of BocLeuAsp(OBzl)ValProSer(
Bzl)Thr(Bzl)OBzl(NO2)(33.
0g, 29mmol), BocIle (Kokusan Kagaku Co., Ltd.)
) (6.7 g, 29 mmol), DCC (6.7 g, 29 mmol),
8g, 33mmol), HOBt (3.8g, 25mm
ol), add DMF (150 ml), and add BocProS
er(Bzl)Thr(Bzl)OBzl(NO2)
Synthesis was carried out in the same manner as for . BocIleLeuAsp(O
Bzl) ValProSer(Bzl) Thr(Bzl
) OBzl(NO2) was obtained as a colorless oil (34.1 g). 7) BocGlu(OBzl)IleLeuAsp(
OBzl) ValProSer(Bzl) Thr(Bz
l) Synthesis of OBzl(NO2) BocIleLeuAsp(OBzl) ValProS
er (Bzl) Thr (Bzl) OBzl (NO2) (
33.7g, 27mmol), BocGlu (OBzl
) (purchased from Kokusan Kagaku Co., Ltd.) (9.1g, 27mmo
l), DCC (6.2 g, 30 mmol), HOBt (
Add BocProSer(Bzl)Thr(Bzl)O
Synthesis of Bzl(NO2) was carried out in the same manner. BocGl
u(OBzl)IleLeuAsp(OBzl)Val
ProSer(Bzl)Thr(Bzl)OBzl(N
O2) was obtained as a colorless oil (34.1 g). 8) BocGlu(OBzl)IleLeuAsp(
OBzl) ValProSer(Bzl) Thr(Bz
l) Synthesis of BocGlu(OBzl)IleLeuAsp(OBz
l) ValProSer(Bzl)Thr(Bzl)O
Bzl(NO2) (1.47g, 1mmol) at 90%
Dissolved in acetic acid (40 ml), added zinc powder (0.32 g, 5 m
mol) was added thereto, and the mixture was stirred at 0° C. for 2 hours and at room temperature for 1 hour. The remaining zinc was filtered off, and the filtrate was concentrated under reduced pressure, made acidic by adding citric acid, and extracted with ethyl acetate. After drying with sodium sulfate, it was concentrated under reduced pressure and subjected to silica gel chromatography (eluent: chloroform/methanol 99%).
:1) and purified by BocGlu(OBzl)Ile
LeuAsp(OBzl) ValProSer(Bzl
) Thr(Bzl) was obtained as a colorless oil (0.97 g).

Introduction of alkyl group into protected peptide 9)
BocGlu(OBzl)IleLeuAsp(OBz
l) ValProSer(Bzl)Thr(Bzl)-
Synthesis of O-C16H33 BocGlu(OBzl)IleLeuAsp(OBz
l) ValProSer(Bzl)Thr(Bzl)(
0.93g, 0.7mmol) 1-hexadecanol (
0.17g, 0.7mmol), DCC (0.17g,
0.8 mmol) and chloroform (50 ml) were added.
The reaction was carried out at 0°C for 30 minutes and at room temperature for 24 hours. DCUr
After removing ea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and the mixture was washed with 200 ml each of 1N aqueous sodium bicarbonate solution and saturated brine, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and purified by silica gel chromatography (eluent: chloroform/methanol 99:1).
BocGlu(OBzl)IleLeuAsp(OBz
l) ValProSer(Bzl)Thr(Bzl)-
O-C16H33 was obtained as a white powder (0.98g).

Deprotection and Purification 10) H-(GluIleLeuAspValPro
Synthesis of SerThr)-O-C16H33 BocG
lu(OBzl)IleLeuAsp(OBzl)Va
lProSer(Bzl)Thr(Bzl)-O-C1
6H33 (0.98 g, 0.63 mmol), chloroform (20 ml), and trifluoroacetic acid (20 ml) were added, and the mixture was reacted at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, chloroform (50 ml) was added, washed with 50 ml each of a 1N aqueous sodium bicarbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain white powder H-Glu (OBzl
)IleLeuAsp(OBzl)ValProSer
(Bzl)Thr(Bzl)-O-C16H33 was quantitatively obtained. Dissolve this in acetic acid (50 ml), add 1 g of 10% palladium on carbon, and perform hydrogenolysis at room temperature under normal pressure for 24 hours.
Time went. The catalyst was filtered off using Celite, and the solvent was distilled off under reduced pressure. Silica gel chromatography (eluent
Chloroform/methanol/water 65:25:4)
610 mg of Compound 1 was obtained.

[0020] FAB-MS 1096 (M-
H) Amino acid analysis: Glu (0.97), Ile (1
．． 19), Leu (1.13), Asp (0.98
), Val (0.89), Pro (0.94),
Ser (0.92), Thr (0.91) Example 2 A synthesis example of Compound 2 is shown below.

Compound 2 was prepared according to the method described in Example 1.
was synthesized. Synthesis was performed by changing 1-hexadecanol to 1-eicosanol. FAB-MS 1152 (MH) Amino acid analysis: Glu (1.07), Ile (1.05),
Leu (1.09), Asp (0.92), Val
(0.97), Pro (0.91), Ser (0.
86), Thr (0.82) Example 3 A synthesis example of Compound 3 is shown below.

Compound 3 was prepared according to the method described in Example 1.
was synthesized. 1-hexadecanol to 3,7,11,1
It was synthesized by changing to 5-tetramethylhexadecane-1-ol. FAB-MS 1152 (MH) Amino acid analysis: Glu (1.01), Ile (1.00),
Leu (0.99), Asp (1.04), Val
(1.11), Pro (0.90), Ser (0.
82), Thr (0.81) Example 4 A synthesis example of Compound 4 is shown below.

Compound 4 was prepared according to the method described in Example 1.
was synthesized. The protected peptide was synthesized by the sequential extension method as in Example 1. 1-hexadecanol to 1-
It was synthesized by changing to tetradecanol. FAB-MS 1923 (MH) Amino acid analysis: Glu (1.10), Ile (1.14),
Leu (1.10), Asp (1.02), Val
(1.09), Pro (0.94), Ser (0.
79), Thr (0.81) Example 5 A synthesis example of Compound 5 of the present invention is shown below.

Compound 5 was prepared according to the method described in Example 1.
was synthesized. Synthesis was performed by changing 1-hexadecanol to 1-octadecylamine. FAB-MS 1123 (MH) Amino acid analysis: Glu (1.07), Ile (1.00),
Leu (1.05), Asp (0.94), Val
(1.12), Pro (0.89), Ser (0.
78), Thr (0.83) Example 6 A synthesis example of Compound 6 of the present invention is shown below.

Compound 6 was prepared according to the method described in Example 1.
was synthesized. Synthesis was performed by changing 1-hexadecanol to 1-tetradecylamine. FAB-MS 1067 (MH) Amino acid analysis: Glu (0.99), Ile (1.04),
Leu (1.12), Asp (0.99), Val
(1.02), Pro (0.94), Ser (0.
85), Thr (0.77) Example 7 A synthesis example of Compound 7 of the present invention is shown below. Compound 8 can also be synthesized by the method exemplified here.

Compound 7 was prepared according to the method described in Example 1.
was synthesized. Protected peptide BocGlu (OBzl)I
leLeuAsp(OBzl)ValProSer(B
The order of deprotection of zl)Thr(Bzl)OBzl(NO2) was changed, first removing the Boc group with trifluoroacetic acid. Next, the protected peptide was heated under reflux in chloroform in the presence of 1-bromohexadecane and triethylamine to introduce an alkyl group into the protected peptide. Subsequently, the benzyl group and the nitrobenzyl group were removed by hydrolysis to synthesize Compound 7.

[0027] FAB-MS 1096 (M-
H) Amino acid analysis: Glu (0.97), Ile (1
．． 01), Leu (1.09), Asp (1.06
), Val (1.08), Pro (0.98),
Ser (0.81), Thr (0.83) Example 8 A synthesis example of Compound 8 of the present invention is shown below.

Compound 8 was prepared according to the method described in Example 7.
was synthesized. Synthesis was performed by changing 1-bromohexadecane to 1-bromotetradecane. FAB-MS 1068 (MH) Amino acid analysis: Glu (1.07), Ile (1.09),
Leu (1.04), Asp (1.10), Val
(1.05), Pro (0.94), Ser (0.
80), Thr (0.80) Example 9 A synthesis example of Compound 9 of the present invention is shown below. Compound 10 can also be synthesized by the method exemplified here.

Compound 9 was prepared according to the method described in Example 1.
was synthesized. Protected peptide BocGlu (OBzl)I
leLeuAsp(OBzl)ValProSer(B
The order of deprotection of zl)Thr(Bzl)OBzl(NO2) was changed, first removing the Boc group with trifluoroacetic acid. Next, a condensation reaction was carried out with 3,7,11,15-tetramethylhexadecanoic acid by DCC in a chloroform solvent to introduce an alkyl group into the protected peptide. Subsequently, the benzyl group and the nitrobenzyl group were removed by hydrolysis to synthesize Compound 9.

[0030] FAB-MS 1066 (M-
H) Amino acid analysis: Glu (1.09), Ile (1
．． 11), Leu (1.20), Asp (1.09
), Val (1.04), Pro (1.00),
Ser (0.92), Thr (0.87) Example 10 A synthesis example of compound 10 of the present invention is shown below.

Compound 1 was prepared according to the method described in Example 9.
0 was synthesized. Compound 10 was synthesized by changing 3,7,11,15-tetramethylhexadecanoic acid to tetradecanoic acid. FAB-MS 1082 (MH) Amino acid analysis: Glu (1.05), Ile (1.01),
Leu (1.22), Asp (1.07), Val
(1.09), Pro (1.03), Ser (0.
82), Thr (0.77) Example 11 A synthesis example of Compound 11 of the present invention is shown below.

Compound 1 was prepared according to the method described in Example 3.
1 was synthesized. A condensation reaction of 3,7,11,15-tetramethylhexadecanoic acid was performed on the synthetic intermediate of compound 8 before hydrolysis by DCC in a chloroform solvent to add an alkyl group to the N-terminal side of the protected peptide. Introduced. Subsequently, the benzyl group was removed by hydrolysis to synthesize Compound 11.

[0033] FAB-MS 1447 (M-
H) Amino acid analysis: Glu (1.05), Ile (1
．． 01), Leu (1.15), Asp (1.13
), Val (0.99), Pro (0.98),
Ser (0.91), Thr (0.89) Example 12 A formulation example of Compound 1 of the present invention is shown below. Compounds 2 to 11 can also be formulated by the methods exemplified here.

Compound 1 of the present invention (10 mg) was dissolved in chloroform, and the solvent was distilled off under reduced pressure to form a thin film. After drying at room temperature for 1 hour, add physiological saline (10 ml) and use a Branson ultrasonic homogenizer model 25.
Dispersion was carried out using Type 0 at 20W for 15 minutes. The dispersion was sterilized by filtration using Millex GV manufactured by Millipore to prepare an injectable preparation. This preparation can be used as an adhesion inhibitor for animal cells and a platelet aggregation/adhesion inhibitor. Example 13 A formulation example of Compound 10 of the present invention is shown below. Compounds 1 to 9 and 11 can also be formulated by the methods exemplified here.

Compound 10 of the present invention (5 mg) and dipalmitoylphosphatidylcholine (5 mg) were dissolved in chloroform, and the solvent was distilled off under reduced pressure to form a thin film. After drying for 1 hour at room temperature, add physiological saline (10 ml) and use a Branson ultrasonic homogenizer model 250.
Dispersion was performed in a mold at 20 W for 15 minutes. The dispersion was sterilized by filtration using Millex GV manufactured by Millipore to prepare an injectable preparation. This preparation can be used as an adhesion inhibitor for animal cells and a platelet aggregation/adhesion inhibitor. Example 14 A formulation example of Compound 5 of the present invention is shown below. Compounds 1 to 4 and 6 to 11 can also be formulated by the methods exemplified here.

Compound 5 of the present invention (5 mg), egg yolk lecithin (5 mg), and cholesterol (5 mg) were dissolved in chloroform, and the solvent was distilled off under reduced pressure to form a thin film. After drying at room temperature for 1 hour, add physiological saline (10 ml) and use Branson Ultrasonic Homogenizer Model 25.
Dispersion was carried out using Type 0 at 20W for 15 minutes. The dispersion was sterilized by filtration using Millex GV manufactured by Millipore to prepare an injectable preparation. This preparation can be used as an adhesion inhibitor for animal cells and a platelet aggregation/adhesion inhibitor. Example 15 Test examples of compounds of the present invention (measurement of cell adhesion inhibitory activity) are shown below.

The peptide derivatives of the present invention inhibit cell adhesion to fibronectin. The activity measurement method is basically a competition method widely used in the field of biochemistry, such as JP-A-1-309682 and JP-A-2-
No. 174797, Methods in Enzymo
82, 803 (1981). 1) Preparation of adsorption plate Commercially available human-derived fibronectin (purchased from Cosmo Bio Co., Ltd.) was added at 10 μg/m in PBS (phosphate buffer).
1 of the solution, and 50 μl of the solution was placed in a 96-well polystyrene plate and coated by incubating at 4° C. overnight. Next, bovine serum albumin (BSA 1%) was added for the purpose of preventing non-specific adsorption, and the mixture was kept at 37° C. for 1 hour, followed by a washing operation (PBS) and sufficient water was drained to prepare an adsorption plate. 2) Adhesion inhibition experiment Dulbecco's Modified Eagle
Peptide derivative dispersion liquid 5 dispersed in s Medium
0 μl was placed in the plate prepared by the above method, 50 μl of NRK49F (1×10 6 cells/ml) suspension was added thereto, and the mixture was incubated at 37° C. for 1 hour to allow the cells to adhere. After washing three times with PBS to remove non-adherent cells,
Adhered cells were detached with a 0.025% EDTA trypsin solution, and the number of cells was counted by staining with 0.2% trypan blue. The results are shown in Table 1. In the table, EILDVPST represents an octapeptide of glutamic acid-isoleucine-leucine-aspartic acid-valine-proline-serine-threonine.

[0038]
Table 1 ────────────────
──────────────────
Adhesion rate of cells to fibronectin (
%) Peptide derivative 0
0.25 0.5 1.0
2.0 (mg/ml) ──────────
────────────────────────
EILDVPST 100
70 25 22
19 Compound 1
100 62 3
0 22 12
Compound 2 100
60 26 20
14 Compound 3
100 66 29
23 17 Compound 4 100 61
22 18 11
Compound 5 100
65 29 20
14 Compound 6
100 70 2
3 16 10
Compound 7 100
66 24 16
11 Compound 8
100 69 22
17 13 Compound 9 100 68
29 25 15
Compound 10 100
66 26 20
14 Compound 11
100 70
30 25 17
────────────────────────
────────── Example 16 A test example (measurement of platelet aggregation inhibitory activity) of the compound of the present invention is shown below.

[0039] IN VIT of the peptide derivative of the present invention
The platelet aggregation inhibitory effect in the RO system was assayed using human platelet-rich plasma. Experimental method 1/9 volume of 3.8% sodium citrate was added to fresh human blood, centrifuged (1000 rpm, 10 minutes), and the upper layer was collected as platelet-rich plasma. This plasma 2
00μl to 25μl of peptide derivative (max 1.5m
g/ml) and incubated at 37°C for 3 minutes, then add 25 μM of 20-50 μM ADP (adenosine diphosphate) solution or 200 μl/ml collagen solution.
In addition, the degree of aggregation was assayed by measuring permeability using an aggregometer. The results are shown in Table 2.

Aggregation inhibition rate (%) (1-T/T0)×
100T0: Transmittance T when no peptide derivative is added
: Permeability when adding peptide derivative

[0041]

[Sequence list]

SEQ ID NO: 1 Array length: 8 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: N-terminal fragment array

[0043] SEQ ID NO: 2 Array length: 16 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: N-terminal fragment array

[0044] SEQ ID NO: 3 Array length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: C-terminal fragment array

SEQ ID NO: 4 Array length: 8 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: middle fragment array

Claims (5)

[Claims] Claim 1: A peptide derivative represented by the following general formula [I] or a salt thereof. R1-W-([X]-Glu-Ile-Leu-
Asp-Val-Pro-Ser-Thr-[Y]
)n-Z-R2 [I] In the formula, Glu, Il
e, Leu, Asp, Val, Pro, Se
r and Thr represent glutamic acid, isoleucine, leucine, aspartic acid, valine, proline, serine, and threonine residues, respectively. [X] and [Y] represent amino acid residues that are present or absent. n represents an integer from 1 to 5. W does not exist or -C
Represents an O- group. Z represents an -O- group or an -NH- group. R
1 and R2 represent hydrogen or a linear or branched alkyl group having 8 to 24 carbon atoms, and may have a substituent or an unsaturated group. Claim 2: In general formula [I], [X], [Y
] represents an amino acid residue present, and [X] and [Y] are amino acid residues selected from serine, glycine, valine, asparagine, proline, cysteine, and threonine residues, or That salt. Claim 3: In general formula [I], [X], [Y
] The peptide derivative or its salt according to claim 1, wherein represents an amino acid residue that does not exist. 4. An animal cell adhesion inhibitor comprising the peptide derivative or its salt according to claim 1, 2 or 3 as an active ingredient. 5. A platelet aggregation/adhesion inhibitor comprising the peptide derivative or its salt according to claim 1, 2 or 3 as an active ingredient.