JP2745353B2 - Immunosuppressants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunosuppressant containing a peptide derivative or a pharmaceutically acceptable salt thereof as an active ingredient. More particularly, a peptide comprising an arginine-glycine-aspartic acid tripeptide unit.
And a pharmaceutically acceptable salt thereof.

[0002]

2. Description of the Related Art Fibronectin is a protein involved in cell-extracellular matrix adhesion, and is considered to be involved in platelet aggregation and cancer metastasis. These interactions are mediated by a series of cell surface receptors, and despite fibronectin being a macromolecule with a molecular weight of about 500,000, these receptors have their arginine-glycine-aspartate (hereinafter Arg-Gly-Asp). (Abbreviated as)), and has been reported to be important for the interaction with the receptor (Nature, Vol. 309, p. 30, 1984). Since then
Studies using an oligo or polypeptide having an Arg-Gly-Asp sequence have been made. For example, a method of inhibiting platelet aggregation using various linear and cyclic oligopeptides having an Arg-Gly-Asp sequence (see Proceedings of the Society of Polymer Science, Japan)
lymer Preprints, Japan), Volume 38, 3149, 1
989, JP-A-2-174797), Arg-Gly-Asp
A method using a peptide having a sequence as a cell migration inhibitor (Japanese Patent Application Laid-Open No. 2-4716), a method using an Arg-Gly-Asp-immobilized PMMA membrane as a cell adhesion membrane (Polymer Preprints, Japan ), Vol. 37, 705
1988). In addition, polymer
A method in which a peptide containing Arg-Gly-Asp as an essential constituent unit is covalently bonded and used as a substrate for animal cell culture or a substrate for a living complex artificial organ (JP-A-1-309682, JP-A-H 1-3)
No. 05960), and a method of using a polypeptide having an Arg-Gly-Asp-Ser sequence as a platelet protective agent for extracorporeal blood has also been disclosed (Japanese Patent Application Laid-Open No. 64-6217). Furthermore, Ar
A method of suppressing cancer metastasis using an oligopeptide having a g-Gly-Asp sequence or a polypeptide having a repeating structure thereof is also known (Int. J. Biol. Macromo
l., Vol. 11, p. 23, 1989, The Journal, Vol. 11,
226, 1989, Jpn. J. Cancer Res., No. 60.
Vol. 722, 1989). The inventors of the present invention
As a result of examining the physiological activity of an acyl derivative or an alkyl ester derivative of the peptide containing the g-Gly-Asp sequence, it was found that the peptide has an immunosuppressive activity, and the present invention was completed.

[0003]

Accordingly, an object of the present invention is to provide a novel peptide derivative having immunosuppressive activity or an immunosuppressant containing a pharmaceutically acceptable salt thereof. The immunosuppressive agent of the present invention is useful for treating and preventing autoimmune diseases, transplant rejection, and graft-versus-host disease.

[0004]

The present invention provides an arginine-
Derivation of peptides containing the glycine-aspartic acid sequence
An immunosuppressant containing the body or a pharmaceutically acceptable salt thereof as an active ingredient. As a result of further investigating various peptide derivatives in accordance with the above purpose, in particular, Arg-Gly-As
It has been found that acyl derivatives or alkyl ester derivatives of peptides containing the p sequence have useful immunosuppressive activity. Specific examples of acyl derivatives or alkyl ester derivatives of the peptide containing the Arg-Gly-Asp sequence and preferred embodiments thereof will be described in detail below. One group of the peptide derivatives used in the present invention comprises peptide derivatives represented by the following general formulas (I) and (II) or salts thereof.

HO ₂ C- (CH ₂ ) _m -C (O)-([X] -Arg-Gly-Asp- [Y]) _n -O-CH ₂ CH (OR ₁ ) CH ₂ OR ₂ (I )

R ₃ -([X] -Arg-Gly-Asp- [Y]) _n -Z (II)

In the formula, Arg, Gly and Asp represent arginine, glycine and aspartic acid residues, respectively. [X],
[Y] indicates an amino acid residue or a peptide residue.
[X] and [Y] are amino acid residues selected from serine, glycine, valine, asparagine, proline, cysteine, threonine and aspartic acid residues or peptide residues composed of these amino acid residues Is preferred. Particularly, [Y] is preferably a serine residue. [X] and [Y] may not both be present. m represents an integer of 1 to 5, preferably an integer of 1 to 3. n represents an integer of 1 to 5, preferably an integer of 1 to 3. R ₁ and R ₂ are each hydrogen or a linear or branched acyl group or alkyl group having 8 to 24 carbon atoms, and may have a substituent or may contain an unsaturated bond. The preferred number of carbon atoms is from 12 to 18. As the acyl group,
Dodecanoyl, myristoyl, palmitoyl and stearoyl groups are shown as preferred examples. Examples of the alkyl group include a dodecyl group, a myristyl group, a palmityl group,
Stearyl and phytanyl groups are shown as preferred examples. R ₁ and R ₂ may be the same or different. R ₃
Is a linear or branched acyl group having 6 to 24 carbon atoms,
It may have a substituent or an unsaturated group. Preferably, it has 8 to 18 carbon atoms. For example, preferred examples include a myristoyl group, a palmitoyl group, a stearoyl group, a 3,7,11,15-tetramethylhexadecanoyl group, and a 3,7,11-trimethyl dodecanoyl group. Z represents a hydroxyl group or an amino group. 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 salt, ammonium salt, magnesium salt, hydrochloride, sulfate, nitrate, acetate and the like.

[0008] Preferred specific examples of the compound of the present invention are shown below, but the present invention is not limited thereto. Compound (1) HO ₂ CCH ₂ CH ₂ C (O) -Arg-Gly-Asp-Ser-O-CH ₂ CH (OC ₁₆ H ₃₃ ) CH ₂
(OC ₁₆ H ₃₃ ) Compound (2) HO ₂ CCH ₂ CH ₂ C (O) -Arg-Gly-Asp-Ser-O-CH ₂ CH (OC ₁₄ H ₂₉ ) CH ₂
(OC ₁₄ H ₂₉ ) Compound (3) HO ₂ CCH ₂ CH ₂ CH ₂ C (O) -Arg-Gly-Asp-Ser-O-CH ₂ CH (OC ₁₆ H ₃₃ )
CH ₂ (OC ₁₆ H ₃₃ ) compound (4) HO ₂ CCH ₂ CH ₂ C (O)-(Arg-Gly-Asp) ₂ -O-CH ₂ CH (OC ₁₄ H ₂₉ ) CH
₂ (OC ₁₄ H ₂₉ ) Compound (5) C ₉ H ₁₉ CO-Arg-Gly-Asp-OH Compound (6) C ₁₃ H ₂₇ CO-Arg-Gly-Asp-OH Compound (7) C ₁₅ H ₃₁ CO -Arg-Gly-Asp-OH compound (8) CH ₃ -[(CH (CH ₃ )-(CH ₂ ) ₃ ] ₃ -CH (CH ₃ ) -CH ₂ -CO-Arg-Gly-Asp-
OH compound (9) C ₁₅ H ₃₁ CO-Arg-Gly-Asp-Ser-OH compound (10) C ₁₇ H ₃₅ CO-Arg-Gly-Asp-Ser-OH compound (11) CH _3- (CH (CH _{3) - (CH 2) 3} ] 3 -CH (CH 3) -CH 2 -CO-Arg-Gly-Asp-S
er-OH compound (12) C ₁₅ H ₃₁ CO-Gly-Arg-Gly-Asp-Ser-OH compound (13) C ₁₃ H ₂₇ CO-Gly-Arg-Gly-Asp-Ser-Pro-OH compound (14 _{) C 13 H 27 CO-Arg} -Gly-Asp-NH 2 compound _{(15) CH 3 - [(} CH (CH 3) - (CH 2) 3] 2 -CH (CH 3) -CH 2 -CO-Arg -Gly-Asp-
NH ₂ compound (16) C ₁₅ H ₃₁ CO-Arg-Gly-Asp-Ser-NH ₂ compound (17) CH ₃ -[(CH (CH ₃ )-(CH ₂ ) ₃ ] ₃ -CH (CH ₃ ) -CH ₂ -CO-Arg-Gly-Asp-
Ser-NH ₂ compound (18) C ₁₉ H ₃₉ CO-Arg-Gly-Asp-Ser-NH ₂ compound (19) C ₁₅ H ₃₁ CO- (Arg-Gly-Asp-Ser) ₂ -OH

Next, a method for synthesizing the compound of the present invention will be described. The compound (I) of the present invention can be basically synthesized by a method comprising the following paragraphs. Synthesis of the following dialkyl (diacyl) glycerol (III)

R ₁ O—CH ₂ —CH (OR ₂ ) —CH ₂ OH (III)

Synthesis of Protected Peptide from Glycerol Derivative (III) by Sequential Extension of Protected Amino Acids Selective deprotection of the N-terminal amino group of the protected peptide followed by malonylation, succinylation or glutarylation. Removal and purification of side-chain protecting groups

Hereinafter, each step will be described in detail. The dialkyl (diacyl) glycerol represented by the formula (III) can be synthesized by a known method (for example, Biochemistry Vol. 2, 3).
94, 1963), or commercially available products can be purchased.

[0013] The method of successively extending protected amino acids is a known method, that is, "Basic and Experimental Peptide Synthesis" by Izumiya et al. (Maruzen) and "PRINCIPLES OF PEPTI" by Bodanszky.
DE SYNTHESIS "," THE PRACTICE OF PEPTIDE SYNTHESI
S "(Springer Verlag, New York) are all effective. In the stage of the condensation reaction, DCC-addi
tive method, azide method, mixed acid anhydride method, active ester method may be adopted, but DC using 1-hydroxybenzotriazole and dicyclohexylcarbodiimide in combination
The C-additive method gave the best results. In the step of introducing the first protected amino acid into the glycerol derivative (III), dicyclohexylcarbodiimide and a catalytic amount of N,
A method using N-dimethylaminopyridine is effective.

In the malonylation, succinylation or glutarylation reaction of the amine, it is preferable to use the corresponding cyclic acid anhydride or acid dichloride, respectively. The conditions used to deprotect the protecting group will depend largely on the type of protecting group used. Commonly used deprotection conditions include hydrogenolysis, trifluoroacetic acid, hydrogen anhydride, trifluoromethanesulfonic acid-thioanisole mixed system, trifluoroacetic acid-thioanisole mixed system, etc., depending on the type of protecting group. A variety of means are possible.

Next, a method for synthesizing the compound (II) of the present invention will be described. Compound (II) can be synthesized, for example, by the following process. Preparation of Protected Protected Peptide Fragment Condensation of Protected Peptide Fragment with R ₃ -W (R ₃ represents a corresponding acyl group; W represents, for example, a hydroxyl group or a halogen atom)

Hereinafter, each step will be described in detail. Protected peptide fragments are prepared by a known method of successively extending protected amino acids, such as Izumiya et al., “Basic and Experimental Peptide Synthesis” (Maruzen) and Bodanszky, “PRINCI
PLES OF PEPTIDE SYNTHESIS "," THE PRACTICE OF PEPT
All of the methods described in “IDE SYNTHESIS” (Springer Verlag, New York) are effective. In the stage of the condensation reaction, any of the DCC-additive method, azide method, mixed acid anhydride method, and active ester method is employed. The DCC-additive method using 1-hydroxybenzotriazole and dicyclohexylcarbodiimide in combination gave the best results.

It is to be noted that particularly preferred protected amino acids are those wherein the α-amino acid is protected with a Boc group and the side chain and α-carboxyl group are protected with a benzyl-based protecting group such as benzyl ester, benzyl ether or benzyl thioether. Gave. For condensation with the corresponding R ₃ —W, the usual methods are used. When W is a hydroxyl group, for example, DC
The C method, DCC-additive method, CDI method and the like are frequently used.
When W is a halogen, for example, a corresponding acid chloride may be used. The conditions used to deprotect the protecting group will depend largely on the type of protecting group used. Commonly used deprotection conditions include hydrogenolysis, trifluoroacetic acid, hydrogen fluoride, trifluoromethanesulfonic acid-thioanisole mixed system, trifluoroacetic acid-thioanisole mixed system treatment, and the like. Depending on the situation, more various means are possible. For purification of the target substance, a reprecipitation method, a gel filtration method, or the like can be employed.

The synthesis examples of the compounds of the present invention are shown below.

Synthesis Example 1 A synthesis example of the compound (1) of the present invention is shown. Compound (1) was synthesized by the following synthetic route. In addition, amino acids, various protecting groups, deprotecting reagents and the like are usually used and represented by using abbreviations. Further, compounds (2) to (4) can also be synthesized by the methods exemplified here. Bzl: benzyl group TFA: trifluoroacetic acid t-Boc: t-butoxycarbonyl group HOBt: 1-hydroxybenzotriazole Z: benzyloxycarbonyl group DCC: dicyclohexylcarbodiimide DC urea: dicyclohexylurea

[0020]

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References (Synthesis, p. 503, 1985)
12.0 g of (1a) prepared by the method described in above was dissolved in 300 ml of toluene, and powdered potassium hydroxide 1
6.0 g and 1-bromohexadecane 82 g were added, and the reaction mixture was heated to reflux for 8 hours. The reaction solution was allowed to cool to room temperature and diluted with 400 ml of hexane. 200 ml of water
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. This was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 40/1) to obtain 41.2 g of (1b) (yield 95.5%). Physical properties are described in the literature (Biochemistry Vol. 2, p. 394, 1963).
Year). The obtained (1b) was dissolved in 250 ml of ethyl acetate, and 10% palladium-carbon 1.5
g was added and the reaction mixture was reacted for 8 hours under a hydrogen atmosphere. The insoluble material was removed by filtration through Celite, and the Celite layer was washed with ethyl acetate. The filtrate and the washing were combined and concentrated under reduced pressure. The residue was recrystallized from ethyl acetate to give (1c) as colorless crystals (34.4 g). Physical properties are described in the literature (Bioche
mistry, Vol. 2, p. 394, 1963).

(1c) 3.25 g (6 mmol), t-Boc-Se
Dissolve 2.05 g (7 mmol) of r (Bzl) and 73 mg of dimethylaminopyridine in 50 ml of methylene chloride, and cool with ice-cooling.
1.5 g of C were added. After stirring under ice-cooling for 2 hours and at room temperature overnight, the generated DCC urea was separated by filtration, the filtrate was concentrated, and the residue was purified by silica gel chromatography (n-hexane / ethyl acetate = 6/1) to give a colorless product. Oily (1d) 4.
7 g (5.74 mmol) were obtained. 97% yield. (1d)
4.7 g (5.74 mmol) was dissolved in 20 ml of methylene chloride, 10 ml of trifluoroacetic acid (TFA) was added, and 30
Minutes. After evaporating the solvent under reduced pressure, ethyl acetate and a 4% aqueous sodium carbonate solution were added to the residue, and the mixture was extracted and separated.
(At this time, it was confirmed that the pH of the aqueous layer was alkaline.) The ethyl acetate layer was washed with brine, dried over magnesium sulfate, and evaporated under reduced pressure. The obtained deprotected compound (1d) was dissolved in a mixed solvent of 30 ml of methylene chloride and 15 ml of DMF, and 2.2 g (6.8 mmol) of t-Boc-Asp (OBzl) and HO were dissolved.
1 g (6.5 mmol) of Bt / H ₂ O was added and the mixture was stirred under ice-cooling. To this, 1.4 g (6.8 mmol) of DCC was added, and the mixture was stirred under ice cooling for 2 hours and at room temperature overnight. The generated DCC urea is filtered off,
The filtrate was concentrated under reduced pressure. Ethyl acetate and a 4% aqueous sodium carbonate solution were added to the residue for extraction and liquid separation. The ethyl acetate layer was washed with water and brine, concentrated under reduced pressure, and the residue was purified by silica gel chromatography (n-hexane / ethyl acetate = 4 / 1-2 / 1) to give a white solid (1e).
5.36 g (5.24 mmol) were obtained. Yield 91%.

(1e) 5.24 g (5.12 mmol) was dissolved in 20 ml of methylene chloride, 10 ml of TFA was added, and the mixture was stirred at room temperature for 1 hour. The solvent was concentrated under reduced pressure, and the residue was recrystallized from a mixed solvent of ethyl acetate and acetonitrile (1: 2) to obtain 4.93 g (4.75 mmol) of (1f) as a white solid. Yield 93%. (1f) 4.8g (4.62mmo
l), t-Boc-Gly 0.96 g (5.5 mmol), HOBt.H ₂ O
842 mg (5.5 mmol) of methylene chloride (30 ml) and DMF
It was dissolved in 15 ml of a mixed solvent. Here, 630 mg (4.9 mmol) of diisopropylethylamine and 1.1 g of DCC
(5.3 mmol), and the mixture was stirred under ice cooling for 2 hours and at room temperature overnight. Thereafter, the same post-treatment as in (1d) was performed, and the mixture was purified by silica gel column chromatography (n-hexane / ethyl acetate = 3/2) to give 3.95 g of a white solid (1 g).
(3.66 mmol) were obtained. 79% yield. (1 g) 2.0
g (1.85 mmol) in 10 ml of methylene chloride and TF
A 5 ml was added, and the mixture was stirred at room temperature for 30 minutes. After the solvent was left under reduced pressure, ethyl acetate and a 4% aqueous sodium carbonate solution were added to the residue, followed by extraction and liquid separation. (At this time, it was confirmed that the pH of the aqueous layer was alkaline.) The ethyl acetate layer was washed with brine, dried over magnesium sulfate, and then ethyl acetate was distilled off under reduced pressure. The obtained deprotected compound (1 g) was dissolved in a mixed solvent of 20 ml of methylene chloride and 5 ml of DMF, and t-Boc-Ar
g (Z) ₂ 1.2 g (2.15 mmol) and HOBt / H ₂ O 330 mg
(2.15 mmol) and the mixture was stirred under ice-cooling. Where DCC
460 mg (2.2 mmol) was added, and the mixture was stirred under ice cooling for 2 hours and at room temperature overnight. Thereafter, the same post-treatment as (1f) was carried out, and the mixture was purified by silica gel column chromatography (chloroform / ethyl acetate = 3/1) to obtain a white solid (1h).
2.09 g (1.48 mmol) were obtained. Yield 80%.

(1h) 1.0 g (0.664 mmol) was dissolved in 10 ml of methylene chloride, and 5 ml of TFA was added thereto.
Stirred for hours. After the solvent was distilled off under reduced pressure, chloroform and a 4% aqueous solution of sodium carbonate were added to the residue for extraction and separation. (At this time, it was confirmed that the pH of the aqueous layer was alkaline.) The chloroform layer was washed with brine, dried over magnesium sulfate, and evaporated under reduced pressure. (1
The det-Boc form of h) was dissolved in 10 ml of methylene chloride, 120 mg of succinic anhydride was added, and the mixture was stirred at room temperature. As the reaction proceeded, white precipitate was formed. After 1 hour, the solvent was concentrated under reduced pressure,
The residue was recrystallized from a mixed solvent of acetonitrile and chloroform (5: 1) to give 920 mg of a white solid (1i).
(0.61 mmol) was obtained. Yield 92%. (1i) 800
mg (0.532 mmol) was dissolved in 20 ml of acetic acid at 55 ° C.
100 mg of 10% palladium-carbon was added, and normal pressure hydrogenation was performed at this temperature for 4 hours. The catalyst was filtered off through celite, the filtrate was concentrated under reduced pressure, and acetonitrile was added to the residue to precipitate a white solid. This was collected by filtration to obtain 390 mg (0.369 mmol) of compound (1). Yield 69
%. ^{(M-H) - 1055 (} FAB, NEGA) amino acid analysis: Arg (1.03), Gly ( 1.0
1), Asp (1.02), Ser (0.91)

Synthesis Example 2 Synthesis of Compound (2) In the synthesis of Compound (1a), 1-bromotetradecane was used in place of 1-bromohexadecane, and the same operation was performed thereafter to synthesize Compound (2). ^{(M-H) - 999 (} FAB, NEGA) amino acid analysis: Arg (1.10), Gly ( 1.0
3), Asp (0.98), Ser (0.89)

Synthesis Example 3 Synthesis of Compound (3) In the synthesis of compound (1i), glutaric anhydride was used in place of succinic anhydride, and thereafter the same operation was performed to synthesize compound (3). ^{(M-H) - 1069 (} FAB, NEGA) amino acid analysis: Arg (1.02), Gly ( 0.9
8), Asp (1.00), Ser (0.95)

Synthesis Example 4 Synthesis of Compound (5) Boc-Asp (OBzl) purchased from Kokusan Kagaku Co., Ltd. by the method described in the literature (Chem. Pharm. Bull., 24, 3025 (1976)).
32.3 g, triethylamine 14 ml, benzyl bromide 17.1
g and 200 ml of ethyl acetate were heated under reflux for 3 hours.
After allowing the reaction solution to cool to room temperature, it was washed with a 1N aqueous solution of sodium hydrogencarbonate and 200 ml of saturated saline each 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. The reaction mixture was purified by silica gel chromatography (eluent hexane / ethyl acetate 95: 5), and Boc-Asp (OBzl) -O
36 g of Bzl were obtained. Dissolve in dichloromethane (20 ml), add trifluoroacetic acid (20 ml), stir at room temperature for 30 minutes, and add TFA-Asp
(OBzl) -OBzl was obtained quantitatively.

8.5 g of TFA-Asp (OBzl) -OBzl was dissolved in dichloromethane, 6.7 g of anhydrous Boc-glycine and 2.5 g of dimethylaminopyridine (DMAP) were added, and the mixture was stirred at room temperature for 6 hours.
The reaction solution was washed with water and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. Dissolve the residue in dichloromethane (20 ml) and add trifluoroacetic acid
20 ml was added and the mixture was stirred at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, chloroform (100 ml) was added, and the mixture was washed several times with a 1N aqueous solution of sodium hydrogen carbonate and saturated saline (100 ml each), and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain Gly-Asp (OBzl) -OBzl. Boc-Arg (Z) ₂ (Kokusan Chemical Co., Ltd.) without purification
10.83g, DCC 4.54g, HOBt 2.76
g, DMF (80 ml) and stirred for 24 hours. After removing DC urea, the solvent was distilled off under reduced pressure and chloroform (100 ml) was added, 1N aqueous sodium hydrogen carbonate solution and saturated saline were added each at 200 ml.
And dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform / ethyl acetate 6: 4) to obtain 13.2 g of Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -OBzl as a white powder. F
AB-MASS (M + H) ⁺ 894

Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -OBzl 1.34
g, dissolved in methylene chloride 10 ml, trifluoroacetic acid 10m
l was added and stirred at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, chloroform (100 ml) was added, and the mixture was washed several times with a 1N aqueous solution of sodium hydrogen carbonate and saturated saline (100 ml each), and dried over anhydrous sodium sulfate. Filter off the sodium sulfate,
The filtrate was concentrated under reduced pressure to give Arg (Z) ₂ -Gly-Asp (OB
zl) -OBzl was obtained quantitatively. 172 mg of dodecanoic acid was dissolved in 50 ml of dichloromethane, 309 mg of DCC was added at room temperature, and the mixture was stirred for 1 hour. Arg (Z) ₂ -Gly-Asp (OBzl) -OBzl 1.19g
Was added and stirred for 10 hours. The reaction solution was filtered, and the filtrate was washed with water, and the organic layer was dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: chloroform / ethyl acetate = 1: 1) to give C ₉ H ₁₉ CO-Arg
620 mg of (Z) ₂ -Gly-Asp (OBzl) -OBzl were obtained. C ₉ H ₁₉ CO-Arg
Dissolve 500 mg of (Z) ₂ -Gly-Asp (OBzl) -OBzl in 50 ml of acetic acid,
100 mg of 10% palladium-carbon was added, and hydrogenolysis under atmospheric pressure was performed at room temperature for 24 hours. The catalyst was filtered off using celite, the solvent was distilled off under reduced pressure, and the residue was purified by a reprecipitation method (acetic acid-ethyl acetate) to obtain 182 mg of compound (5). ^{(M + H) + 501 (} FAB, positive) amino acid analysis: Arg (0.94), Gly ( 0.9
3), Asp (0.99) Synthesis Example 5 Synthesis of Compound (6) Compound (6) was synthesized in the same manner as in Synthesis Example 4 using myristic acid. (M + H) ⁺ 557 (FAB, positive) Amino acid analysis: Arg (0.97), Gly (0.9
4), Asp (0.92)

Synthesis Example 6 Synthesis of Compound (7) Compound (7) was synthesized in the same manner as in Synthesis Example 4 using palmitic acid. (M + H) ⁺ 584 (FAB, positive) Amino acid analysis: Arg (1.02), Gly (0.9
9), Asp (0.91) Synthesis Example 7 Synthesis of compound (8) Dissolve 50 g of synthetic phytol in ethanol and hydrogenate with 500 mg of platinum dioxide at room temperature to perform 3,7,11,15-
Tetramethylhexadecanol was obtained quantitatively. 3,7,1
Oxidation of 1,15-tetramethylhexadecanol with Jones reagent gave 3.7.11.15-tetramethylhexadecanoic acid. It was synthesized in the same manner as in Synthesis Example 4 using 3,7,11,15-tetramethylhexadecanoic acid synthesized as described above. (M + H) ⁺ 641 (FAB, positive) Amino acid analysis: Arg (0.97), Gly (0.9
2), Asp (1.01)

Synthesis Example 8 Synthesis of Compound (9) Boc-Ser (Bzl) 2 purchased from Kokusan Chemical Co., Ltd. by the method described in the literature (Chem. Pharm. Bull., 24, 3025 (1976)).
9.5 g, triethylamine 14 ml, benzyl bromide 17.1 g,
A mixture of 200 ml of ethyl acetate was heated under reflux for 3 hours. After allowing the reaction solution to cool to room temperature, it was washed with a 1N aqueous solution of sodium hydrogencarbonate and 200 ml of saturated saline each 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. The reaction mixture was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 40: 1), and Boc-Ser (Bzl) -OBzl 3
6 g were obtained.

Next, 7.71 g of Boc-Ser (Bzl) -OBzl was dissolved in 20 ml of methylene chloride, 20 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, chloroform (100 ml) was added, and the mixture was washed several times with a 1N aqueous solution of sodium hydrogen carbonate and saturated saline (100 ml each), 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. A mixture of 6.47 g of this and Boc-Asp (OBzl) (purchased from Kokusan Chemical Co., Ltd.), 4.54 g of dicyclohexylcarbodiimide (DCC), 2.76 g of hydroxybenzotriazole (HOBt), and 80 ml of DMF was added to 0 ml
The mixture was stirred at C for 3 hours and at room temperature for 12 hours. After removing DC urea, the solvent was distilled off under reduced pressure, and chloroform 100 ml was removed.
And then add 1N aqueous sodium hydrogen carbonate solution and saturated saline
Washed with 0 ml and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and 20 ml of dichloromethane and 20 ml of trifluoroacetic acid were added thereto.
Stirred for minutes. After evaporating the solvent under reduced pressure, chloroform 10
0 ml was added, and the mixture was washed several times with 1N aqueous sodium hydrogencarbonate solution and 100 ml each of saturated saline, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain Asp (OBzl) -Ser (Bzl) -OBzl. Furthermore, Boc-Gly
(Purchased from Kokusan Chemical Co., Ltd.) 3.50 g, DCC 4.54 g, H
2.76 g of OBt and 80 ml of DMF were added, and the mixture was added at 0 ° C for 3 hours.
The mixture was further stirred at room temperature for 12 hours.

After removing DC urea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and the mixture was washed with a 1N aqueous solution of sodium hydrogencarbonate and 200 ml of saturated saline each and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, 20 ml of dichloromethane and 20 ml of trifluoroacetic acid were added, and the mixture was stirred at room temperature for 30 minutes. After the solvent was distilled off under reduced pressure, chloroform (100 ml) was added, and the mixture was washed several times with a 1N aqueous solution of sodium hydrogen carbonate and saturated saline (100 ml each), and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and Gly-Asp (OBzl) -Ser (Bzl) -OBzl
I got Without purification, BocArg (Z) ₂ (purchased from Kokusan Chemical Co., Ltd.) 10.83 g, DCC 4.54 g, HOBt
2.76 g and DMF (80 ml) were added, and the mixture was stirred all day and night. After removing DC urea, the solvent was distilled off under reduced pressure and chloroform (100 ml) was added.
l, add 1N aqueous sodium bicarbonate solution and saturated saline
Washed with 200 ml and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 99: 1),
Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Ser (Bzl) -O as white powder
14.4 g of Bzl was obtained. FAB-MASS (M + H) ⁺ 1071

Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Ser (Bzl) -OBz
1.03 g 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, chloroform (100 ml) was added, and the mixture was washed several times with a 1N aqueous solution of sodium hydrogen carbonate and saturated saline (100 ml each), and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to give Arg (Z) ₂ -Gly-As as a white powder.
p (OBzl) -Ser (Bzl) -OBzl was obtained quantitatively. Palmitic acid
Dissolve 256 mg in 50 ml of dichloromethane and add DCC at room temperature.
206 mg was added and stirred for 1 hour. Arg (Z) ₂ -Gly-Asp
0.97 g of (OBzl) -Ser (Bzl) -OBzl was added and stirred for 10 hours. The reaction solution was filtered, and the filtrate was washed with water, and the organic layer was dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (eluent: chloroform / ethyl acetate =
1: 1) and purified by C ₁₅ H ₃₁ CO-Arg (Z) ₂ -Gly-Asp (OBz
l) -Ser (Bzl) -OBzl 640 mg was obtained. C ₁₅ H ₃₁ CO-Arg (Z) ₂ -Gly
-Asp (OBzl) -Ser (Bzl) -OBzl 500 mg was dissolved in acetic acid 50 ml,
100 mg of 10% palladium-carbon was added, and hydrogenolysis under atmospheric pressure was performed at room temperature for 24 hours. The catalyst was filtered off using celite, the solvent was distilled off under reduced pressure, and the residue was purified by a reprecipitation method (acetic acid-ethyl acetate) to obtain 120 mg of compound (9). (M + H) ⁺ 671 (FAB, positive) Amino acid analysis: Arg (0.99), Gly (0.9
9), Asp (0.93), Ser (0.78)

Synthesis Example 9 Synthesis of Compound (10) Compound (10) was synthesized in the same manner as in Synthesis Example 8 using stearic acid. (M + H) ⁺ 700 (FAB, positive) Amino acid analysis: Arg (0.98), Gly (1.0
2), Asp (0.95), Ser (0.62) Synthesis Example 10 Synthesis of Compound (11) Using 3,7,11,15-tetramethylhexadecanoic acid obtained in the same manner as in Synthesis Example 7. The compound was synthesized in the same manner as in Synthesis Example 8. (M + H) ⁺ 728 (FAB, positive) Amino acid analysis: Arg (0.96), Gly (0.9
7), Asp (0.96), Ser (0.71)

Synthesis Example 11 Synthesis of Compound (18) Boc-Ser (Bzl) -OH (29.5 g, 0.1 mol), p-nitrophenol (13.9 0.1 mol) in methylene chloride (20 ml) and DMF
(20 ml), and DCC (20.6 g, 0.1 mol) was added thereto while cooling with ice. The reaction mixture was stirred under ice-cooling for 2 hours and further at room temperature overnight. The precipitate formed by filtration through celite was removed, and the filtrate was concentrated under reduced pressure to obtain a crude p-nitrophenyl ester. This was dissolved in THF (150 ml), and 25% aqueous ammonia (10 ml) was added. The reaction mixture was stirred at room temperature for 5 hours and concentrated under reduced pressure. The resulting oil was purified by silica gel column chromatography,
27.7 g of c-Ser-NH ₂ was obtained as colorless crystals. Yield 94%.
6.47g of Boc-Asp (OBzl) purchased from Domestic Chemical Co., Ltd., Bo
4.1 g of c-Ser-NH ₂ , dicyclohexylcarbodiimide (D
CC) 4.54 g, hydroxybenzotriazole (HO
Bt) 2.76 g, N, N-diisopropylethylamine 2.6
g and DMF (80 ml) was stirred at 0 ° C. for 3 hours and further at room temperature for 24 hours. After removing DC urea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and the mixture was washed with a 1N aqueous solution of sodium hydrogencarbonate and 200 ml of saturated saline each and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, 20 ml of dichloromethane and 20 ml of trifluoroacetic acid were added, and the mixture was stirred at room temperature for 30 minutes.The solvent was distilled off under reduced pressure to remove TFA-Asp (OBzl) -Ser-NH _2. I got

TFA-Asp (OBzl) -Ser-NH ₂ is converted to dichloromethane 8
After dissolving in 0 ml, Boc-glycine anhydride 6.7 g and dimethylaminopyridine (DMAP) 2.5 g were added, followed by stirring at room temperature for 6 hours. The reaction solution was washed with water and dried over anhydrous sodium sulfate.
The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure.
The residue was dissolved in dichloromethane (20 ml), trifluoroacetic acid (20 ml) was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure to give a TFA-Gly-Asp (OBzl) -Ser-NH 2. This was purified without purification using Boc-Arg (Z) ₂ (purchased from Kokusan Chemical Co., Ltd.) 10.83 g, DCC 4.54 g, HOBt 2.76 g, N, N
2.6 g of diisopropylethylamine and 80 ml of DMF were added, and the mixture was stirred overnight. After removing DC urea, the solvent was distilled off under reduced pressure, 100 ml of chloroform was added, and the mixture was washed with 1N aqueous sodium hydrogen carbonate solution and 200 ml of saturated saline each, and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 1: 0 to 9: 1) to give Bo as a white powder.
10.6 g of c-Arg (Z) ₂ -Gly-Asp (OBzl) -Ser-NH ₂ was obtained. FAB-
Was dissolved ^{MASS (M + H) + 861} Boc-Arg (Z) 2 -Gly-Asp (OBzl) -Ser-NH 2 1.29g, the methylene chloride 10ml, stirred at room temperature for 30 minutes with trifluoroacetic acid 10ml did. After the solvent was distilled off under reduced pressure, chloroform (100 ml) was added, and the mixture was washed several times with a 1N aqueous solution of sodium hydrogen carbonate and saturated saline (100 ml each), and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain Arg (Z) ₂ -Gly-Asp (OBzl) -Ser-NH ₂ as a white powder.
Was quantitatively obtained.

468 mg of arachidic acid is added to dichloromethane 5
The solution was dissolved in 0 ml, DCC (309 mg) was added at room temperature, and the mixture was stirred for 1 hour. Arg (Z) ₂ -Gly-Asp (OBzl) -Ser-NH ₂
Stirred for 0 hours. The reaction solution was filtered, and the filtrate was washed with water, and the organic layer was dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 95: 5 to 8: 2) to obtain C ₁₉ H ₃₉ CO-A.
820 mg of rg (Z) ₂ -Gly-Asp (OBzl) -Ser-NH ₂ were obtained. C ₁₉ H ₃₉ CO
500 mg of -Arg (Z) ₂ -Gly-Asp (OBzl) -Ser-NH ₂ was dissolved in 50 ml of acetic acid, 100 mg of 10% palladium-carbon was added, and hydrogenolysis under atmospheric pressure was performed at room temperature for 24 hours. The catalyst was filtered off using celite, the solvent was distilled off under reduced pressure, and the residue was reprecipitated (acetic acid-
Purification by ethyl acetate) gave 190 mg of compound (18). (M + H) ⁺ 727 (FAB, positive) Amino acid analysis: Arg (0.98), Gly (1.0
2), Asp (0.95), Ser (0.62)

SYNTHESIS EXAMPLE 12 Synthesis of Compound (19)
c- (Arg (Z) ₂ -Gly-Asp (OBzl)) ₂ -OBzl was prepared. Then, dissolve in dichloromethane and add trifluoroacetic acid
TFA- (Arg (Z) ₂ -Gly-Asp (OBzl)) ₂ -OBzl. 456 mg of myristic acid was dissolved in 100 ml of chloroform, 412 mg of DCC was added at room temperature, and the mixture was stirred for 1 hour.

TFA- (Arg (Z) ₂ -Gly-Asp (OBzl)) ₂ -OBzl 3.1
9 g was added and the mixture was stirred for 10 hours. The reaction solution was filtered, and the filtrate was washed with water, and the organic layer was dried over anhydrous sodium sulfate. Removed by filtration sodium sulfate, the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 95: 5) to give C ₁₃ H ₂₇
950 mg of CO- (Arg (Z) ₂ -Gly-Asp (OBzl)) ₂ -OBzl were obtained. C
The _{13 H 27 CO- (Arg (Z} ) 2 -Gly-Asp (OBzl)) 2 -OBzl 500mg was dissolved in acetic acid 50 ml, 10% palladium-carbon 100mg addition,
The hydrogenolysis under normal pressure was performed at room temperature for 24 hours. The catalyst was filtered off using celite, the solvent was distilled off under reduced pressure, and the residue was purified by a reprecipitation method (acetic acid-ethyl acetate) to obtain 130 m of the compound (19)
g was obtained. (M + H) ⁺ 885 (FAB, positive) Amino acid analysis: Arg (2.21), Gly (2.1
1), Asp (1.98)

The compound used in the present invention has an immunosuppressive effect. Therefore, the immunosuppressive agent of the present invention is used for rejection of transplantation of organs or tissues such as kidney, liver, bone marrow and skin, and graft-versus-host reaction. And systemic lupus erythematosus, type I diabetes, multiple sclerosis, Hashimoto's thyroiditis,
It is useful for treating and preventing autoimmune diseases such as rheumatoid arthritis. The immunosuppressive agent of the present invention is a derivative of a peptide containing the sequence of arginine-glycine-aspartic acid ,
Or it can contain a pharmaceutically acceptable salt thereof as an active ingredient, use in the form of a pharmaceutical formulation of a liquid dispersion suitable for parenteral administration. In addition, it may be used by mixing with a generally pharmaceutically acceptable carrier or other immunosuppressants.
Carriers that can be used include water, glucose, lactose, gum acacia, gelatin, starch, dextran, salt and other physiological inorganic salts. It can also be dispersed and used together with known immunosuppressants, for example, cyclosporin A, corticosteroids, Imran, FK506 and the like. The dosage of the immunosuppressant of the present invention can vary depending on the type of disease to be treated, the patient, the state of the disease, and the like, but is usually 0.05 mg / kg as a peptide, a peptide derivative or a salt thereof. Weight / day to 10 m
The dose is about g / kg body weight / day. In order to show the usefulness of the present immunosuppressant, pharmacological test results are shown in the following examples.

[0042]

【Example】

Example 1 T cell proliferation inhibitory effect (dispersion of compound) 30 mg of a peptide derivative to be used was added to a mixed solvent of 10 ml of chloroform and 10 ml of methanol at 60 ° C.
And the solvent was distilled off under reduced pressure at 60 ° C. using a rotary evaporator. Thereafter, it was further dried at room temperature for 30 minutes using a vacuum pump. 3 ml of physiological saline is added to the formed thin film of the compound, and 1N while monitoring with a pH test paper.
The pH was adjusted to about 7 with NaOH or HCl. This solution was heated to 80 ° C. and dispersed by a vortex mixer. When dispersion was incomplete, pH adjustment, heating and dispersion were repeated. Then, a uniform dispersion was obtained by repeatedly passing through a microfilter having a diameter of 0.2 μm. The dispersion of the peptide derivative is RP used for cell culture.
It was diluted with MI1640 medium and sterilized by filtration at about 50 ° C. before use.

(T Cell Proliferation Test) 50 μl of various concentrations of the peptide derivative to be tested prepared in the same manner as above
And concanavalin A (8 μg / ml, RPMI164
0) 50 μl of a BALB / C mouse spleen cell dispersion (4 × 1)
0 ⁶ cells / ml, RPMI1640 containing 10% FCS) 10
The cells were cultured in a CO ₂ incubator at 37 ° C. After about 48 hours, ³ H-thymidine (0.4 μC
The pulse was applied for 5 hours in i), and the amount of ³ H-thymidine incorporated into the cells was measured. Table 1 shows the results. The amount of thymidine incorporation is 100% of the amount when the peptide derivative concentration is 0.
And expressed as a percentage (%) thereof. As can be seen from the results in Table 1, the compounds of the present invention suppressed the proliferation of T cells.

Table 1-Concentration (μg / ml) Compound (2) Compound (4) Compound (11) Compound (12) ０ 0 100% 100% 100% 100% 0.1 76 91 96 92 171 163 613 82 10 3 10 25 46 30 30 1---100 100 10 { ───────────────

Example 2 Inhibitory effect on in vitro mixed lymphocyte reaction (MLR) The compound used was dispersed in the same manner as in Example 1. (Mixed lymphocyte test) As stimulator cells, C57BL / 6
Mouse spleen cells were incubated with 25 μg / ml mitomycin C
The cells were treated at 7 ° C. for 30 minutes and washed three times with RPMI1640 medium. This C57BL / 6 mouse spleen cell 3 × 10 ⁶
Cells / ml and 75 μl of BALB / C
The same amount of mouse spleen cells was mixed, 50 μl of the test sample dispersion was added, and the mixture was cultured in a CO ₂ incubator at 37 ° C. for 66 hours. ³ H-thymidine (0.4 μC
After the pulse for 6 hours in i), the amount of ³ H-thymidine incorporated into the cells was measured. Table 2 shows the results. The results were expressed as in Example 1. As can be seen from the results in Table 2, the compounds of the present invention inhibited allo-MLR in mice.

Table 2-Concentration (μg / ml) Compound (2) Compound (4) Compound (11) Compound (12) ０ 0 100% 100% 100% 100% 0.1 54 92 97 98 1 49 70 65 72 358 58---10 52 25 57 63 30 30 41---100 18 11 31 35 ───────────────────────

Example 3 Acute toxicity test of compound (2) Compound (2) was dispersed in physiological saline according to the method of Example 1. This dispersion was intravenously and intraperitoneally administered to ICR mice, and an acute toxicity test was performed for 7 days. No death was observed at a dose of 100 mg / kg body weight or less, and a control in which only saline was administered. It showed the same weight gain as the group. No abnormalities were observed in appearance and anatomical findings.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Akiko Suzuki 210 Nakanuma, Minamiashigara-shi, Kanagawa Fujisha Shin Film Co., Ltd. (56) References International Publication 90/6943 (WO, A) International Publication 92/995 (WO , A)

Claims (4)

(57) [Claims] 1. An immunosuppressant comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. General formula _{(I) HO 2 C- (CH} 2) m -C (O) - ([X] -Arg-Gly-Asp- [Y]) n -O-CH 2 CH (OR 1) CH 2 OR 2 (I) In the formula, Arg, Gly and Asp represent arginine, glycine and aspartic acid residues, respectively. [X] and [Y] indicate amino acid or peptide residues. m represents an integer of 1 to 5. n represents an integer from 1 to 5. [X] and [Y] may or may not be present. R ₁ and R ₂ are each hydrogen or a linear or branched acyl group or alkyl group having 8 to 24 carbon atoms, and may have a substituent or may contain an unsaturated bond.
The asymmetric carbon present in the molecule may be either a racemic form or an optically active form. 2. In the general formula (I), [X], [Y]
Is an amino acid residue selected from serine, glycine, valine, asparagine, proline, cysteine, threonine, and aspartic acid residues or a peptide residue composed of such amino acid residues. Immunosuppressants. 3. An immunosuppressant comprising a compound represented by the following general formula (II) or a pharmaceutically acceptable salt thereof as an active ingredient. Formula (II) R ₃ -([X] -Arg-Gly-Asp- [Y]) _n -Z (II) In the formula, Arg, Gly and Asp represent arginine, glycine and aspartic acid residues, respectively. [X] and [Y] indicate amino acid or peptide residues. n represents an integer from 1 to 5. [X] and [Y] need not be present. R ₃ is a linear or branched acyl group having 6 to 24 carbon atoms, and may have a substituent or an unsaturated group. Z represents a hydroxyl group or an amino group. 4. In the general formula (II), [X], [Y]
Is an amino acid residue selected from serine, glycine, valine, asparagine, proline, cysteine, threonine, and aspartic acid residues, or a peptide residue composed of such amino acid residues. Immunosuppressants.