JPH07228565A - Spergualin analog and its use - Google Patents

Abstract

PURPOSE:To obtain a new compound having low toxicity, chemical stability and excellent immunosuppressive action. CONSTITUTION:A compound of the formula GU-X1-(CH2)a-CONH-X2- CONH-(CH2)4-NH-(CH2)3-NH-Y [Gu is guanidino; X1 is (CH2)5 or phenylene; (a) is 4-13; (X2 is (CH2)0-2-CH(R00)0-2; R00 is H, OH, OCH3 or CH2OH; Y is H or a residue after removal of OH from carboxyl of an amino acid or a peptide] or its salt such as 10-{N-(11-guanidinoundecanoyl)-r-amino-beta- hydroxybutanoyl}-1,5,10-triazadecane. The compound is obtained by removing a protecting group of a compound of the formula Gu-X1-(CH2)a-CONH-X2- CONH-(CH2)4-NR1-(CH2)3-NH-R2-R2 by reduction, acidolysis, hydrolysis, etc. The compound is useful for suppressing rejection in organ transplantation and skin graft and treating autoimmune diseases such as multiple sclerosis, hemolytic anemia, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a spagarin analog and its use. The spagarin analog of the present invention has low toxicity, has effects such as immunosuppression, and is expected as a medicine.

[0002]

2. Description of the Related Art Spagarine is Bacillus.
antibiotics produced by microorganisms belonging to the genus s) (Japanese Patent Laid-Open No. 57-48)
957), and many related compounds have been synthesized thereafter (JP-A-59-42356;
-185758; Peptide Chemistry (Pepti)
de chemistry) 1987, 671-67
4 pages, published by Protein Research Encouragement Society; JP-A-63-4524
(See No. 7).

[0003]

In the conventionally known spagarin and its analogs, a compound having a strong activity lacks chemical stability, while a chemically stable compound has a drawback that the activity is not so strong. Have. Therefore, there is a demand for the development of a spagarin analog that is chemically stable, highly active, and weakly toxic. Therefore, an object of the present invention is to provide a spagarin analog which is low in toxicity, chemically stable, and has an excellent immunosuppressive action.

[0004]

That is, when the present invention is outlined, the first invention is represented by the general formula [I]

Embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NH— (CH ₂ ) ₃ —NH—Y [I] [In the formula, Gu is a guanidino group. , X ₁ is — (CH ₂ ) ₅ —
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ) And Y represent a hydrogen atom or a residue obtained by removing a hydroxyl group from a carboxyl group of an amino acid or a peptide. ] It is related with the spagarin analog represented by these, and its pharmacologically acceptable salt.

The second aspect of the present invention is the general formula [II]

Embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NR ₁ — (CH ₂ ) ₃ —NH—R ₂ [II] [wherein Gu is guanidino group, X ₁ is - (CH _{2) 5} -
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ), R ₁ is an amino-protecting group, R ₂ is a residue obtained by removing a hydroxyl group from a carboxyl group of an amino-protecting group or an amino acid or peptide having a protected amino group (these side chains are protected. May have been). ] The protecting group is removed from the compound having the protecting group represented by

General formula [I]

Embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NH— (CH ₂ ) ₃ —NH—Y [I] [In the Formula, Gu is a guanidino group. , X ₁ is — (CH ₂ ) ₅ —
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ) And Y represent a hydrogen atom or a residue obtained by removing a hydroxyl group from a carboxyl group of an amino acid or a peptide. ] It is related with the manufacturing method of the spagarin analog represented by these, and its pharmacologically acceptable salt.

A third aspect of the present invention is the general formula [I]

Embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NH— (CH ₂ ) ₃ —NH—Y [I] [In the formula, Gu is a guanidino group. , X ₁ is — (CH ₂ ) ₅ —
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ) And Y represent a hydrogen atom or a residue obtained by removing a hydroxyl group from a carboxyl group of an amino acid or a peptide. ] It is related with the immunosuppressive agent which uses the spagarin analog represented by these, and its pharmacologically acceptable salt as an active ingredient.

The present invention will be described in more detail. When X in the general formula [1] represents a phenylene group, the phenylene group may have a substituent, and as the substituent, a halogen atom such as chlorine, fluorine or bromine; methyl,
Lower alkyl groups such as ethyl, propyl, t-butyl and pentyl; lower alkoxy groups such as methoxy, ethoxy, propoxy, t-butoxy, pentoxy and the like.

Examples of Y include a hydrogen atom or a residue obtained by removing a hydroxyl group from the carboxyl group of the following amino acid or the following peptide. The configuration of amino acid residues is S, R or RS except for glycine, β-alanine and γ-aminobutyric acid.

(1) Amino acids alanine, arginine, ornithine, aspartic acid,
Asparagine, cysteine, cystine, glutamic acid,
Glutamine, pyroglutamic acid, glycine, histidine, lysine, proline, hydroxyproline, isoleucine, leucine, methionine, phenylalanine, phenyl-substituted phenylalanine, serine, threonine, tryptophan, homoserine, tyrosine, valine, phenylglycine, parahydroxyphenylglycine, 4- Hydroxymethyl-3-hydroxyphenylglycine, β-
Alanine, γ-aminobutyric acid, 3-amino-2-hydroxy-4-phenylbutyric acid and the like.

(2) Peptide Two or more of the amino acids of the above (1) may be used alone or in combination.
Di- or tripeptides in which 3 to 3 amino acids are condensed are preferable. For example, alanylalanine, leucylleucine, valilvaline, phenylalanylphenylalanine, tyrosyltyrosine, phenylglycylphenylglycine, glycylglycine, isoleucylisoleucine,
Examples include leucylphenylalanine, phenylalanylleucine, leucylphenylglycine, phenylglycylleucine, glycylglycylglycine, phenylglycylphenylglycylphenylglycine, phenylalanylphenylalanylphenylalanine and leucylleucylleucine. .

Preferred amino acids or peptides include phenylglycine, phenylalanine, leucine,
Examples include aspartic acid, tryptophan, alanine, and peptides in which 2 to 3 of these amino acids are condensed. Preferred as Y are a hydrogen atom, an amino acid such as phenylglycine, and a dipeptide such as leucyl leucine.

The spagarin analog represented by the general formula [I] may form a salt with an acid. The acid for forming the salt may be an inorganic acid or an organic acid as long as it is non-toxic. The inorganic acid is not particularly limited, but hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like are preferable, and the organic acid is also not particularly limited, acetic acid, propionic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, Glutaric acid, citric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, aspartic acid, glutamic acid and the like are preferable.

In the general formula [I] of the present invention, the steric configuration of the carbon atom to which R ₀₀ is bonded is S, R or RS. The main compounds of the present invention are shown in Tables 1 to 6 below. The steric configuration of the carbon atom marked with * ₂ in the table is S, R or RS type as described above.

[0015]

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

The compound of the general formula [I] of the present invention is synthesized as follows. The following general formula [II]

Embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NR ₁ — (CH ₂ ) ₃ —NH—R ₂ [II] [wherein Gu is guanidino group, X ₁ is - (CH _{2) 5} -
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ), R ₁ is an amino-protecting group, R ₂ is a residue obtained by removing a hydroxyl group from a carboxyl group of an amino-protecting group or an amino acid or peptide having a protected amino group (these side chains are protected. May have been). ] It is obtained by removing a protecting group from a compound having a protecting group.

The removal of the protecting group can be carried out by a method known per se such as reduction, acid decomposition, hydrolysis and the like. Reactions such as reduction, acid decomposition and hydrolysis are usually carried out in an inert solvent.
It is carried out at 60 ° C to the boiling point of the solvent, preferably about -50 ° C to 100 ° C. As the inert solvent, water and hydrophilic organic solvents such as lower alcohols such as methanol and ethanol; ketones such as acetone and methyl ethyl ketone; amides such as dimethylformamide and dimethylacetamide; cyclic ethers such as tetrahydrofuran and dioxane; acetic acid, Lower fatty acids such as trifluoroacetic acid; liquid ammonia, liquid hydrogen fluoride and the like.

From the reaction solution from which the protecting group has been removed, a compound of the general formula [I]
Isolation of the spagarin analog of, for example, when the protecting group is removed by catalytic reduction with palladium black, the catalyst is filtered off,
The filtrate was concentrated under reduced pressure, and the residue was purified by a known purification method using CM-Sephadex (registered trademark) C-25 (Na ⁺ ) and Sephadex (registered trademark) LH-20 (T. Take).
uchi et al. J. Antibiotic
s, 34, 1619 (1981)). When the protecting group is removed with trifluoroacetic acid, the reaction solution can be concentrated under reduced pressure, and the residue can be isolated by purifying by a known purification method similar to the above.

By the above-mentioned purification method, the spagarin analog of the general formula [I] can be obtained as a salt such as hydrochloride.
When these salts are converted to other salts, for example, the hydrochloride is dissolved in water and the aqueous solution is passed through a strongly basic ion exchange resin, and the fractions containing the desired product are collected to obtain the desired acid, an aqueous solution containing it or methanol. , Ethanol, acetone,
It can be carried out by adding a solution of a hydrophilic organic solvent such as tetrahydrofuran or dioxane to neutralize, and drying the neutralized solution under reduced pressure, or when an organic solvent is included, the organic solvent is distilled off under reduced pressure and then freeze-dried. . Alternatively, for example, a hydrochloric acid salt of a compound of the general formula [I] is added with an aqueous solution of silver hydroxide or silver oxide to neutralize hydrochloric acid, insoluble silver chloride is filtered off, and a desired acid is added to the filtrate to form a salt, which is then lyophilized. It is done by doing.

The target product obtained by the above method may have a hydrate depending on the treatment conditions, and these hydrates are also one form of the compound of the present invention. The protected spagarin analog of the general formula [II], which is a raw material of the spagarin analog of the present invention, is synthesized as follows.

A) When R ₁ and R ₂ both represent an amino-protecting group, the compound of the general formula [II] is synthesized as follows. As described in JP-A-59-42356, the general formula [III]

Embedded image H ₂ N— (CH ₂ ) ₄ —NR ₁ — (CH ₂ ) ₃ —NH—R ₂ ′ [III] (In the formula, R ₁ and R ₂ ′ represent a protecting group for an amino group. To the diprotected spermidine represented by the formula [IV]

Embedded image P—NH— (CH ₂ ) _0-2- CH (R ₀₀ )-(CH ₂ ) _0-2- COOH [IV] (In the formula, P is an amino group different from R ₁ and R ₂ ′. The protecting group R ₀₀ has the same meaning as described above.) And a reactive derivative of the protected amino acid represented by

Embedded image P—NH— (CH ₂ ) _0-2 —CH (R ₀₀ ) —
_{(CH 2) 0-2 -CONH- (CH} 2) 4 -NR 1 -
(CH ₂ ) ₃ —NH—R ₂ ′ (wherein P, R ₁ and R ₂ ′ have the same meanings as described above), and then the protecting group P of the amino group is removed. V]

Embedded image A reactive derivative of ω-guanidino fatty acid represented by Gu—X ₁ — (CH ₂ ) _a —COOH [V] (wherein X ₁ and a have the same meanings as described above) is reacted. To obtain a compound represented by the general formula [II].

(B) Of the compounds represented by the general formula [II], residues in which a hydroxyl group is removed from the carboxyl group of an amino acid or peptide having an amino group in which R ₂ is protected (these side chains are protected by these residues). The compound having the formula (1) may be synthesized as follows. A compound obtained by removing the protecting group for R ₂ from a compound synthesized by the method (a), wherein R ₁ and R ₂ are both amino groups (of the compounds represented by the general formula [I], Y is a hydrogen atom). Compound represented by the formula [VI]

Embedded image A reactive derivative of a protected amino acid or a protected peptide represented by Y—OH [VI] (wherein Y has the same meaning as described above) is reacted,
It can be a compound represented by the general formula [II].

The condensation in the above (a) and (b) is
Conventional methods used for peptide conjugation can be used. That is, N, N'-dicyclohexylcarbodiimide, 1-
Carbodiimide method using ethyl-3- (3-dimethylaminopropyl) -carbodiimide, azide method from hydrazide, mixed acid anhydride method using ethyl chlorocarbonate, isobutyl chlorocarbonate, cyanomethyl ester, vinyl ester, substitution and Active ester method using unsubstituted phenyl ester, thiophenyl ester, hydroxysuccinimide ester, etc., acetoxime,
Examples thereof include an O-acyl hydroxylamine derivative method using cyclohexanone oxime and the like, an N-acyl compound method using carbonyldiimidazole and the like, and a carboxylic acid activation method using 1,3-thiazolidine-2-thione.

As a solvent used for condensation, a solvent used for ordinary peptide bonds can be used. For example, ethers such as diethyl ether, tetrahydrofuran, dioxane; esters such as ethyl acetate; ketones such as acetone and methyl ethyl ketone; halogenated hydrocarbons such as methylene chloride and chloroform; amides such as dimethylformamide, dimethylacetamide; acetonitrile. In the case of a solvent in which nitriles and the like are mixed alone or with water, they can be used as a mixed solvent with water.

Examples of the amino group-protecting group that can be used in the present invention include substituted benzyloxycarbonyl groups such as benzyloxycarbonyl group and p-methoxybenzyloxycarbonyl group, t-butyloxycarbonyl group, t-amyloxycarbonyl group, Formyl group, trityl group, O-nitrophenylsulfenyl group and the like can be mentioned.

The protective group for a carboxyl group, which is a protective group for an amino acid side chain, is a lower alkyl group such as t-butyl group, benzyl group, substituted benzyl group, etc .; The protective group for a hydroxyl group is t-butyl group, benzyl group. Etc .; benzyl group, p-methoxybenzyl group etc. as a mercapto group protecting group;
Examples of the protective group for the imidazole group include benzyloxycarbonyl group, benzyl group and tosyl group; and examples of the protective group for guanidine group include nitro group, tosyl group and t-butyloxycarbonyl group, but they are not limited thereto. Not a thing.

When the compound of the present invention is used as an immunosuppressant, it is administered alone or in admixture with an excipient or carrier as an injection, an oral preparation, a suppository or the like. As the excipient and the carrier, those which are pharmaceutically acceptable are selected, and the type and composition thereof depend on the administration route and administration method. For example, water, alcohols or soybean oil, animal or vegetable oils such as peanut oil, sesame oil, mineral oil or synthetic oil is used as a liquid carrier, and lactose, maltose, sugars such as sucrose, amino acids, hydroxypropyl cellulose and the like are used as a solid carrier. A cellulose derivative, an organic acid salt such as magnesium stearate, or the like is used.

Excipients used in injections are mannitol,
Maltose, dextran, lactose, cyclodextrin, chondroitin sulfate, gelatin, human serum albumin, but maltose, lactose, chondroitin sulfate,
Gelatin and human serum albumin are preferred. A lyophilized preparation together with these excipients, and a suitable solvent for injection at the time of administration, such as sterile water, physiological saline, glucose solution,
It can also be administered by dissolving it in an ordinary liquid for intravenous administration such as an electrolyte solution or an amino acid solution.

An acid, an alkali or an appropriate amount of a buffer may be added to the composition of the preparation of the present invention for the purpose of adjusting pH and the like.

The content of the compound of the present invention in the preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1 to 98% by weight. For example, in the case of injection solution,
It is preferable to contain the active ingredient in an amount of usually 0.1 to 30% by weight, preferably 1 to 10% by weight. For oral administration, tablets together with the solid carrier or liquid carrier,
It is used in the form of capsules, powders, granules, liquids, dry syrups and the like. Capsules, tablets, granules and powders generally contain 5-100% by weight, preferably 25-98% by weight, of the active ingredient.

The dose is determined depending on the patient's age, body weight, symptoms, therapeutic purpose, etc.
01-100 mg / kg · day, preferably 1-50 mg
/ Kg · day, orally administered 5-1000 mg / kg · day,
It is preferably 2 to 500 mg / kg · day. The compounds of the present invention are characterized by relatively low toxicity and small accumulation of toxicity upon continuous administration. Therefore, the compounds of the present invention are various diseases thought to be caused by hyperimmunopathy, for example, suppression of rejection in organ transplantation or skin transplantation, autoimmune diseases thought to be mainly caused by various autoimmunity, such as multiple sclerosis. , Hemolytic anemia, type I diabetes, myasthenia gravis, Hashimoto's thyroiditis, Behcet's syndrome, rheumatism and allergic diseases, and renal diseases based on autoimmune diseases, such as nephrosis, are also therapeutic agents for those diseases It is an immunosuppressant that can be effectively used as.

[0032]

The stability and physiological activity of the spagarin analog represented by the general formula [I] in an aqueous solution will be specifically described with reference to test examples. Test example 1. Stability test method of the compound of the present invention in aqueous solution The compound of the present invention is dissolved in water so as to be 0.5 (w / w)%. This aqueous solution was kept at 40 ± 1 ° C., sampled at regular intervals, the peak area ratio was determined by high performance liquid chromatography, and the residual rate was calculated. Start at 100
Table 7 shows the residual rate (%) of the compound of the present invention after the lapse of a certain period of time when it is defined as%. As the control substance, in the general formula _{[I], X 1 = -} (CH 2) 5 -, a = 1, X
_{2 = - (CH 2) 2} -CH (OH) -CO-, using a known compound of Y = H.

[0033]

[Table 7] As shown in Table 7, the compounds of the present invention are almost stable even after 1 week.

2. Immunosuppressive effect in rat skin transplantation Test method (a) Rat skin transplantation experiment was performed using male WKAH (RT1 ^k )
It was carried out by implanting a piece of tail skin (5 × 10 mm) of a rat (7 to 10 weeks old) on the back of a male F344 (RT1 ^1v1 ) rat (7 to 10 weeks old). The immunosuppressive effect when the compound of the present invention is intraperitoneally administered every day for 10 days from the day after transplantation is shown by the average number of days of engraftment of transplanted skin pieces. As the control substance, the same known compound as that used in Test Example 1 was used. The results of the rat skin transplantation experiment are shown in Table 8.

[0035]

[Table 8]

[0036]

EXAMPLES Next, the production method of the compound represented by the general formula [I] and the preparation of the present invention will be explained concretely with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1 10- {N- (11-guanidinoundecanoyl) -γ
-Amino-β- (R) -hydroxybutanoyl} -1,
5,10-Triazadecane-3-hydrochloride (Compound No. 1)
Synthesis of (a) 10- {N- (11-guanidinoundecane
) -Γ-Amino-β- (R) -hydroxybutanoi
Ru} -1,5-di-benzyloxycarbonyl-1,
5,10-Triazadecane / hydrochloride 11-guanidinoundecanoic acid / hydrochloride 6.04 g (2
1.58 mmol) was dissolved in 60 ml of dimethylformamide, and N-hydroxysuccinimide 2.98 under ice cooling.
g (25.89 mmol) and N, N'-dicyclohexylcarbodiimide (5.35 g, 25.89 mmol) are added, and the mixture is reacted overnight at room temperature. The precipitate is filtered off and the filtrate is used as it is in the next reaction.

Oily 10- (γ-amino-β- (R)-
Hydroxybutanoyl) -1,5-di-benzyloxycarbonyl-1,5,10-triazadecane hydrochloride 9.95 g (equivalent to 8.0 mmol) was dissolved in 60 ml of dimethylformamide, and triethylamine 2.0 was added under ice cooling.
g (19.77 mmol) was added, then 11-
A solution of guanidinoundecanoic acid hydrochloride N-hydroxysuccinimide ester in dimethylformamide is added, and the mixture is reacted at room temperature overnight. The reaction solution is concentrated under reduced pressure, the oily residue is washed twice with 100 ml of n-hexane, and then concentrated under reduced pressure.

The resulting oily substance was subjected to column chromatography on silica gel 60 (manufactured by Merck), and chloroform-methanol 17% aqueous ammonia (6: 2.5:
When developed with a mixed solution of 0.5 v / v), 8.0 g (yield 57.26%) of an oily substance is obtained.

NMR (CD ₃ OD + D ₂ O, exter
nal TMS) delta = 1.2-2.2 (m, 22H), 2.2-2.8.
(M, 4H), 3.1 to 3.8 (m, 12H), 4.0
~ 4.6 (m, H), 5.25 (S, 2H), 5.28
(S, 2H), 7.56 (S, 10H). TLC (chloroform: methanol: 17% aqueous ammonia = 6: 2.5: 0.5%) Rf = 0.45

(B) 10- {N- (11-guanidinoundecanoyl) -γ-amino-β- (R) -hydroxybutanoyl} -1,5-di-benzyloxycarbonyl-1,5,10 -Triazadecane / hydrochloride 8.0 g
(10.30 mmol) was dissolved in 60 ml of methanol, 3 ml of acetic acid and 1.0 g of palladium black were added, and the mixture was stirred at room temperature.
Catalytic reduction is performed at atmospheric pressure for 6 hours.

After the reaction, the catalyst was filtered off and the filtrate was concentrated under reduced pressure to obtain 7.7 g (yield quantitative) of an oily substance. This oily substance was dissolved in 100 ml of distilled water and applied to a column packed with 500 ml of CM-Sephadex (registered trademark) C-25 (Na ⁺ ), and a column between 2500 ml of distilled water and 2500 ml of 0.8 M aqueous sodium chloride solution was added. Elute by the gentle elution method, collect the fractions containing the desired product, concentrate to dryness under reduced pressure, add methanol to the dried product, and filter off the insoluble sodium chloride. The desired product is purified from the obtained oily substance as follows.

The oil obtained in order to remove the small amount of residual sodium chloride is dissolved in 40 ml of methanol,
Apply to a column packed with 330 ml of Sephadex (registered trademark) LH-20, elute with methanol, collect fractions containing the target substance, and concentrate under reduced pressure. The obtained oily substance is dissolved in 45 ml of distilled water, and the insoluble matter is filtered off and freeze-dried to obtain 4.90 g of the desired product (yield 81.93%).

NMR (D ₂ O, 200 MHz) δ = 1.18 to 1.46 (m, 12H), 1.47 to
1.83 (m, 8H), 1.98 to 2.19 (m, 2)
H), 2.21 to 2.54 (m, 4H), 3.03 to
3.4 (m, 12H), 4.03 to 4.20 (m,
H). IR (KBr) ν (cm ⁻¹ ) = 3310, 3160, 2930, 285
0, 1640, 1545, 1455, 1410. [Α] ²⁰ _D −1.4 ° (C = 1.00, H ₂ O)

Example 2 10- {N- (11-guanidinoundecanoyl) -γ
-Amino-β- (R) -hydroxybutanoyl} -1-
Synthesis of L-leucyl-L-leucyl-1,5,10-triazadecane-3-hydrochloride (Compound No. 2) (a) 10- {N- (11-guanidinoundecaneoi
) -Γ-Amino-β- (R) -hydroxybutanoi
Ru} -1-benzyloxycarbonyl-L-leucyl-
L-leucyl-1,5,10-triazadecane dihydrochloride
Salt white crystals of 10- {N- (11-guanidinoundecanoyl) -γ-amino-β- (R) -hydroxybutanoyl} -1,5,10-triazadecane-3-hydrochloride (Compound No. 1) 1 0.1 g (1.89 mmol) was dissolved in 30 ml of methanol, and N-methylmorpholine 0.2 was added under ice cooling.
1 g (2.07 mmol) is added. Then N-benzyloxycarbonyl-L-leucyl-L-leucine and N
-Ester with hydroxysuccinimide 1.08 g
(2.27 mmol) is added, and the mixture is reacted under ice cooling for 1.5 hours.

The reaction solution was concentrated under reduced pressure, the oily residue was suspended in 60 ml of acetone, and the supernatant was decanted. This procedure was repeated twice, and then concentrated under reduced pressure to obtain 2.2 g of the desired product as white crystals (yield). Quantitative) is obtained. TLC (chloroform: methanol: 17% aqueous ammonia = 6: 2.5: 0.5 v / v) Rf = 0.4

(B) 10- {N- (11-guanidino
Undecanoyl) -γ-amino-β- (R) -hydroxy
Cibutanoyl} -1-L-leucyl-L-leucyl-
1,5,10-Triazadecane-3-hydrochloride 10- {N- (11-guanidinoundecanoyl) -γ
-Amino-β- (R) -hydroxybutanoyl} -1-
Benzyloxycarbonyl-L-leucyl-L-leucine-1,5,10-triazadecane dihydrochloride 2.2
The desired product can be obtained by using g in the same manner as in (b) of Example 1.

NMR (D ₂ O, 200 MHz) δ = 0.83 to 1.07 (m, 12H), 1.19 to
1.42 (m, 12H), 1.46 to 1.82 (m, 1
4H), 1.82 to 2.01 (m, 2H), 2.19 to
2.53 (m, 4H), 2.93 to 3.12 (m, 4)
H), 3.12 to 3.41 (m, 8H), 3.91
4.04 (t, H, J = 7.11), 4.04 to 4.2
0 (m, H), 4.26 to 4.42 (t, H, J = 7.
37). [Α] ²⁰ _D −1.3 ° (C = 1.00, H ₂ O)

Example 3 10- {N- (15-guanidinopentadecanoyl)-
γ-amino-β- (R) -hydroxybutanoyl}-
Synthesis of 1,5,10-triazadecane-3-hydrochloride (Compound No. 17) (a) 10- {N- (15-guanidinopentadecano
Yl) -γ-amino-β- (R) -hydroxybutanoi
Ru} -1,5-di-benzyloxycarbonyl-1,
5,10-Triazadecane / hydrochloride 15-guanidinopentadecanoic acid / hydrochloride 2.82 g
(8.39 mmol) was used in the same manner as in (a) of Example 1 to obtain 3.1 g of the oily target product (yield 5
2.45%) is obtained.

NMR (CD ₃ OD + D ₂ O, exter
nal TMS) delta = 1.0-2.8 (m, 34H), 2.9-3.7.
(M, 12H), 3.9 to 4.4 (m, H), 5.17
(S, 4H), 7.4 (S, 10H). IR (KBr) ν (cm ⁻¹ ) = 3300, 2920, 2860, 165
0,1540,1445,1380,1250,109
0. TLC (chloroform: methanol: 17% aqueous ammonia = 6: 2.5: 0.5 v / v) Rf = 0.48

(B) 10- {N- (15-guanidino
Pentadecanoyl) -γ-amino-β- (R) -hydro
Xybutanoyl} -1,5,10-triazadecane / 3
Hydrochloride 10- {N- (15-guanidinopentadecanoyl)-
γ-amino-β- (R) -hydroxybutanoyl}-
1,5-di-benzyloxycarbonyl-1,5,10
-Triazadecane hydrochloride 3.1 g (3.72 mmo
l) is dissolved in 40 ml of methanol and 10 ml of acetic acid, 0.7 g of palladium black is added, and catalytic reduction is performed at room temperature and atmospheric pressure for 6 hours.

After the reaction, the catalyst was filtered off, and the filtrate was concentrated under reduced pressure to obtain 4.1 g (yield quantitative) of an oily substance. This oily substance was dissolved in 30 ml of distilled water, and Mitsubishi Kasei (registered trademark) H
Apply to a column packed with 300 ml of P-20, and elute by a gradient elution method between 1500 ml of distilled water and 1500 ml of 40% methanol. Fractions containing the desired product were collected and concentrated to dryness under reduced pressure to obtain 1.9 g of a dried product.

1.9 g of this dry solid was dissolved in 70 ml of distilled water, and CM-Sephadex (registered trademark) C-25 (N
a ⁺ ) A column filled with 200 ml of distilled water 10
00 ml and 1.2 M sodium chloride aqueous solution 1000 ml
Elute by the gradient elution method between and, collect the fractions containing the desired product, concentrate to dryness under reduced pressure, add methanol to the dry product, and filter off the insoluble sodium chloride. To remove a small amount of sodium chloride remaining in the obtained oil, the oil was dissolved in 30 ml of methanol and applied to a column packed with 200 ml of Sephadex (registered trademark) LH-20 and eluted with methanol, Fractions containing the desired product are collected and concentrated under reduced pressure. The obtained oily substance is dissolved in 10 ml of distilled water, and the insoluble substance is filtered off and freeze-dried to obtain 0.83 g of the desired product (yield 35.02%).

NMR (D ₂ O, 200 MHz) δ = 1.12 to 1.46 (m, 20H), 1.46 to
1.82 (m, 8H), 1.97 to 2.17 (m, 2)
H), 2.18 to 2.53 (m, 4H), 2.97 to
3.38 (m, 12H), 3.99 to 4.20 (m,
H). IR (KBr) ν (cm ^-1 ) = 3320, 3160, 2930, 286
0, 1645, 1545, 1460. [Α] ²⁰ _D −0.9 ° (C = 1.00, H ₂ O)

Using the compounds corresponding to the general formula [II] below, the corresponding compounds of the general formula [I] were obtained in the same manner as in the step (b) of Example 1 above. In the compound of the general formula [II], the amino-protecting group is te
In the case of rt-butyloxycarbonyl group, 4N hydrochloric acid-
Deprotection was performed using dioxane solution.

Further, a benzyl group was used as a protective group for a hydroxyl group such as serine, and the treatment method in that case was catalytic reduction. When the protective groups for the amino group and the hydroxyl group are different, first, 4N hydrochloric acid-
Treatment with dioxane followed by catalytic reduction.

Next, in the case of producing a compound such as compound No. 2, the corresponding compound of the present invention (in the same manner as in step (a) of Example 2 and then step (b) of Example 1) General formula [I]) was obtained.

Example 4 Production of Tablets 30 parts by weight of Compound No. 1 or 2, 120 parts by weight of crystalline lactose, 147 parts by weight of crystalline cellulose and 3 parts by weight of magnesium stearate were tabletted with a V-type mixer to give 1 tablet 300 m.
g tablets were obtained.

[0058]

As is clear from the results shown in Tables 7 and 8, the compound represented by the general formula [I] of the present invention is stable in an aqueous solution, and the number of days of skin engraftment after skin transplantation is Has a prolonging action. Therefore, the compound represented by the general formula [I] in the present invention is extremely useful for suppressing rejection in organ transplantation and treating autoimmune diseases.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C07K 5/062 8318-4H (72) Inventor Fumiko Kobayashi 3-13-9 Koishi Ward, Koto-ku, Tokyo 602 (72) Inventor Takesako Kazuo 3-4-1 Seta, Otsu City, Shiga Prefecture

Claims (7)

[Claims] 1. A compound represented by the general formula [I]: embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NH— (CH ₂ ) ₃ —NH—Y [ I) (wherein, Gu is guanidino group, X ₁ is - (CH _{2) 5} -
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ) And Y represent a hydrogen atom or a residue obtained by removing a hydroxyl group from a carboxyl group of an amino acid or a peptide. ] The spagarin analog represented by these, and its pharmacologically acceptable salt. 2. General formula [II] embedded image Gu-X _1- (CH ₂ ) _a -CONH-X ₂ -CONH- (CH ₂ ) ₄ -NR _1- (CH ₂ ) ₃ -NH-R ₂ [II] [In the formula, Gu is a guanidino group, and X ₁ is-(CH ₂ ) _5-.
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ), R ₁ is an amino-protecting group, R ₂ is a residue obtained by removing a hydroxyl group from a carboxyl group of an amino-protecting group or an amino acid or peptide having a protected amino group (these side chains are protected. May have been). ] The protecting group is removed from the compound having the protecting group represented by the formula [I] embedded image Gu-X _1- (CH ₂ ) _a -CONH-X ₂ -CONH- (CH _{2) 4 -NH- (CH 2)} 3 -NH-Y (I) wherein, Gu is guanidino group, X ₁ is - (CH _{2) 5} -
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ) And Y represent a hydrogen atom or a residue obtained by removing a hydroxyl group from a carboxyl group of an amino acid or a peptide. ] The manufacturing method of the spagarin analog represented by these, and its pharmacologically acceptable salt. 3. A compound represented by the general formula [I]: embedded image Gu—X ₁ — (CH ₂ ) _a —CONH—X ₂ —CONH— (CH ₂ ) ₄ —NH— (CH ₂ ) ₃ —NH—Y [ I) (wherein, Gu is guanidino group, X ₁ is - (CH _{2) 5} -
Or a phenylene group which may have a substituent, a is 4 to 13
, X ₂ is-(CH ₂ ) _0-2 -CH (R ₀₀ )-(C
H ₂ ) _0-2- (wherein R ₀ is -H, -OH, -OCH
₃ or an -CH ₂ OH. ) And Y represent a hydrogen atom or a residue obtained by removing a hydroxyl group from a carboxyl group of an amino acid or a peptide. ] The immunosuppressive agent which uses the spagarin analog represented by these, and its pharmacologically acceptable salt as an active ingredient. 4. A spagarin analog represented by the general formula [I] and a salt thereof are 10- {N- (11-guanidinoundecanoyl) -γ-amino-β-hydroxybutanoyl} -1,5. The immunosuppressive agent according to claim 3, which is 10-triazadecane and a salt thereof. 5. The spagarin analogue represented by the general formula [I] and its salt are 10- {N- (11-guanidinoundecanoyl) -γ-amino-β-hydroxybutanoyl} -1-L-. Leucyl-L-leucyl-1,5,10
-The immunosuppressive agent according to claim 3, which is triazadecane and a salt thereof. 6. Spagarins represented by the general formula [I]
Rim and its salt X ₂-CH (R₀₀)-Is -C
H (OH)-and the optical configuration is S, RS or R
The immunosuppressive agent according to claim 3, which is only one kind. 7. The immunosuppressive agent according to claim 3, wherein the immunosuppressive agent is a rejection inhibitor in tissue transplantation or an autoimmune disease therapeutic agent.