JPH04295497A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE:To provide the subject derivative containing the octapeptide unit of formula Glu-I le-Leu-Asp-Val-Pro-Ser-Thr, having activity to inhibit the adhesion of animal cell and the coagulation and cohesion of platelet and useful for suppressing cancer metastasis. CONSTITUTION:The objective peptide derivative of formula II ((n) is 1-3) or its salt can be produced by reacting a dialkyl (diacyl) glycerol (e.g. 1,2- dihexadecylglycerol) with a cyclic acid anhydride (e.g. succinic anhydride) in an organic solvent capable of dissolving the above components (e.g. methylene chloride) in the presence of a base at room temperature to obtain a dialkyl (diacyl) glycerol derivative of formula I (R<1> and R<2> are H, 8-24C acyl or alkyl;(m)is 1-5), separately preparing a protected peptide by successively bonding protected amino acids according to the amino acid sequence of the objective peptide in the presence of a condensation agent and condensing the above compound with the protected peptide in the presence of a carbodiimide in an organic solvent (e.g. chloroform) at room temperature.

Description

Detailed Description of the Invention [0001] [Industrial Application Field] The present invention provides a glutamic acid-isoleucine-leucine-aspartic acid-valine-proline-serine-threonine octapeptide unit.
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] 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 smallest unit of adhesive activity is an octapeptide having a glutamic acid-isoleucine-leucine-aspartic acid-valine-proline-serine-threonine (hereinafter abbreviated as EILDVPST) sequence (J. Cell Biol., 107, 2
189 (1988)). [0004] On the other hand, peptide derivatives that are optimal for forming molecular aggregates such as liposomes or micelles that have oligopeptides having the EILDVPST sequence or repeating structures thereof are not known, 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] An object of the present invention is to solve the problem of EI
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. [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 forms a salt with an appropriate ion. You can. General formula [I] R1OCH2-CH(OR2)-CH2-OOC-(C
H2) m -CO-([Gly]-Glu-Ile-
Leu-Asp-Val-Pro-Ser-Thr)
n in the formula, Glu, He, Leu, Asp,
Val, Pro, Ser, and Thr represent glutamic acid, isoleucine, leucine, aspartic acid, valine, proline, serine, and threonine residues, respectively. [Gly] represents a glycine residue, present or absent. m represents an integer from 1 to 5, and integers from 1 to 3 are particularly preferred. n represents an integer from 1 to 3, with 1 or 2 being particularly preferred. R1 and R2
is hydrogen or a linear or branched acyl group or alkyl group having 8 to 24 carbon atoms, and may have a substituent or an unsaturated group. The preferred carbon number is from 12 to 18. Preferred examples of the acyl group include myristoyl group, palmitoyl group, and stearoyl group. Alkyl groups include myristyl group, palmityl group,
Preferred examples include a stearyl group and a 3,7,11,15-tetramethylhexadecyl group. R1, R2
may be the same or different. In the present invention, the amino acid residue may be L-, D-, or racemic, but 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 present invention include sodium salts, potassium salts, ammonium salts, magnesium salts, hydrochlorides, sulfates, nitrates, and acetates. Preferred examples of compounds of the present invention are listed below, but the present invention is not limited thereto. [0008] The compound of the present invention is expected to enhance binding ability with receptors and stabilize in blood, and is effective in EILDV.
Utilizing the fact that the PST site can bind to fibronectin receptors present on the surfaces of cancer cells, platelets, lymphocytes, etc., it can be used for the purposes of suppressing cancer metastasis, platelet aggregation, and lymphocyte activation. Next, a method for synthesizing the compound of the present invention will be explained. The compound of the present invention can basically be synthesized by the following method. ■ Synthesis of dialkyl (diacyl)glycerol represented by general formula [II] General formula [II] R1OCH2-CH(OR2)-CH2OH In the formula, R1
and R2 are as defined above. ■ Synthesis of a dialkyl (diacyl)glycerol derivative represented by the general formula [III]. General formula [III] R1OCH2-CH(OR2)-CH2-OOC-(C
H2) m -COOH In the formula, R1, R2 and m are as defined above. ■ Synthesis of protected peptide by sequential elongation of protected amino acids ■ Condensation of protected peptide and dialkyl (diacyl)glycerol derivative ■ Removal and purification of protecting groups Each step will be explained in detail below. ■ The dialkyl (diacyl)glycerol represented by the general formula [II] can be prepared by a known method (for example, Biochem
istry, 2, 394 (1963)), or commercially available products. ■ The synthesis of the dialkyl (diacyl) glycerol derivative represented by the general formula [III] is carried out by stirring dialkyl (diacyl) glycerol and a cyclic acid anhydride or acid dichloride in an organic solvent in which they are dissolved at room temperature in the presence of a base. It can be synthesized by ■ 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. (2) Condensation of a protected peptide and a dialkyl (diacyl) glycerol derivative can be synthesized by stirring the dialkyl (diacyl) glycerol derivative, the protected peptide, and a condensing agent such as dicyclohexylcarbodiimide in an organic solvent in which they are dissolved at room temperature. ■ The conditions used to deprotect a protecting group are highly dependent on the type of protecting group used. Commonly used deprotection conditions include hydrolysis, trifluoroacetic acid, anhydrous hydrogen fluoride, trifluoromethanesulfonic acid-thioanisole mixed system, trifluoroacetic acid-thioanisole mixed system, etc., but depending on the type of protecting group. Even more diverse methods are possible. Further, the target product can be purified by using silica gel chromatography, gel filtration, or the like. [0012] 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). Further, additives such as sterols (for example, cholesterol, β-sitosterol, stigmasterol, carbenseterol, etc.) may be mixed. [0014] 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. [0015] 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, for example, if it is incorporated into a microcapsule in a liposome, or if it is in the form of a suppository, sublingual agent, 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 the core sequence in a similar mechanism to that of 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. [0017] Therefore, the peptide derivative of the present invention is
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. EXAMPLES Example 1 A synthesis example of Compound 1 of the present invention is shown below. Compounds 2 to 7 can also be synthesized by the methods exemplified here. Synthesis of dialkylglycerol C16H33OCH2CH(OC16H33)CH2O
Synthesis, 503, (19
12.0 g of 1-benzylglycerol prepared by the method described in 85)) was dissolved in 300 ml of toluene, 16.0 g of powdered potassium hydroxide and 82 g of 1-bromohexadecane were added to this solution, and the reaction mixture was heated under reflux for 8 hours. After cooling the reaction solution to room temperature, hexane 40
Diluted with 0ml. After washing twice with 200 ml of water, it was 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. This was purified by silica gel chromatography (eluent: hexane/ethyl acetate = 40:1) to obtain 41.2 g of 1,2-dihexadecyl-3-benzylglycerol (
Yield: 95.5%). The physical property values are from the literature (Biochem
isrry, 2, 394 (1963)). The obtained 1,2-dihexadecyl-3-benzylglycerol was dissolved in 250 ml of ethyl acetate.
1.5 g of 10% palladium-carbon was added and the reaction mixture was allowed to react under hydrogen atmosphere for 8 hours. Insoluble materials were removed by filtration through Celite, and the Celite layer was washed with ethyl acetate. The filtrate and washing solution were combined and concentrated under reduced pressure. The residue was recrystallized from ethyl acetate to give dialkylglycerol C16H33O.
CH2CH(OC16H33) CH2OH as colorless crystals (
34.4g). The physical property values are from the literature (Bioche
Misrry, 2, 394 (1963)). Synthesis of dialkylglycerol derivative C1
6H33OCH2CH(OC16H33)CH2OOC
Synthesis of (CH2)2COOH 1,2-dihexadecylglycerol (27g, 50mmol), succinic anhydride (7.5g, 75mmol), diisopropylethylamine (9.7g, 75mmol), dimethylaminopyridine (1g), chloride A mixture of methylene (300ml) was stirred at room temperature for 12 hours. The reaction solution was washed several times with 1N aqueous sodium bicarbonate solution, and the organic layer was dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain dialkylglycerol derivative C16H3.
3OCH2CH(OC16H33)CH2OOC(CH
2) 2COOH was obtained as colorless crystals (32 g). Synthesis of protected peptide BocSer(Bzl)Thr(Bzl)OBzl(N
Synthesis of BocThr (Bzl) (Kokusan Kagaku Co., Ltd.)
A mixture of (15.5 g, 50 mmol) diisopropylethylamine (6.46 g), p-nitrobenzyl bromide (10.8 g), and ethyl acetate (200 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 give a colorless oil, BocThr(Bzl)OBzl(NO
2) was obtained quantitatively. To this, chloroform (100ml
), add trifluoroacetic acid (50 ml) and stir at room temperature for 30 min.
Allowed to react for minutes. After removing the solvent under reduced pressure, ethyl acetate (
250 ml), 1N aqueous sodium hydrogen carbonate solution,
It was washed with two 200 ml portions of 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)OBzl(N
O2) was obtained quantitatively. BocSer (Bzl
) (purchased from Kokusan Kagaku Co., Ltd.) (14.8g, 50mm
ol), DCC (11.4 g, 55 mmol), HOB
t (6.9g, 45mmol), DMF (150ml)
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 a 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). BocProSer(Bzl)Thr(Bzl)OBz
Synthesis of l(NO2)BocSer(Bzl)Thr(
Bzl) OBzl(NO2) (28.0g, 45mmo
l), BocPro (purchased from Kokusan Kagaku Co., Ltd.) (9.
69g, 45mmol), DCC (10.3g, 50m
mol), HOBt (6.1 g, 40 mmol), DM
Add F (150 ml) and incubate at 0°C for 30 min, then at room temperature for 24 min.
Allowed time to react. 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 a 1N aqueous sodium bicarbonate 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). BocValProSer(Bzl)Thr(Bzl)
Synthesis of OBzl(NO2) BocProSer(Bzl
)Thr(Bzl)OBzl(NO2)(28.8g,
40 mmol), BocVal (purchased from Kokusan Kagaku Co., Ltd.) (8.7 g, 40 mmol), DCC (9.3 g,
45 mmol), HOBt (5.4 g, 35 mmol)
, DMF (150 ml) was added, and BocProSer (
Synthesis of Bzl)Thr(Bzl)OBzl(NO2) was performed in the same manner. BocValProSer(Bzl)
Thr(Bzl)OBzl(NO2) is converted into colorless oil (2
9.4 g). BocAsp(OBzl) ValProSer(Bzl
)Thr(Bzl)OBzl(NO2) synthesis Boc
ValProSer(Bzl)Thr(Bzl)OBz
l(NO2) (28.6 g, 35 mmol), BocA
sp(OBzl) (purchased from Kokusan Kagaku Co., Ltd.) (11.
3g, 35mmol), DCC (8.3g, 40mmol)
l), HOBt (4.6 g, 30 mmol), DMF (
150ml) and BocProSer(Bzl)T
Synthesis of hr(Bzl)OBzl(NO2) was carried out in the same manner. BocAsp(OBzl) ValProSer(
Bzl)Thr(Bzl)OBzl(NO2) was obtained as a colorless oil (33.7g). BocLeuAsp(OBzl)ValProSer(
Bzl) Thr (Bzl) OBzl (NO2) Synthesis 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). BocIleLeuAsp(OBzl)ValProS
er(Bzl)Thr(Bzl)OBzl(NO2)
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). BocGlu(OBzl)IleLeuAsp(OBz
l) ValProSer(Bzl)Thr(Bzl)O
Synthesis of Bzl(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). Glu(OBzl)IleLeuAsp(OBzl)V
alProSer(Bzl)Thr(Bzl)OBzl
(NO2) Synthesis of BocGlu(OBzl)IleLeuAsp(OBz
l) ValProSer(Bzl)Thr(Bzl)O
Bzl(NO2) (1.47 g, 1 mmol) was dissolved in 10 ml of methylene chloride, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, it was added to 100 ml of chloroform, washed several times with 100 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 Glu(OBzl)Ile.
LeuAsp(OBzl) ValProSer(Bzl
) Thr(Bzl)OBzl(NO2) was obtained as a colorless oil (1.37 g). Condensation of protected peptide and dialkylglycerol derivative C16H33OCH2CH(OC16H33)CH2O
OC(CH2)2CO-Glu(OBzl)IleLe
uAsp(OBzl)ValProSer(Bzl)T
Synthesis of hr(Bzl)OBzl(NO2) Glu(O
Bzl) IleLeuAsp(OBzl) ValPro
Ser(Bzl)Thr(Bzl)OBzl(NO2)
(1.37g, 1mmol) and C16H33OCH2C
H(OC16H33)CH2OOC(CH2)2COO
A mixture of H (0.64 g, 1 mmol), dicyclohexylcarbodiimide (0.23 g, 1.1 mmol), and 20 ml of chloroform was stirred at room temperature for 24 hours. Silica gel chromatography (eluent chloroform/
Purified with ethyl acetate 90:10) and purified with C16H3
3OCH2CH(OC16H33)CH2OOC(CH
2) 2CO-Glu(OBzl)IleLeuAsp(
OBzl) ValProSer(Bzl) Thr(Bz
l) OBzl(NO2) was obtained as a white powder (1.83 g). Deprotection and Purification Synthesis of Compound (1) C16H33OCH2CH(OC16H33)CH2O
OC(CH2)2CO-Glu(OBzl)IleLe
uAsp(OBzl)ValProSer(Bzl)T
hr(Bzl)OBzl(NO2) (1.83 g) was dissolved in 50 ml of acetic acid, 1 g of 10% palladium on carbon was added, and atmospheric pressure hydrogenolysis was performed at room temperature for 24 hours. The catalyst was filtered off using Celite, and the solvent was distilled off under reduced pressure. The compound (1) was purified by silica gel chromatography (eluent chloroform/methanol/water 65:25:4).
) was obtained. [0025] FAB-MS 1495 (M-
H) Amino acid analysis: Glu (0.91), Ile (1
．． 20), Leu (1.13), Asp (0.98
), Val (0.89), Pro (0.94),
Ser (0.92), Thr (0.89) Example 2 A synthesis example of compound (2) is shown below. According to the method described in Example 1, the compound (
2) was synthesized. Glycerol derivatives were synthesized by changing 1-bromohexadecane to 1-bromooctadecane and changing succinic anhydride to glutaric anhydride. FAB-MS 1565 (MH) Amino acid analysis: Glu (1.09), Ile (1.05),
Leu (1.06), Asp (0.92), Val
(0.97), Pro (0.91), Ser (0.
83), Thr (0.82) Example 3 A synthesis example of compound (3) is shown below. According to the method described in Example 1, the compound (
3) was synthesized. A glycerol derivative was synthesized by changing 1-bromohexadecane to 1-iodo-3,7,11,15-tetramethylhexadecane. FAB-MS 1607 (MH) Amino acid analysis: Glu (0.97), Ile (1.05),
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. According to the method described in Example 1, the compound (
4) was synthesized. 1-bromohexadecane to 1-bromododecane, succinic anhydride to malonyl dichloride,
Glycerol derivatives were synthesized by changing each. FAB-MS 1369 (MH) Amino acid analysis: Glu (1.10), Ile (1.09),
Leu (1.00), Asp (1.02), Val
(1.09), Pro (0.94), Ser (0.
79), Thr (0.81) Example 5 A synthesis example of compound (5) is shown below. In addition, the compound (6
) and (7) can also be synthesized by the method exemplified here. Literature (Synthesis, 503(1)
985)) Dissolve 18 g of 1-benzylglycerol prepared by the method described in 100 ml of methylene chloride,
55 g of palmitoyl chloride, 20 g of triethylamine.
2 g was added and stirred at room temperature for 12 hours. This was washed several times with 100 ml each of 1N aqueous sodium bicarbonate 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 subjected to silica gel chromatography (eluent: hexane: ethyl acetate).
40:1). 1,2-dipalmitoyl-
60 g of 3-benzyl-glycerol was obtained as colorless crystals. The following was synthesized by the method described in Example 1. [0030] FAB-MS 1523 (M-
H) Amino acid analysis: Glu (1.09), Ile (1
．． 03), Leu (1.05), Asp (0.94
), Val (1.11), Pro (0.99),
Ser (0.78), Thr (0.83) Example 6 A synthesis example of compound (6) is shown below. According to the method described in Example 5, the compound (
6) was synthesized. Synthesis of the protected peptide was performed using BocGlu
(OBzl)IleLeuAsp(OBzl)ValP
roSer(Bzl)Thr(Bzl)OBzl(NO
Glu(OBzl)IleLeuAsp(OBzl) obtained by treating 2) with trifluoroacetic acid to remove the Boc group
ValProSer(Bzl)Thr(Bzl)OBz
l(NO2) and BocGlu(OBzl)IleLe
uAsp(OBzl)ValProSer(Bzl)T
BocGlu(O
Bzl) IleLeuAsp(OBzl) ValPro
Ser(Bzl)Thr(Bzl) to DCC-HOB
Fragment condensation was performed by the t method. [0032] FAB-MS 2378 (M-
H) Amino acid analysis: Glu (0.97), Ile (1
．． 08), Leu (1.02), Asp (0.99
), Val (1.07), Pro (0.94),
Ser (0.85), Thr (0.79) Example 7 A synthesis example of compound (7) is shown below. According to the method described in Example 5, the compound (
7) was synthesized. Glycerol derivatives were synthesized by changing palmitoyl chloride to stearoyl chloride. Synthesis of protected peptide Glu(OBzl)IleLe
uAsp(OBzl)ValProSer(Bzl)T
BocG from hr(Bzl)OBzl(NO2)
ly was sequentially extended by the DCC-HOBt method. FAB-MS 1650 (MH) Amino acid analysis: Gly (1.08), Glu (0.97),
Ile (1.01), Leu (1.09), Asp
(1.06), Val (1.08), Pro (0.
98), Ser (0.88), Thr (0.89) Example 8 A formulation example of the compound (1) of the present invention is shown below. Compounds (2) to (7) 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 for 1 hour at room temperature, PBS (10 ml)
In addition, Branson ultrasonic homogenizer model 2
Dispersion was carried out using a model 50 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 9 A formulation example of Compound 5 of the present invention is shown below. Compounds (1) to (4), (6), and (7) can also be formulated by the methods exemplified here. Compound (5) 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 Branson Ultrasonic Homogenizer Model 25.
Dispersion was performed for 15 minutes at 20 W using type 0. 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 10 A formulation example of the compound (3) of the present invention is shown below. Compounds (1), (2), (4) to (7) can also be formulated by the methods exemplified here. Compound (3) 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, physiological saline (10 ml
) was added and dispersed for 15 minutes at 20 W using an ultrasonic homogenizer model 250 manufactured by Branson. 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 11 Test examples (measurement of cell adhesion inhibitory activity) of the compounds of the present invention 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 63 3
1 22 13
Compound 2 100
65 25 20
14 Compound 3
100 63 29
23 17 Compound 4 100 62
25 18 10
Compound 5 100
68 29 21
13 Compound 6
100 66 2
1 16 10
Compound 7 100
64 24 15
11 ────────────────
────────────────── Example 12 Test examples (measurement of platelet aggregation inhibitory activity) of the compounds of the present invention are 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 upon addition of peptide derivative 0041 [Sequence Listing] 0042 Sequence number: 1 Sequence length: 8 Sequence type: Amino acid topology: Linear sequence type: Peptide fragment type: C-terminal fragment [ SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide fragment type: C-terminal fragment sequence Glu Ile Leu Asp Val Pro S
er Thr Glu Ile Leu Asp Va
l Pro Ser Thr 1
5
10 15 [
SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide fragment Type: C-terminal fragment

Claims (3)

[Claims] Claim 1: A peptide derivative represented by the following general formula [I] or a salt thereof. General formula [I] R1OCH2-CH(OR2)-CH2-OOC-(C
H2) m -CO-([Gly]-Glu-Ile-
Leu-Asp-Val-Pro-Ser-Thr)
n in the formula, Glu, He, Leu, Asp,
Val, Pro, Ser, and Thr represent glutamic acid, isoleucine, leucine, aspartic acid, valine, proline, serine, and threonine residues, respectively. [Gly] represents a glycine residue, present or absent. m represents an integer from 1 to 5. n is 1
Represents an integer from to 3. R1 and R2 represent hydrogen or a linear or branched acyl group or alkyl group having 8 to 24 carbon atoms, and may have a substituent or an unsaturated group. Furthermore, the asymmetric carbon present in the molecule may be either a racemic form or an optically active form. 2. An animal cell adhesion inhibitor comprising the peptide derivative or its salt according to claim 1 as an active ingredient. 3. A platelet aggregation/adhesion inhibitor comprising the peptide derivative or its salt according to claim 1 as an active ingredient.