KR800000076B1 - Process for the preparation of polypeptides - Google Patents

Abstract

Polypeptides (I; R1 = H, basic or neutral amino acid residue ; R2 = neutral amino acid residue ; R3 = NH2, TyrNH2, tyrocylpeptide reidue having 1-5 amino acid residue, amide residue of tyrocylpeptide) were prepd. by condensation polymn. of one part of polypeptide consisted with amino acid residue, or arginine (R1 = H) with the remains of polypeptide or by isolation of protecting group of condensation products.

Description

[Name of invention]

Preparation of Polypeptides

Detailed description of the invention

The present invention relates to the preparation of novel polypeptides useful as pharmaceuticals.

The object of the present invention is represented by the following formula.

Where

R ₁ is hydrogen or a basic or neutral amino acid residue,

R ₂ is a neutral amino acid residue,

R ₃ represents NH ₂ , Tyr-NH ₂ or a tyrosyl peptide residue of 1 to 5 amino acid residues or an amide residue of a tyrosyl peptide. However, when R ₁ mentioned above is hydrogen, R ₂ represents Pro.

In the present specification and claims, amino acids, peptides and their activators, protecting groups and activators, protecting group introducing agents are denoted by biological nomenclature by abbreviations by the IUPAC-IUB Commission or by conventions commonly used in the art.

Such an abbreviation is illustrated below.

α-Abu: α-aminobutyric acid

γ-Abu: γ-aminobutyric acid

ε-Acap: ε-aminocaproic acid

Ala: Alanine

β-Ala: β-alanine

Arg: Arginine

δ-Aval: δ-amino valeric acid

α, γ-Dab: a.r-diaminobutyric acid

Gly: glycine

His: histidine

Leu: leucine

Lys: Lysine

Nle: norleucine

Phe: Phenylalanine

Pro: Proline

Sar: sarcosine

Ser: Serine

Thr: Threonine

Tyr: tyrosine

Val: Valine

Aoc: t-amyloxycarbonyl

Bzl: Benzyl Ether

D.CC: N, N'-dicyclohexylcarbodiimide

HONB: N-hydroxy-5-norbornene-2, 3-dicarboxyimide

OBzl: Benzil Star

ODNP: 2, 4-dinitrophenyl ester

OEt: ethyl ester

ONB: HONB Ester

Opcp: Chlorophenyl Ester

Osu: N-hydroxysuccinimide ester

OtBu: t-butyl ester

Tos: Tossil

Z: benzyloxycarbonyl

In the description of the present specification and claims, when an optically active substance is present for an amino acid, all amino acids thereof mean an optically active substance and a racemate, unless otherwise specified.

When it comes to the following structural formula (Ⅰ), R ₁ complete sweep, group R ₁ -Arg; means a group ginil are as follows.

In the description of the present specification and claims, the term "amino acid residue" means a group capable of forming a peptide bond, that is, a group in which one or both of the hydroxyl groups of hydrogen and carboxyl groups of the amino group or amino group of the amino acid and the imino group are missing. do.

The basic or neutral amino acid represented by R ₁ is preferably 2 to 10 carbon atoms. As such a basic amino acid, (alpha)-amino acid (for example, (alpha),-diamino propionic acid, (alpha), (gamma)-diaminobutyric acid, arginine, lysine, or histidine) can be illustrated, for example. Neutral amino acids represented by R ₁ include, for example, α-neutral amino acids (e.g., glycine, alanine, serine, threonine, α-aminobutyric acid, prolyl, leucine, isoleucine, norleucine, phenylalanine or tyrosine) and other neutrals. Amino acids (eg, β-alanine, γ-aminobutyric acid, δ-aminovaleric acid or ε-aminocaproic acid) can be exemplified. With respect to the amino acid represented by R ₁ , when there is an optically active substance, all of the amino acids respectively represent the optically active substance and the racemate. Among the amino acids corresponding to R ₁ , β-alanine is preferred for certain applications.

The neutral amino acid represented by R ₂ preferably has 2 to 10 carbon atoms, for example, glycine, alanine, β-alanine, sarcosine, serine, proline, valine, leucine, isoleucine, phenylalanine or tyrosine. have. Among the amino acids corresponding to R ₂ , glycine and proline are preferred for certain applications.

In the case where R ₂ is a proline residue, certain uses are achieved even when R ₁ is hydrogen.

Each amino acid capable of constituting the tyrosyl peptide represented by R ₃ or an amide thereof is preferably 2-10 carbon atoms, for example, glycine, alanine, serine, threonine, valine, proline, leucine, isoleucine, lysine , Phenylalanine or tyrosine can be exemplified. These amino acids combine in any order to form tyrosyl peptides or amides of 1-5 amino acid residues. A tyrosyl peptide is illustrated below. Their amides are exemplified as tyrosyl peptide amides.

When the obtained body is optically active with respect to all the amino acid residues other than R _1, which can configure the polypeptide (Ⅰ) is, these amino acids are all preferable because it means that each optically active substance and racemate, but, L body of low toxicity .

Polypeptide (Ⅰ) of the present invention, to form a polypeptide (Ⅰ) the amino acid or the R ₁ corresponding to R ₁ which is hydrogen-arginine, or as an N- terminal amino acid of amino acid residues R ₁ or R ₁ is a hydrogen group are carbonyl groups A part of the peptide component to be prepared is prepared by peptide condensation with the rest of the polypeptide (I). The two kinds of raw materials for peptide condensation may be appropriately protected from the amino group and the carboxyl group that do not participate in the predetermined condensation reaction, and the group involved in the predetermined condensation reaction may be appropriately activated. Therefore, when the condensation product has a protecting group, the protecting group is appropriately removed.

The condensation reaction of the present invention can be carried out according to a conventional method capable of forming a peptide bond.

The condensation reaction of the present invention can be carried out at any position of the polypeptide (I). When the condensation reaction of the present invention is carried out between the third amino acid and the fourth phenylalanine corresponding to R ₂ , the polypeptide (I) can be produced industrially advantageously.

Similarly to known polypeptides, the polypeptide (I) of the present invention is prepared by sequentially condensing each of the amino acids that can constitute it according to its amino acid sequence. Usually, the final condensation step is performed between two constituents, namely, a peptide and a terminal amino acid or two peptides. These two components are raw materials for producing the polypeptide (I) of the present invention. Their raw materials or final intermediates are prepared by known peptide synthesis means.

One of the raw materials which can be used for this invention, for example, the N-terminal amino acid of polypeptide (I), or each of the amino acids which comprise polypeptide (I) in the N-terminal amino acid thereof is sequentially according to the amino acid sequence. The combined thing can be illustrated. Another raw material corresponding thereto is the remainder of the polypeptide (I) except the above raw material. The basic combination of such raw materials is listed in the following table.

TABLE 1

rder and Lubke, Academic press, New 4York, U.S.A.]을 들 수가 있다.In the present invention, the condensation reaction can be carried out by a known condensation method that can be used for peptide bond formation. As the condensation method, DCC / HONB method (especially in Belgium 763.399), azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method active ester method, Woodward reagent K is used. Carbodiimidazole method, redox method ["The peptides"). Vol. 1 (1966), Sch

rder and Lubke, Academic press, New 4York, USA.

Prior to the condensation reaction, a known method may protect the carboxyl group or amino group which will not be involved in the predetermined condensation reaction, or may activate the carboxyl group or (and) amino group which will be involved in the predetermined condensation reaction. Carboxyl groups not involved in certain condensation reactions are protected in the form of metal salts (eg sodium, potassium salts) or esters (eg esters such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl or t-amyl) You may also

As a protecting group of the raw amino group, an amino protecting group is used in known peptide synthesis. Examples thereof include benzyloxy carbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, and the like. There is a number. These protecting groups can also be used for protecting the imino group of proline. The imidazole functional group of histidine may be protected with a known protecting group, for example benzyl, tosyl, 2,4-dinitrophenol, t-butoxycarbonyl, or carbobenzox. The hydroxyl group of serine, tyrosine or threonine may be protected by a known protecting group, for example, ether type such as benzyl and t-butyl. The guanidino group of arginine may be protected with, for example, a nitro, toxin, carbobenzoxyl, isobornyloxycarbonyl or adamantyloxycarbonyl group. Examples of activated carboxyl groups of the raw materials include the corresponding acid anhydrides, azides, active esters (eg pentachlorophenyl, 2,4,5-trichlorophenyl, 2,4-dinitrophenyl, cyanomethyl alcohol and alcohols such as p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboxyimide, N-hydroxysuccinimide N-hydroxyphthalimide or N-hydroxybenztriazole Esters). As an activated amino group of a raw material, the corresponding phosphate amide is mentioned, for example.

The combination example of the above state of the carboxyl and amino group of two raw materials A and the raw material B is shown in the following table.

TABLE 2

Note: In the case of ^* , it is preferred to have a dehydrating agent (eg a carbodiimide reagent such as dicyclohexyl-carbodiimide) in the reaction system.

This reaction can be carried out in the presence of a solvent. The solvent may be appropriately selected from those known to be used for the peptide condensation reaction. For example, anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran or a suitable mixture thereof can be mentioned.

The reaction temperature is appropriately selected from the ranges known to be used for the peptide bond formation reaction, and is usually appropriately selected in the range of about -20 ° C to about 30 ° C. In addition, the precursor of the compound of the present invention: a protective peptide) can be easily produced by solid phase synthesis.

After the completion of the condensation reaction, when the product has a protecting group, it can be separated by a conventional method. As such a conventional method, for example, catalytic reduction using palladium black, palladium-carbon, platinum, or the like as a catalyst, hydrolysis by hydrogen fluoride, trifluoroacetic acid or the like, reduction by metal sodium in liquid ammonia, or the like can be used.

The polypeptide (I) thus prepared can be recovered by means of separation of the peptide from the mixture after the reaction is completed, for example, extraction, chromatography, chromatography and the like.

Polypeptide (I) can also be recovered as a salt or a metal complex.

As the acid capable of forming a salt of the polypeptide (I), for example, inorganic acids such as hydrochloric acid, hydrochloric acid, perchloric acid, acetic acid, thiocyanic acid, sulfuric acid, phosphoric acid, or for example, acid, acetic acid, propionic acid, glycolic acid, Organic acids such as lactic acid, pyruvic acid, oxalic acid, malonic acid, citric acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid, and the like.

As a metal which can form the metal complex compound of polypeptide (I), zinc, nickel, cobalt, copper iron, etc. are mentioned, for example. Such metal complexes can be prepared by known means, for example by reacting polypeptide (I) with a hydroxide or oxide of the metal at a pH of about 6-8.

Polypeptide (I) of the present invention has the effect of lowering high concentrations of glucose (glucose) in blood and urinary tract to normal levels not only by parenteral but also by oral administration. Polypeptide (I) has an unexpected and distinctive action that does not lower the blood or urine equivalent to normal levels, and this action is remarkably superior to insulin causing hypoglycemia. Polypeptide (I) is safely administered with low toxicity. Polypeptide (I) also has an action of promoting fat metabolism. Due to the peculiar action as described above, polypeptide (I) is used for the treatment of glucose metabolism or fat metabolism in humans or other animals (eg rats or birds), in particular in the treatment of diabetes mellitus or hypercholesteremia. It is administered effectively without causing side effects. Polypeptide (I) may be administered by itself, or may be administered as a formulation (e.g., tablet, pellet, granule, powder, liquid, injection) prepared by a conventional method.

Prescription examples of each formulation are listed below.

Injection

Powder

The daily dose of polypeptide (I) is the type of disease. Although it changes with a symptom, the age of a target animal, etc., it normally selects suitably from the range of 50 micrograms-about 100 mg per kilogram of body weight.

Pharmacological Experiment of Diabetes Treatment Effect by Polypeptide (I) Artificial diabetic rats (KKA ^y, yellow KK rats, 8 weeks old, cut) are placed in metal cages one by one and urine is collected.

Three groups of polypeptides (I) of formula (β-Ala-L-Arg-Gly-L-Phe-L-Phe-L-Tyr-NH ₂ (Compound A) were used in three different amounts (1.10 and 100 mg). / Kg / day) The treatment is as described in Table 3. The control group is administered with saline, and blood glucose and urine glucose are treated every four days (Huggett, A. St. G. and Nixon, DA). , "Lamcet" Volume II. 368 to 370 pages (1957)].

The results are shown in Table 3. In the control group, blood glucose and urine glucose are very high after 24 hours. In contrast, blood glucose and urine glucose are gradually decreased in the polypeptide-administered group. Thus, drug dependence is observed in the antidiabetic effect. At the post-administration stage, blood glucose and urine glucose levels are increased to the three groups, but drug dependence is recognized in the persistence of the antidiabetic effect even after administration.

From the findings, the polypeptide can be concluded to be able to treat diabetic rats.

TABLE 3

Example 1

Preparation of Arg-Pro-Phe-Phe-Tyr-NH ₂ .

a) Preparation of Z-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂ .

10.0 g of Z-Phe-Phe-Tyr-NH ₂ was dissolved in 50 ml of DMF (dimethylformamide), and catalytic reduction was performed for 10 hours with Pd black as a catalyst. The catalyst was filtered off, and the filtrate was ice-cooled, and 7.37 g of Z-Arg (NO ₂ ) -Pro-OH and 3.53 g of HONB were added.

Also, after adding 4.60 g of DCC, the mixture was stirred for 16 hours. The formed DC (dicyclohexyl) urea was filtered off and the DMF was distilled off under reduced pressure. 200 ml of ethyl acetate was added to the residue, and the resulting powder was collected by filtration. This powder was purified by reprecipitation from DMF-Methanol-water. Quantity 14.5 g (95.6)

500 mg of Z-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 20 hours using Pd-black as a catalyst.

The catalyst is filtered off and the filtrate is dried under reduced pressure, and the residue is dissolved in 50 ml of water. After a small amount of insolubles are filtered off, the filtrate is lyophilized. The product was dissolved in 5 ml of water, and this solution was used for a column of carboxymethyl-sepadex (1.8 × 9 cm) in a gradient elution with 0.1 M aqueous ammonium acetate solution (400 ml) and 1 M aqueous ammonium acetate solution (400 ml). (gradient elution) was performed. 250-370 mL of the eluate was collected and lyophilized to give 250 mg (52.3%) of whitef luffy product.

Example 2

Preparation of Gly-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

a) Preparation of Z-Gly-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

800 mg of Z-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 8 hours using Pd black as a catalyst. The catalyst is filtered off and the filtrate is dried under reduced pressure. The residue is dissolved in ml of water and freeze dried.

The resulting powder was dissolved in 10 ml of DMF, and 147 mg of p-toluenesulfonic acid monohydrate was added thereto. 349 mg of A-Gly-ONB was added to the mixture, followed by stirring for 10 hours. DMF was distilled off under reduced pressure and 50 ml of ethyl acetate was added. The resulting powder was collected by filtration. Yield 858mg (89.5%)

b) Preparation of Gly-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

500 mg of p-toluenesulfonate of Z-Gly-Argo-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 5 hours using Pd black as a catalyst. This catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 20 ml of water and the solution was subjected to ion exchange chromatography on a column of Amberlite IRA 410 (acetic acid type) (strong base anion exchange resin from Tom & Haaz Corporation, USA). The eluate was lyophilized to dissolve the resulting powder in 5 ml of water. This solution was passed through a column of carboxymethyl cellulose (2.6 x 23 cm) and eluted by a decanting method using 0.005 M to 0.2 M ammonium acetate aqueous solution (700 ml each). 840-970 ml of fractions were collected and lyophilized. Yield 270 mg (63.6%),

Example 3

Preparation of γ-Abu-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

The target object 324 mg was obtained by performing the method of Example 2 using Z-γ-Abu-ONB instead of Z-Gly-ONB.

Example 4

Preparation of ε-Acap-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

a) Preparation of Z-ε-Acap-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

250 mg of Z-ε-Acap-OH was dissolved in 10 ml of DMF under ice-cooling, and HONB186 mg and DCC214 mg were added thereto. The mixture was stirred for 16 hours and the DC-urea formed was filtered off. This filtrate was added to a DMF solution of p-toluenesulfonate of H-Arg-Pro-Phe-Phe-Tyr-NH ₂ prepared in the same manner as described in Example 2a). The mixture was stirred for 12 hours and the DMF was distilled off under reduced pressure. 30 ml of ethyl acetate was added to the residue, and the resulting powder was collected by filtration. Yield 810 mg (79.1%). Melting point (90-92 캜).

b) Preparation of ε-Acap-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

500 mg of the Z-derivative prepared in a) was treated in the same manner as in Example 2-b) to obtain 316 mg of the target compound.

Amino acid analysis: Arg, 1.02; Pro, 1.03 Tyr, 0.9; h. 13; ε-Acap (Measured) (peptide content: 81.5%)

Example 5

Preparation of Lys-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

a) Preparation of di-Z-Lys-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

390 mg of di-Z-Lys-OH was used instead of Z-ε-Acap-OH and Example 4-a) was carried out to obtain 932 mg of the target substance. Melting Point 103 ~ 105 ℃

b) Preparation of Lys-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

500 mg of the di-Z-derivative prepared in a) was treated in the same manner as in Example 2-b) to obtain 336 mg of the target compound.

Amino acid analysis: Lys, 1.02; Arg, 0.98; Pro, 1.00; Tyr, 0.94; Phe, 2.04 (peptide content: 91%)

Example 6

Preparation of α, γ-Cab-Arg-Pro-Phe-Phe-Tyr-NH ₂ .

In exactly the same manner as in Examples 4 and 5, 535 mg of di-Z-α, γ-Dab-dicyclohexylamine salt was synthesized as the target product. Yield 325 mg.

Amino acid analysis; α, γ-Dab, 1.00; Arg, 0.98; Pro, 1.00; Tyr, 0.87; Phe, 1.96 (peptide content: 84.2%)

Example 7

Preparation of β-Ala-Arg-Pro-Phe-Tyr-NH ₂ .

a) Preparation of Z-β-Ala-Arg (NO ₂ ) -Pro-OMe.

To 3.50 g of Z-Arg (NO ₂ ) -Pro-OMe, 35 ml of 25% HBr-acetic acid was added and the mixture was shaken for 35 minutes at room temperature. 300 ml of ethyl ether was added to the mixture, and the resulting precipitate was collected by filtration, washed with ether and dried.

This powder was dissolved in 20 mL of DMF and the solution was neutralized with triethylamine under ice cooling. Then 2.9 g of Z-Ala-ONB was added and the mixture was stirred for 16 hours.

DMF was distilled off under reduced pressure and the residue was dissolved in 300 ml of ethyl acetate. The solution was washed three times using 100 ml of 4% aqueous sodium hydrogen carbonate solution once. After washing with water the solution was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and ethyl ether was added to the residue. The resulting powder was collected by filtration. Yield 3.05 g (74.3% (. Melting point 62-65 ℃)

b) Preparation of Z-β-Ala-Arg (NO ₂ ) -Pro-OH

2.82 g of Z-β-Ala-Arg (NO ₂ ) -Pro-OMe was dissolved in 30 ml of acetone, and 10.7 ml of 1N aqueous sodium hydroxide solution was added under ice-cooling. The mixture was stirred at rt for 2 h. The mixture was neutralized with 11 mL of 1N hydrochloric acid, and acetone was distilled off under fitting. 30 ml of cold water was added to the oily residue, and this water was removed by decantation. The residue was washed once with cold water and dried. Then ethyl acetate-pyridine-acetic acid-water (60: 20: 6: 11) was chromatographed on a column of silica gel (90 g) using a developing solvent. Fractions contained in a large amount in the target were collected and dried under reduced pressure. The residue was washed with ether and collected by filtration. Quantity 2.62g, melting point 65-67 degreeC

c) Preparation of Z-β-Ala-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂ Dissolve 1.50 g of Z-Phe-Phe-Tyr-NH _{2 in} 15 ml of DMF and catalyze Pd black as a catalyst. Contact reduction was carried out for 4 hours. The catalyst was filtered off and the filtrate was ice-cooled, and 1.21 g of Z-β-Ala-Arg (NO ₂ ) -Pro-OH, 0.53 g of HONB and 0.61 g of DCC were added. This mixture was stirred for 18 hours. The formed DC-urea was filtered off and the DMF was distilled off under reduced pressure. 50 ml of water was added to the residue, and the resulting powder was collected by filtration and dried. This powder was reprecipitated with DMF-ethyl acetate. Yield 1.99 g (80.9%), melting point 135-138 ° C,

d) Preparation of β-Ala-Arg-Pro-Phe-Phe-Tyr-NH ₂

1.50 g of Z-β-Ala-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂ was dissolved in 40 ml of glacial acetic acid, and catalytic reduction was performed for 8 hours using Pd black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 50 ml of water, a small amount of insoluble material was filtered off and the filtrate was lyophilized. The product was further dissolved in 20 ml of water and passed through a column of carboxymethyl-cellulose (3.0 × 28 cm), which was eluted with 0.005 M and 0.2 M aqueous ammonium acetate solution (1.5 L each) by gradient method. Elution fractions of 1490-1840 ml were collected and lyophilized.

By the same method as c) and d), it is possible to produce the same object according to the following reaction scheme.

Example 8

Preparation of β-Ala-Arg-Pro-Phe-Phe-NH ₂

In the method of Example 7-c), Z-Phe-Phe-NH ₂ is used instead of Z-Phe-Phe-Tyr-NH ₂ and the same method as described in Examples 7-c) to d). 1.24 g of the target substance was obtained as a white feather material.

Example 9

Preparation of β-Ala-Arg-β-Ala-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-β-Ala-Phe-Phe-Tyr-NH ₂

2.00 g of Z-Phe-Phe-Tyr-NH ₂ was dissolved in 40 mL of DMF, and catalytic reduction was performed for 5 hours using Pd black as a catalyst. The catalyst was filtered off and 1.39 g of Z-β-Ala-ONB was added to the filtrate. The mixture was stirred for 9 hours and the DMF was distilled off under reduced pressure. 50 ml of water was added to the residue, and the resulting powder was collected by filtration and dried. This product was reprecipitated with DMF-ethyl acetate. Yield 2.02 g (88.0%), melting point 232 ° C,

b) Preparation of Z-Arg (NO ₂ ) -β-Ala-Phe-Phe-Tyr-NH ₂

1.0 g of Z-β-Ala-Phe-Phe-Tyr-NH ₂ was dissolved in 30 mL of DMF, and catalytic reduction was performed for 6 hours using Pd black as a catalyst. A solution of Z-Arg (NO ₂ ) -ODNP dissolved in a tetrahydride lofuran by filtration of the catalyst (0.52 g of Z-Arg (NO ₂ ) -OH in 10 ml of tetrahydrofuran by the DCC method and 2, Dinitrophenyl ester prepared from 0.30 g of 4-dinitrophenol). The mixture was stirred for 13 hours and the solvent was distilled off. 50 ml of water was added to the residue, and the resulting powder was collected by filtration and dried. Yield 1.01 g (74.9%), melting point 192-194 degreeC,

c) Preparation of Z-β-Ala-Arg (NO ₂ ) -β-Ala-Phe-Phe-Tyr-NH ₂

To 700 mg of Z-Arg (NO ₂ ) -β-Ala-Phe-Phe-Tyr-NH ₂ 0.2 ml of anizol was added followed by 7 ml of 25% HBr-acetic acid. This mixture was shaken for 40 minutes at room temperature. 100 ml of ethyl ether was added to the reaction mixture, and the resulting precipitate was collected by filtration and washed with ethyl ether. After drying well, the precipitate was dissolved in 100 mL of DMF and the solution was neutralized with triethylamine under ice cooling. 336 mg of Z-β-Ala-ONB was added and then the solution was stirred for 18 hours. DMF was distilled off under reduced pressure and 50 ml of water was added to the residue. The resulting precipitate was collected by filtration, dried and then reprecipitated with DMF and ethyl acetate. Yield 560 mg (77%), melting point 200-202 ° C (decomposition),

d) Preparation of β-Ala-Arg-β-Ala-Phe-Phe-Tyr-NH ₂

400 mg of Z-β-Ala-Arg (NO ₂ ) -β-Ala-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of glacial acetic acid, and catalytic reduction was performed for 8 hours using Pb black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 5 ml of water and the solution passed through a column of carboxymethyl-cellulose (2.6 x 22 cm). The solution was decanted using 0.005 M and 0.2 M ammonium acetate aqueous solutions (700 mL each). Fractions of 720 to 870 ml were collected and lyophilized to obtain 270 mg (69.1%) of the title compound as a white wool material.

Example 10

Preparation of Ala-Arg-β-Ala-Phe-Phe-Tyr-NH ₂

In the method of Example 9-c), Z-β-Ala-ONB was replaced with 340 mg of Z-Ala-ONB, and the same method as described in Examples 9-c) to d) was carried out to give a white feathery appearance. 297 mg of the target substance was obtained as a substance.

Example 11

Preparation of β-Ala-Arg-Sar-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-Sar-Phe-Phe-Pyr-NH ₂

2.02 g of Z-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of DMF, and catalytic reduction was performed for 5 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was cooled. On the other hand, 810 mg of Z-Sar-OH was dissolved in 10 ml of tetrahydrofuran, and then 0.71 g of HONB was added. The mixture was cooled to 0 ° C. and 0.82 g of DCC was added. This mixture was stirred for 3 hours. The DC-urea formed was filtered off and this filtrate was added to the DMF solution. After the mixture was stirred for 20 minutes, the solvent was distilled off under reduced pressure, and 50 ml of water was added to the residue. The resulting powder was collected by filtration and reprecipitated with DMF-ethyl acetate. Yield 1.90 g (83.8%), melting point 246 to 248 ° C (decomposition),

b) Preparation of Z-Arg (NO ₂ ) -Sar-Phe-Phe-Tyr-NH ₂ .

1.0 g of Z-Sar-Phe-Phe-Tyr-NH ₂ was dissolved in 10 mL of DMF, and catalytic reduction was performed for 7 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was cooled.

On the other hand, 0.52 g of Z-Arg (NO ₂ ) -OH was dissolved in 10 ml of tetrahydrofuran, and 0.3 g of 2,4-dinitrophenol and DCC-0.33 g were added under ice cooling. The mixture was stirred for 3 hours to filter off the DC-urea formed, and the filtrate was added to the above DMF solution. The mixture was stirred for 20 hours and the solvent was distilled off under reduced pressure. 50 ml of water was added to the residue, and the resulting powder was collected by filtration. After drying, the powder was reprecipitated with DMF-ethyl acetate-ethyl ether. Yield 1.04 g (77.2%), melting point 96-98 degreeC.

c) Preparation of Z-β-Ala-Arg- (NO ₂ ) -Sar-Phe-Phe-Tyr-NH ₂

700 mg of Z-Arg (NO ₂ ) -Sar-Phe-Phe-Tyr-NH ₂ and 0.2 ml of anisole were dissolved in 7 ml of 25% HBr-acetic acid, and the solution was stirred at room temperature for 40 minutes. 100 ml of ethyl ether was added to the reaction mixture, and the resulting precipitate was collected by filtration, washed with ether, and dried over sodium hydroxide pellets for 2 hours.

The dried precipitate was dissolved in 10 ml of DMF and the solution was neutralized with triethylamine under ice cooling. After adding 335 mg of Z-β-Ala-ONB, the solution was stirred for 20 minutes. DMF was distilled off under reduced pressure and 50 ml of water was added. The resulting powder was collected by filtration, dried and then reprecipitated with DMF-ethyl acetate. Yield 500 mg (76.5%) and melting | fusing point 135-138 degreeC.

d) Preparation of β-Ala-Arg-Sar-Phe-Phe-Tyr-NH ₂

450 mg of Z-β-Ala-Arg (NO ₂ ) -Sar-Phe-Pre-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 8 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was solid under reduced pressure. The residue was dissolved in 5 ml of water and chromatographed with a column of carboxymethyl-cellulose (2.6 x 22 cm). After eluting with 0.005 M and 0.2 M ammonium acetate solutions (700 ml each), 770-880 ml of the elution fractions were collected and freeze-dried to obtain 309 mg (70.4%) of the target product as a white feather.

Example 12

Preparation of Leu-Arg-Pro-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-Leu-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂

20 mL of DMF dissolved 700 mg of Z-Phe-Phe-Tyr-NH ₂ and catalytic reduction was performed for 6 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was cooled. Z-Leu-Arg (NO ₂ ) -Pro-OH was added to the filtrate, followed by HONB247 mg and DCC 285 mg. The mixture was stirred for 20 hours to filter off the DC-urea formed, and the DMF was distilled off under reduced pressure. 50 ml of water was added to the residue, the resulting powder was collected by filtration, dried, and reprecipitated with DMF-ethyl acetate-ether. Yield 1.05 g (86.5%), melting point 101-111 degreeC.

b) preparation of Leu-Arg-Pro-Phe-Phe-Tyr-NH ₂

500 mg of Z-Leu-Arg (NO ₂ ) -Pro-Phe-Phe-Tyr-NH ₂ was dissolved in 20 ml of acetic acid, and catalytic reduction was performed for 10 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was dried under reduced pressure. The residue was dissolved in 5 ml of water and chromatographed with a column of carboxymethyl-cellulose (2.6 x 20 cm). After eluting with 0.005 M and 0.2 M ammonium acetate solutions (700 ml each), 700-790 ml of the elution fractions were collected and lyophilized to obtain 300 mg (63.6%) of the target product as a white feather.

Example 13

Preparation of β-Ala-Arg-Phe-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-β-Ala-Arg (NO ₂ ) -Phe-Phe-Phe-Tyr (Bzl) -resin

4.33 g of BOC-Tyr (Bzl) -resin (Tyr content, 2.18 mM) was added to a reaction tank of a solid peptide synthesis apparatus (Shimazu APS-800, manufactured by Shimazu Seisakusho, Japan), and swelled with dichloromethane. BOC-Phe-OH, BOC-Phe, OH, BOC-Phe-OH, BOC-Arg (NO ₂ ) and Z-β-Ala by DCC method according to Merrifield method (see JACS 86,304 (1964)) -OH, was introduced in order. After completion of all reactions, the resin was washed with dichloromethane, DMF ethanol, glacial acetic acid and methanolol and dried. 6.55 g of a peptide resin was obtained by the above method.

b) Preparation of Z-β-Ala-Arg (NO ₂ ) -Phe-Phe-Phe-Tyr (Bzl) -NH ₂

6.04 g of the resin obtained in a) was suspended in 50 ml of 17% ammonia-methanol, and the suspension was stirred at room temperature for 48 hours. The resin was filtered off and the filtrate was dried under reduced pressure. The powder produced by adding ether to the residue was collected by filtration and dried. Quantity 2.43 g.

c) Preparation of β-Ala-Arg-Phe-Phe-Phe-Tyr-NH ₂

509 mg of the protective peptide-amide preparation obtained in b) was dissolved in 30 ml of glacial acetic acid, and catalytic reduction was performed for 10 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was dried under reduced pressure. The residue was dissolved in 5 ml of water and the solution passed through a column of carboxymethyl-cellulose (2.6 x 24.5 cm). The solution was decanted with an aqueous solution of 0.005 M and 0.2 M ammonium acetate (800 ml each), and 1,180-1,545 ml of elution fractions were collected and freeze-dried. Yield 237 mg (64%).

Example 14

Preparation of β-Ala-Arg-Leu-Phe-Phe-Tyr-Gly-NH ₂

a) Preparation of Z-β-Ala-Arg (Tos) -Leu-Phe-Phe-Tyr (Bzl) -Gly-resin

4.0 g of BOC-Gly-resin (containing Gly of 1.7 mM equivalent) was added to a reaction vessel of a solid phase peptide synthesizer (Shimazu APS-800), followed by swelling with dichloromethane for 20 hours. BOC-Phe-OH, BOC-Phe-OH, BOC-Leu-OH, Aoc-Arg (Tos) -OH and Z-β-Ala-OH were introduced in this reactor in order, and DCC and BOC as condensing agents. Trifluoroacetic acid-dichloromethane (1: 1) was introduced as a releasing agent. After completion of all reactions, the resin was washed with dichloromethane, DMF, methanol, glacial acetic acid and methanol and then dried. Quantity 5.09 g.

b) Preparation of Z-β-Ala-Arg (Tos) -Leu-Phe-Phe-Tyr (Bzl) -Gly-NH ₂

4.4 g of the resin prepared in a) was suspended in 50 ml of 17% ammonia-methanol, and the vessel was tightly sealed, and then the suspension was stirred at room temperature for 48 hours.

The resin was filtered off and washed with DMF. The filtrate was mixed and dried under reduced pressure. Ether was added to the residual oil and the resulting powder was collected by filtration. Yield 950 mg.

c) Preparation of β-Ala-Arg-Leu-Phe-Phe-Tyr-Gly-NH ₂

0.5 ml of aniazole was added to 500 mg of the protective peptide-amide prepared in b (b), and the mixture was added to 10 ml of hydrogen fluoride. The mixture was stirred at 0 ° C. for 60 minutes and hydrogen fluoride was reduced under reduced pressure. The residue was dissolved in 50 ml of water and the solution was washed twice with 20 ml of ether, introduced into a column (1 × 10 cm) of Amberlite IRA-410 (acetic acid type) and lyophilized. Dissolved in 20 mL and passed through a column of carboxymethyl-cellulose (2.6 x 25 cm) in a gradient elution using 0.005 M and 0.2 M ammonium acetate aqueous solutions (800 mL each) to produce 1,200-1,530 mL. The elution fractions were collected and freeze-dried to give 167 mg of a white feather material.

Example 15

Preparation of β-Ala-Arg-Ser-Phe-Phe-Tyr-Gly-NH ₂

a) Z-β-Ala-Arg (Tos) -Ser (Bzl) -Phe-Phe-Tyr (Bzl) -Gly-resin

4.02 g of BOC-Gly-resin (containing 2.17 mM Gly) was introduced into a reaction tank of a solid peptide synthesis apparatus (model APS-800, manufactured by Seisakusho, Shimazu, Japan), and BOC-Tyr (Bzl) was obtained by the Mary Feid method. ) -OH, BOC-Phe-OH, BOC-Phe-OH, BOC-Ser (Bzl) -OH, Aoc-Arg (Tos) -OH and Z-β-Ala-OH were introduced in order. After completion of all reactions, the resin was washed with dichloromethane, methanol, DMF, glacial acetic acid and methanol and then dried. Quantity 6.4g.

b) Preparation of Z-β-Ala-Arg (Tos) -Ser (Bzl) -Phe-Phe-Tyr (Bzl) -Gly-NH ₂

5.9 g of the resin prepared in a) was suspended in 50 ml of 17% ammonia-methanol, and the suspension was stirred at room temperature for 48 hours. The resin was filtered off and washed with DMF. The wash was mixed with the filtrate and the mixed solution was dried under reduced pressure. The powder produced by adding ether to the residue was collected by filtration. Quantity 2.02 g.

c) Preparation of β-Ala-Arg-Ser-Phe-Phe-Tyr-Gly-NH ₂

1.0 g of the protective peptide-amide preparation prepared in b) and 1 ml of anisole were dissolved in 10 ml of anhydrous hydrogen fluoride, and the solution was stirred at 0 ° C. for 60 minutes. Hydrogen fluoride was distilled off under reduced pressure and the residue was dissolved in 20 ml of water. The solution was washed twice with 10 ml ether and introduced into a column of Amberlite IRA-410 (1.0 × 15 cm) and then freeze dried. The solution in which the product was dissolved in 10 ml of water was passed through a column of carboxymethyl-cellulose (2.5 × 25 cm), followed by decanting with 0.005 M and 0.2 M aqueous ammonium acetate solution (800 ml each), followed by 800-950 ml. The eluate was collected and freeze dried. The product was lyophilized by passing through a column (2 × 10 cm) of Ambolite XAD-2 (polystyrene resin, manufactured by Tom & Haars, USA). In this way, 55 mg of the title compound was obtained as a white, feathery material.

Example 16

Preparation of β-Ala-Arg-Pro-Phe-Phe-Thr-Thr-Pro-Lys-Ala-OH

The Z-Phe-Phe-Tyr- Tyr-Pro-Lys (BOC) -Ala-O t BU1.16g dissolved in methanol 30㎖ and was subjected to catalytic reduction for 4 hours by the Pd black as the catalyst. The catalyst was filtered off and the filtrate was dried under reduced pressure. The residue was dissolved in 15 mL of DMF, and Z-β-Ala-Arg (NO ₂ ) -Pro-OH492 mg and HONB 200 mg were added to the solution, DCC 250 mg was added under cooling, and the mixture was stirred for 18 hours.

The insolubles were filtered off and the DMF was distilled off under reduced pressure. Ethyl acetate was added to the residue, and the resulting powder was dried and dissolved in 10 ml of trifluoroacetic acid. The solution was left to stand for 40 minutes, trifluoroacetic acid was distilled off under reduced pressure, and the residue was dissolved in 30 ml of glacial acetic acid. Catalytic reduction was carried out for 16 hours using Pd black as a catalyst.

The catalyst was filtered off and the filtrate was dried under reduced pressure and then dissolved in 10 ml of water. After ion exchange with Amberlite IRA-410, a small amount of insoluble material was filtered off and the filtrate was passed through a carboxymethyl-cellulose (2.2 x 30 cm) column. The solution was decanted with an aqueous solution of 0.005 M and 0.2 M ammonium acetate (800 ml each), and 820-1,180 ml of elution fractions were collected and freeze-dried. This method yielded 670 mg of a white, feathery material.

Amino acid analysis: Lys, 1.02; Arg, 0.98; Thr, 0.96; Pro, 2.04; Ala, 1.00; Tyr, 0.87; Phe, 2.02; β-Ala, 1.05 (peptide content: 74%)

Example 17

Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Leu-OH

a) Preparation of Z-Phe-Phe-Tyr-OEt

3.42 g of Z-Phe-Tyr-OEt was dissolved in 30 ml of ethanol, and catalytic reduction was performed for 2 hours using Pd black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 15 mL of DMF, and 2.77 g of Z-Phe-OSus was added. The mixture was stirred for 10 hours. 80 ml of water was added to the reaction mixture, and the resulting crystals were collected by filtration and reprecipitated with ethanol-ethyl acetate-ethyl ether. In this manner, 3.72 g (83.4%) of acicular material having a melting point of 185 ° C to 186 ° C was obtained.

b) Preparation of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-OEt

2.0 g of Z-Phe-Phe-Tyr-OEt was suspended in 50 ml of ethanol, and catalytic reduction was performed for 1.5 hours using Pd black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 20 ml of DMF. To this solution was added 1.56 g of Z-β-Ala-Arg (NO ₂ ) -Gly-OH, 0.7 g of HONB, and 0.8 g of DCC under ice-cooling, and the mixture was stirred for 12 hours.

The urea derivative formed was filtered off and the DMF was distilled off under reduced pressure. To the residue was added 60 ml of water, the resulting powder was collected by filtration, dried and reprecipitated with DMF-ethyl acetate. Yield 2.58 g (82%), melting point 83-85 ° C

b) Preparation of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-OH

2.00 g of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-OEt was suspended in 30 ml of acetone, and 6.3 ml of 1N aqueous sodium hydroxide solution was added under ice-cooling. Then at room temperature, the mixture was stirred for 1 hour. After adding 3 ml of 1N hydrochloric acid, acetone was distilled off under reduced pressure. A small amount of insoluble matter was filtered off and 3.8 ml of 1N hydrochloric acid was added. The resulting precipitate was collected by filtration, washed well with cold water and dried. Yield 1.96 g (98%), melting point 70-73 ° C,

d) Preparation of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-Leu-OBzl

275 mg of H-Leu-OBzl-P-toluenesulfonate was dissolved in 5 ml of DMF and the solution was neutralized with 0.9 ml of 10% N-ethylmorpholine-DMF. After cooling to −10 ° C., 670 mg of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-OH, 251 mg of HONB, and 289 mg of DCC were added. The mixture was stirred for 12 hours to filter out DC urea formed. DMF was distilled off under reduced pressure and 30 ml of water was added to the residue. The resulting precipitate was collected by filtration, washed with hot methanol and dried. Yield 703mg (87.9%), melting point 178-181 degreeC,

e) Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Leu-OH

500 mg of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-Leu-OBzl was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 8 hours using Pd black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 5 ml of water and the solution passed through a column of carboxymethyl-cellulose (2.6 x 18 cm). Elution was carried out using a linear gradient method using 0.005 M aqueous ammonium acetate solution (700 ml) and 0.2 M aqueous ammonium acetate solution (700 ml) to elute the compound in fractions of 370 to 720 ml. The fractions were combined and lyophilized. Yield 277 mg (64.2%).

Example 18

Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Ala-NH ₂

Repeating the method described in Examples 17-d) and -e) using H-Ala-NH ₂ hydrochloride instead of H-Leu-OBzl-P-toluenesulfonate, β-Ala-Arg-Gly-Phe -Phe-Tyr-Ala-NH ₂ .2 acetate (quantity 216 mg) was obtained.

Amino acid analysis: Arg, 1.01; Gly, 1.00; Ala, 1.02; Phe, 2.00; Tyr, 0.89, β-Ala, 1.12 (peptide content: 82.4%).

Example 19

Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-OH

Preparation of a) Z-Phe-Phe- Tyr-Thr-Pro-O t Bu

Dissolving ^{Z-Thr-Pro-O t} Bu 1.80g 30㎖ in methanol and subjected to catalytic reduction for 5 hours to the Pd black as the catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 20 mL of DMF, and after cooling the solution to -10 ° C, 2.70 g of Z-Phe-Phe-Tyr-OH, 1.59 g of HONB, and 1.37 g of DCC were added. The mixture was stirred for 18 hours to remove urea derivative formed and 100 ml of water was added. The resulting precipitate was collected by filtration, dried, and dissolved in 100 ml of ethyl acetate. The solution was washed twice with 50 ml of water and dried over sodium sulfate. After distilling off the solvent, ethyl acetate was added to the secretary. The resulting powder was collected by filtration and then dried. Yield 2.51 g (89.8%), melting point 137-139 ° C

Preparation of b) Z-β-Ala- Arg (NO 2) -Gly-Phe-Phe-Tyr-Thr-Pro-O t Bu

Dissolving Z-Phe-Phe-Tyr- Thr-Pro-O t Bu 1.50g in methanol 20㎖ and using Pd black as the catalyst was subjected to catalytic reduction for 4 hours in a hydrogen stream. The catalyst was filtered off and the solvent was distilled off under reduced pressure.

The residue was dissolved in 10 mL of DMF, while 0.87 g of Z-β-Ala-Arg (NO ₂ ) -Gly-OH, HONB, 0.47 g, and 0.54 g of DCC were added to this solution under ice cooling. The mixture was stirred for 16 hours. The formed DC-urea was filtered off and 50 ml of water was added to the filtrate and the gel was separated. The precipitate was collected by filtration, dried and reprecipitated with DMF-ethyl acetate. This precipitate was collected by filtration and then reprecipitated with ethanol. Quantity 1.04 g (67.4%), Melting point 153-156 ° C

c) Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-OH

A Z-β-Ala-Arg ( NO 2) -Gly-Phe-Phe-Tyr-Thr-Pro-O t Bu was added anisole 0.9㎖ the 900㎎ Next, anhydrous hydrogen fluoride was added 10㎖. The mixture was stirred at -4 ° C for 30 minutes. Hydrogen fluoride was distilled off under reduced pressure, and 30 ml of water was added to the residue.

The solution was extracted with 30 ml of ethyl ether, and the aqueous layer was passed through a column of Ambolite IRA-410 (acetic acid type) (1.0 × 10.0 cm), which was then washed with water, and the washing solution was combined with the effluent and lyophilized. This product was dissolved in 10 ml of water and the solution passed through a column of carboxymethyl-cellulose column (2.2 x 21 cm). The solution was decanted with 0.005 M aqueous ammonium acetate solution (700 mL) and 0.3 M aqueous ammonium acetate solution (700 mL), and 235-385 mL elution fractions were collected and lyophilized. Yield 476mg (57.0%)

Example 20

Preparation of ε-Acap-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-OH

a) Preparation of Z-ε-Acap-Arg (NO ₂ ) -Gly-OEt

12 ml of 25% HBr-acetic acid was added to 24.1 g of Z-Arg (NO ₂ ) -Gly-OEt, and the mixture was shaken at room temperature for 40 minutes. The precipitate produced by adding ethyl ether to this solution was collected by filtration and dried over sodium hydroxide.

On the other hand, 1.33 g of Z-ε-Acap-OH and 0.99 g of HONB were dissolved in 5 ml of tetrahydrofuran, and 1.14 g of DCC was added under ice cooling. The mixture was stirred for 4 hours, and the dicyclohexylurea formed was filtered off. The amine component was dissolved in 10 ml of DMF, and the solution was neutralized with triethylamine, and then the active ester solution was added. The mixture was stirred overnight. The solvent was distilled off under reduced pressure and the residue was dissolved in 50 ml of ethyl acetate. The solution was washed with water and then with a saturated aqueous solution of sodium hydrogen carbonate (50 mL × 2). Washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure and petroleum benzine was added to the residue. The resulting crystals were recrystallized from water-ethanol-ethyl acetate. Yield 2.1g (76%), Melting Point 85 ~ 87 ℃,

b) Preparation of Z-ε-Acap-Arg (NO ₂ ) -Gly-OH

1.8 g of Z-ε-Acap-Arg (NO ₂ ) -Gly-OEt was dissolved in 10 ml of acetone, and after cooling with ice, 2.9 ml of 2N sodium oxide was added. At room temperature, the mixture was stirred for 2 hours, 2.5 ml of 1N hydrochloric acid was added to the mixture, and then acetone was distilled off. 10 ml of water was added to the residue, and the insoluble was filtered off, and then the solution was changed to acidic with 1N hydrochloric acid. The resulting crystals were collected by filtration. Yield 1.59 g (93%), melting point 97.5-99 degreeC.

c) Preparation of ε-Acap-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-OH

In the method of Example 19-b), Z-ε-Ala-Arg (NO ₂ ) -Gly-OH is replaced with Z-ε-Acap-Arg (NO ₂ ) -Gly-OH, and Example 19- The same procedure as described in b) and c) was repeated to obtain 428 mg of the title product.

Example 21

Preparation of Lys-Arg-Gly-Phe-Phe-Tyr-Phr-Pro-OH

a) Preparation of di-Z-Lys-Arg (NO ₂ ) -Gly-OEt

Using 2.07 g of di-Z-Lys-OH, the target product was obtained as a gel in the same manner as in Example 20-a). This gel material was reprecipitated with water-ethanol-ethyl acetate. Yield 2.57 g (73%), melting | fusing point 161-162 degreeC.

b) Preparation of di-Z-Lys-Arg (NO ₂ ) -Gly-OH

2.0 g of di-Z-Lys-Arg (NO ₂ ) -Gly-OEt was dissolved in 10 ml of acetone, and 2.1 ml of 2N sodium hydroxide was added under ice-cooling. Then, the mixture was stirred at room temperature for 2 hours. After adding 1.4 mL of 1N hydrochloric acid, acetone was distilled off under reduced pressure, and 10 mL of water was added to the residue.

Filter off the insoluble matter and acidify the filtrate with 1N hydrochloric acid.

did. The resulting gel was collected by filtration. Yield 1.78g (93%), Melting Point 131-132.5 ℃,

c) Preparation of Lys-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-OH

Z-β-Ala-Arg ( NO 2) -Gly-OH in place of di-Z-Lys-Arg ( NO 2) -Gly-OH Example 19-b) how the target product 452 was repeated using the Mg was obtained.

Example 22

Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala-OH

Preparation of a) Z-Lys (BOC) -Ala-O t Bu

In methanol 300㎖ dissolved ^{Z-Ala-O t Bu 14g} , catalytic reduction is carried out for 2 hours using Pd black as the catalyst. The catalyst was filtered off and the methanol was distilled off.

The residue was dissolved in 50 ml of tetrahydroputane. Meanwhile. 28.1 g of Z-Lys (BOC) -OH and dicyclohexylamine salt were suspended in 300 ml of ethyl acetate and washed three times with 200 ml of 0.5 N sulfuric acid. After washing with water it was dried over anhydrous sodium sulfate.

Ethyl acetate was partially distilled off in about 150 ml. The residue was added to the tetrahydrobutane solution followed by 10 g of HONB. 11.3 g of DCC were added under ice cooling, and the mixture was stirred for 16 hours. The DC-urea formed was filtered off and the filtrate was dried. The residue was dissolved in 500 ml of ethyl acetate and washed with 0.5 N sulfuric acid and 4% aqueous sodium hydrogen carbonate solution. After washing well with water, the ethyl acetate layer was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure and crystals were separated by addition of a petroleum engine. These crystals were collected by filtration and reprecipitated with ethyl acetate-petroleum benzine. Quantity 21.9 g (87%). Melting point 55-57 degreeC.

Preparation of b) Z-Pro-Lys ( BOC) -Ala-O t Bu

Dissolving Z-Lys (BOC) -Ala- O t Bu 2.5g 40mI in methanol and subjected to catalytic reduction for 2 hours by using Pd black as the catalyst. The catalyst was filtered off and the filtrate was dried under reduced pressure. The residue was dissolved in 20 ml of ethyl acetate, and 2.45 g of Z-Pro-ONB was added to this solution, and then the mixture was stirred at room temperature for 18 hours. 200 ml of ethyl acetate was added to the solution, and the mixture was washed with 0.5N sulfuric acid and 4% aqueous sodium hydrogen carbonate solution, washed three times with water, dried over anhydrous sodium sulfate, and dried under reduced pressure. The residue was collected by petroleum benzine and the resulting crystals were collected by filtration and reprecipitated with a mixture of ethyl acetate-petroleum benzine. Quantity 2.53 g (85%).

Preparation of c) Z-Thr-Pro- Lys (BOC) -Ala-O t Bu

Dissolving Z-Pro-Lys (BOC) -Ala-O t Bu 2.12g 40㎖ in methanol and was subjected to catalytic reduction for 3 hours using Pd black as the catalyst. This catalyst was filtered off and the solvent was distilled off under reduced pressure.

The residue was dissolved in 20 ml of dioxane. Meanwhile. 977 mg of Z-Thr-OH and 806 mg of HONB were dissolved in 10 ml of ethyl acetate-dioxane (1: 1), and DCC800 mg was added under ice-cooling. The mixture was stirred for 6 hours to filter off the DC-urea formed and this filtrate was added to the dioxane solution. This mixture was stirred for 16 hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in 150 ml of ethyl acetate. The solution was washed with 0.5N sulfuric acid and 4% aqueous sodium hydrogen carbonate solution. After washing with water, it was dried over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure. The resulting powder was collected by adding petroleum benzine to the residue. Yield 2.3 g. (93%). Melting point 64-67 degreeC.

Preparation of d) Z-Phe-Phe- Tyrr-Thr-Pro-Lys (BOC) -Ala-O t Bu

Dissolving Z-Thr-Pro-Lys ( BOC) -Ala-O t Bu 1.41g in methanol 70㎖ and, using Pd black as the catalyst was subjected to catalytic reduction for 2 hours. The catalyst was filtered off and the solvent was distilled off under reduced pressure. This residue was dissolved in 10 ml of DMF. Then 1.1 g of Z-Phe-Phe-OH and 720 mg of HONB were added. Under ice-cooling, 618 mg of DCC was added, and the mixture was stirred for 20 hours. The solvent was distilled off under reduced pressure and the residue was treated with ethyl acetate-ether (1: 1). The resulting powder was collected by filtration and determined from ethanol. Quantity 2.1g (100%). Melting point 160 ~ 163.5 ℃,

_{e) Z-Arg (NO 2} ) Preparation of -Gly-Phe-Phe-Tyr- Thr-Pro-Lys (BOC) -Ala-O t Bu

Dissolving Z-Phe-Phe-Thr- Pro-Lys (BOC) -Ala-O t Bu 2.32g 30㎖ in methanol and subjected to catalytic reduction for 3 hours using Pd black as the catalyst. The catalyst was filtered off and the methanol was distilled off. The residue was dissolved in 5 ml of DMF. To this solution was added 1.26 g of Z-Aly (NO ₂ ) -Gly-ONB. This mixture was stirred at room temperature for 20 hours. DMF was distilled off under reduced pressure, and 50 ml of water was added to the residue. The resulting powder is collected by filtration. Reprecipitated twice with DMF-water. Quantity 2.5g (88%). Melting point 138-141 degreeC (decomposition).

f) Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Pro-Lys-Ala-OH

Methanol and glacial acetic acid (2: 1) mixture and _{30㎖ Z-Arg (NO 2)} -Gly-Phe-Phe-Tur-Thr-Pro-Lys (BOC) was dissolved -Ala-O ^t Bu 710㎎ in, Catalytic reduction was performed for 8 hours using Pd black as a catalyst. The catalyst was allowed to rest and the filtrate was dried under reduced pressure. The residue was dissolved in 10 mL of water and the solution was passed through a column of carboxymethyl-cellulose (130 mL). The solution was decanted with 0.005 M aqueous ammonium acetate solution (800 mL) and 0.2 M aqueous ammonium acetate solution (800 mL). Fractions of 913 mL to 1190 mL were collected and freeze-dried to obtain 350 mg of white feathery material.

This powder was dissolved in 8 ml of DMF, and 41 mg of p-toluenesulfonic acid monohydrate was added thereto. Then 82 mg BOC-β-Ala-ONB was added and the mixture was stirred for 16 h. DMF was distilled off under reduced pressure and 3 ml of trifluoroacetic acid was added to the residue. The mixture was shaken for 30 minutes and the trifluoroacetic acid was distilled off. The residue was dissolved in 10 ml of water and passed through a column (1.0 × 10 cm) of Amberlite IRA-410 (acetic acid type) to convert this compound to acetate. The eluate was lyophilized and passed through a column of carboxymethyl-cellulose (2.2 × 17 cm). The solution was decanted with 0.005 M aqueous ammonium acetate solution (500 mL) and 0.2 M aqueous ammonium acetate solution (500 mL). 430-500 ml of powder was collected and freeze-dried to obtain 250 ml of white wool.

Example 23

Preparation of Gly-rg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala-OH Replace BCC-β-Ala-ONB with BOC-Gly-ONB and repeat the method of Example 22-f) 214 mg of the target substance was obtained as a white feathery substance.

Example 24

Preparation of Leu-Arg-Gly-Phe-Phe-Tyr-Gly-NH ₂

a) Preparation of Z-Leu-Arg (Tos) Gly-Phe-Phe-Tyr (Bzl) -Gly-resin

4.04 g of BOC-Gly-resin (containing 1.75 mmol of Gly) was added to a reaction vessel of a solid-phase peptide synthesizer (Shimadzu APS 800), followed by swelling with dichloromethane for 20 hours. Then, by the Mary Feild method, BOC-Tyr (Bzl) -OH, BOC-Phe-OH, BOC-Phe-OH, BOC-Gly-OH, Aoc-Arg (Tos) -OH and Z-Leu-OH Was introduced in a predetermined order. DCC was used as a condensing agent, and each amino acid was used in 4 equivalents, and the BOC group was removed by trifluoroacetic acid-dichloromethane (1: 1). The resin was taken from the reaction vessel, washed with dichloromethane, DMF, methanol and acetic acid and then dried. In this way, 5.07 g of the resin was obtained.

b) Preparation of Z-Leu-Arg (Tos) -Gly-Phe-Tyr (Bzl) -Gly-NH ₂

4.41 g of the resin prepared in a) was suspended and sealed in 50 ml of 17% ammonia-methanol, and the suspension was stirred for 48 hours at room temperature. This resin was filtered off and washed sufficiently with DMF. The washing solution and the filtrate were collected and the solvent was distilled off under reduced pressure. Ethyl ether was added to the oily residue, and the resulting powder was collected by filtration and washed sufficiently with ether. Yield 950 mg.

c) preparation of Leu-Arg-Gly-Phe-Phe-Tyr-Gly-NH ₂

A solution obtained by dissolving 500 mg of the protective peptide-amide preparation prepared in b) and 0.5 ml of anisole in 10 ml of anhydrous hydrogen fluoride was stirred at 0 ° C. for 60 minutes. Hydrogen fluoride was distilled off and the residue was dissolved in 50 ml of water. The solution was extracted twice with 20 ml of ethyl ether, and the aqueous layer was passed through a column (2 × 10 cm) of Amberide IRA-410 (acetic acid type) and lyophilized. This product was passed through a column of carboxymethyl-cellulose (2.3 x 17 cm) and eluted with 0.005 M aqueous ammonium acetate solution (400 mL) and 0.2 M aqueous ammonium acetate solution (400 mL). Fractions of 490-550 ml were combined and lyophilized. In this manner, 167 mg of the target product was obtained as a white, feathery material.

Amino acid analysis: Arg, 1.02; Gly, 2.00: Leu, 1.03; Tyr, 0.89; Phe, 2.08 (peptide content: 84%)

Example 25

Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Ala-Gly-NH ₂

a) Preparation of Z-β-Ala-Arg (Tos) -Gly-Phe-Phe-Tyr (Bzl) -Ala-Gly-resin

3.03 g of BOC-Gly-resin (Gly content 1.32 mM) was placed in a reactor of a Shimadzu APS-800 synthesis apparatus, followed by swelling with dichloromethane. Then, a symmetric anhydride process [Hoppe-Seyle-r's, Z. Physiol. Chem. 353. See 1973 (1972) in which BOC-Ala-OH, BOC-Tyr (Bzl) -OH, BOC-Phe-OH, AOC-Arg (Tos) -OH and Z-β-Ala-OH Introduced along. Removal of BOC at each step was done with 50% trifluoroacetic acid-dichloromethane.

In each step, the unreacted amino group was acetylated with acetic anhydride. After introduction of all amino acids, the resin was washed with ethanol, DMF, dichloromethane, acetic acid and methanolol and then dried under reduced pressure. This method yielded 3.70 g of peptide resin.

b) Preparation of Z-β-Ala-Arg (Tos) -Gly-Phe-Phe-Tyr (Bzl) -Ala-Gly-N ₂

Suspend and seal 3.28 g of the peptide resin prepared in a) in 50 ml of 17% ammonia-methanol. This suspension was stirred for 40 hours at room temperature. The resin was filtered off and washed with 50 mL of DMF. The filtrate and washings were combined and dried under reduced pressure. The powder produced by adding ether to the residue was collected by filtration and dried. In this way, 433 mg of crude peptide-amide was obtained.

c) Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-Ala-Gly-NH ₂

In a mixture of 8 ml of hydrogen fluoride anhydride and 0.3 ml of anisol, 310 mg of the protective peptide-amide prepared in b) above was dissolved. This solution was stirred at 0 ° C. for 60 minutes.

Hydrogen fluoride was distilled off under reduced pressure, and the residue was dissolved in 50 ml of water. This solution was extracted two times with 10 ml of ether. The aqueous layer was passed through a column (1 × 10 cm) of Amberlite IRA-410 (acetic acid type), which was washed with water. The eluate and wash were combined and lyophilized. The product was dissolved in 10 ml of water and the solution passed through a column of carboxymethyl-cellulose (2.3 x 17 cm). The solution was decanted with an aqueous solution of 0.005 M and 0.2 M ammonium acetate (400 mL each). 495-540 ml of the fractions were collected and freeze-dried to obtain 134 mg of the target substance.

Example 26

Preparation of α, γ-Dab-Arg-Gly-Phe-Phe-Tyr-Ala-Gly-NH ₂

The method of Example 25 was repeated using di-Z-α, γ-Abu-OH instead of Z, β-Ala-OH as the amino acid to be finally introduced. In this manner, the target product was obtained as a white, feathery material.

Example 27

Preparation of Gly-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-Gly-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

To 0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ , 8 ml of 25% HBr acetic acid was added and the mixture was sufficiently shaken at room temperature for 45 minutes. 100 ml of ether was added to the reaction mixture, and the resulting powder was collected by filtration and dried in a dryer for 2 days under reduced pressure with sodium hydroxide granules. The dryness was dissolved in 10 mL of DMF and the solution was neutralized with triethylamine. 0.43 g of Z-Gly-ONB was added to the solution, and the mixture was stirred for 10 hours.

7 ml of water was added to this solution to separate the oil. After decantation and removal of the supernatant, 50 ml of water was added. The resulting powder was collected by filtration and then dried. This crude product was purified by reprecipitation with DMF-ethyl acetate. Quantity 0.66g (77%).

b) Preparation of Gly-Arg-Gly-Phe-Phe-NH ₂

500 mg of Z-Gly-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 7 hours using Pd black as a catalyst in a hydrogen stream. The catalyst was filtered off and acetic acid was distilled off under reduced pressure. The residue was dissolved in 10 ml of water and the solution passed through a column of carboxymethyl-cellulose (1.8 × 10 cm).

Elution was performed by the linear gradient gradient method using 0.1 M aqueous ammonium acetate solution (700 ml) and 1 M ammonium acetate aqueous solution (700 ml). Fractions of 410-515 mL were collected and freeze-dried to give 307 mg (65%) of white feathery material.

Example 28

Preparation of D-Ala-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of ZD-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 8 ml of 25% HBr acetic acid, and the solution was shaken at room temperature for 40 minutes. 100 ml of ethyl ether was added to this mixture to form a precipitate. This precipitate was collected by filtration, washed with ether and dried. The dried product was dissolved in 10 mL of DMF, the solution was neutralized with triethylamine, and then 0.51 g of ZD-Ala-ONB was added. This mixture was stirred for 8 hours. DMF was distilled off under reduced pressure and 50 ml of water was added to the residue.

After the resulting powder was collected by filtration and dried, the powder was washed with hot ethyl acetate and then reprecipitated with DMF-ethyl acetate. Yield 0.799 g (92%). Melting point 223-225 캜,

b) Preparation of D-Ala-Arg-Gly-Phe-Phe-NH ₂

600 mg of ZD-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 30 ml of acetic acid and catalytic reduction was performed for 7 hours using P ^d black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 10 ml of water, and the solution was passed through a column of carboxymethyl-sefadex (1.8 x 11 cm).

Linear gradient elution was performed with 0.1 M aqueous ammonium acetate solution (700 ml) and 1 M aqueous ammonium acetate solution (700 ml) to collect 350-450 ml of the elution fractions and lyophilized. 400 ml (73%) of white feathery material was obtained by this method. Created.

Example 29

Preparation of Phe-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-Phe-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

800 mg of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 10 ml of 25% HBr-acetic acid, and the solution was shaken for 50 minutes. 80 ml of ether was added to the solution and the resulting precipitate was collected by filtration, dried and dissolved in 10 ml of DMF. After neutralizing this solution with triethylamine, 700 mg of Z-Phe-OPCP (pentachlorophenyl ester) was added. The mixture was stirred for 10 hours, and 50 ml of 1N aqueous ammonia solution was added thereto. The larvae were taken by tilt and water was added to this oily substance. The resulting powder was collected by filtration and then dried. 50 ml of ethyl acetate was added to the powder, and the mixture was heated, and the powder was collected by filtration under hot conditions. The powder was purified by reprecipitation with DMF-ethyl acetate. Yield 805 mg (92%).

b) preparation of Phe-Ag-Gly-Phe-Phe-NH ₂

500 mg of Z-Phe-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 6 ml of anhydrous hydrogen fluoride, and the solution was stirred at −10 ° C. for 40 minutes. Hydrogen fluoride was distilled off and the residue was dissolved in 10 ml of water. This solution was passed through a column of Amberlite-lRA-410 (acetic acid type) (0.9 × 15 cm) and washed with water. In this way, 402 mg of the target product was obtained.

Amino acid analysis: Arg, 1.01; Gly, 0.98; Phe, 2.94 (peptide content: 86.2%)

Example 30

Preparation of Leu-Arg-Gly-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

12.2 g of Z-Phe-Phe-Tyr-NH ₂ was dissolved in 100 mL of DMF, and catalytic reduction was performed for 5 hours using Pd black as a catalyst. The catalyst was filtered off, and 8.6 g of Z-Arg (NO ₂ ) -Gly-OH and 4.3 g of HONB were added thereto.

Under ice cooling. 4.95 g of DCC were added and the mixture was stirred for 12 hours. The formed DC-urea was filtered off, and 1 liter of water was added to the filtrate, and the resulting gel was collected by filtration and then reprecipitated with methanol and water. 16.25 g (91.8%),

b) Preparation of Arg-Gly-Phe-Phe-Tyr-NH ₂

600 mg of Z-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 9 hours using Pd black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. This residue was dissolved in 15 ml of water and then purified by column of carboxymethyl sepadex (1.3 x 11 cm). Linear gradient gradient elution was performed with 0.1 M aqueous ammonium acetate solution (500 mL) and 1 M aqueous ammonium acetate solution (500 mL). 250-420 mL of fractions were combined and lyophilized. Yield 416 mg (72.5%).

c) Preparation of Z-Leu-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

To 700 mg of Z-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was added 0.2 ml of anizole and then 7 ml of 25% HBr-acetic acid. The mixture was shaken at room temperature for 40 minutes and then 100 ml of ethyl ether was added. The resulting precipitate was collected by filtration and dissolved in 100 ml of DMF. The solution was neutralized with triethylamine and then 344 mg of Z-Leu-Sou (N-hydroxysuccinamide ester) was added. This mixture was stirred for 12 hours. DMF was distilled off under reduced pressure and 50 ml of water was added to the residue. The resulting powder was collected by filtration and purified by reprecipitation with methanol and water. Yield 560 mg (70.9%). Melting Point 193 ~ 195 ℃

d) preparation of Leu-Arg-Gly-Phe-Phe-Tyr-NH ₂

400 mg of Z-Leu-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 10 hours using Pd black as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 50 ml of water. The solution was passed through a column of carboxymethyl-cellulose (2.6 x 22 cm).

The solution was decanted with 0.005 M aqueous ammonium acetate solution (700 mL) and 0.2 M aqueous ammonium acetate solution (700 mL), and 830-910 mL fractions were collected and freeze-dried. Yield 217 mg (55.5%).

Example 31

Preparation of α-Abu-Arg-Gly-Phe-Phe-Tyr-NH ₂

The target of the above was obtained by repeating the method of Example 30 using Z-α-Abu instead of Z-Leu.

Example 32

Preparation of Nle-Arg-Gly-Phe-Phe-Tyr-NH ₂

The target of the above was obtained by repeating the method of Example 30 using Z-Nle instead of Z-Leu.

Example 33

Preparation of Val-Arg-Gly-Phe-Phe-Tyr-NH ₂

-13.7°(c=0.5, 물)400 mg of Arg-Gly-Phe-Tyr-NH ₂ .2 acetate prepared in Example 30-b) was dissolved in 10 ml of DMF, followed by addition of 300 mg of Z-Val-ONB. The solution was neutralized with triethylamine and stirred for 10 hours. 50 ml of ether was added to the solution, and the resulting precipitate was collected by filtration, washed with ethyl acetate and dried. This product was dissolved in 30 ml of acetic acid-water (1: 1) and catalytic reduction was carried out for 4 hours using Pd black as a catalyst. The catalyst was filtered off and the filtrate was solidified. The residue was dissolved in 10 ml of water and purified by chromatography in the same manner as described in Example 30-c). 360 mg,

-13.7 ° (c = 0.5, water)

Amino acid analysis: Arg, 1.02; Gly, 1.00 Val, 0.97; Phe, 2.04; Tyr, 0.97 (peptide content: 86.2%)

Example 34

Preparation of β-Ala-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-Arg (NO ₂ ) Gly-OEt

70.7 g of Z-Arg (NO ₂ ) -OH was dissolved in 200 mL of tetrahydrofuran, 40.5 g of 2.4-dinitrophenol and 45.4 g of DCC were added under ice-cooling, and the mixture was stirred for 4 hours. On the other hand, 30.7 g of Gly-OEt HC1 was suspended in 200 ml of tetrahydrofuran, and the suspension was neutralized with 30.8 ml of triethylamine under ice cooling. The dicyclohexyl urea formed was filtered off, the active ester solution prepared as mentioned above was added to this filtrate, and it stirred overnight. The solution was concentrated to dissolve the residue in 500 ml of ethyl acetate. The solution was washed in the order of a saturated aqueous solution of sodium bicarbonate (200 mL x 3), 1N hydrochloric acid (200 mL x 3), and a saturated aqueous solution of sodium chloride (200 mL x 3), followed by drying over anhydrous sodium sulfate. This solution was concentrated and determined with ethyl acetate-petroleum benzine. The crystals were collected by filtration and recrystallized with ethyl acetate-petroleum benzine. Yield 70.0 g (79.8%), melting | fusing point 109-110 degreeC.

b) Preparation of Z-Arg (NO ₂ ) -Gly-OH

20.0 g of Z-Arg (NO ₂ ) -Gly-OEt was suspended in 100 ml of acetone, and 64 ml of 1N aqueous sodium hydroxide solution was added under ice-cooling. The mixture was stirred at rt for 2 h.

After neutralizing excess alkali with hydrochloric acid, the solution was concentrated under reduced pressure and 100 ml of water was added to the residue. The insolubles were filtered off, the filtrate was made acidic with hydrochloric acid and the resulting crystals were collected by filtration. Quantity 18.3 g (93.7%), Melting point 103-105 degreeC,

c) Preparation of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

8.91 g of Z-Phe-Phe-NH ₂ was dissolved in a mixture of 60 ml of DMF and 40 ml of methanol, and contact hydrogenation was carried out for 14 hours in the presence of Pd black. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was dissolved in 30 ml of DMF. Meanwhile, 6.80 g of Z-Arg (NO ₂ ) -Gly-OH was added to 50 mL of tetrahydrofuran, and HOB3.23 g and DCC3.71 g were added under ice cooling, followed by stirring for 5 hours. The formed dicyclohexyl urea was filtered out and the amine component (H-Phe-Phe-NH ₂ ) was added to the filtrate. The mixture was stirred overnight, the solvent was distilled off under reduced pressure, and 100 ml of water was added to the residue, and the resulting powder was collected by filtration.

After drying, the product was washed with ethyl acetate and recrystallized with ethanol-ethyl acetate. Yield 8.34 g (72.7%), melting point 112-113 ° C,

d) preparation of Z-β-Ala-ONB

3.35 g of Z-β-Ala-OH and 3.23 g of HONB were dissolved in 40 mL of tetrahydrofuran, and after cooling with ice, DCC3.71 g was added. The mixture was stirred overnight to dicyclohexylurea formed. The solvent was distilled off under reduced pressure and a petroleum engine was added thereto. The resulting crystals were collected by filtration and recrystallized from acetonitrile. Yield 3.83 g (66.4%), melting point 125-126 degreeC.

e) Preparation of Z-β-Ala-Arg) NO ₂ ) -Gly-Phe-Phe-NH ₂

10 ml of 25% HBr-acetic acid was added to 1.06 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ , shaken at room temperature for 40 minutes, and ethyl ether was added thereto. The resulting precipitate was collected by filtration, dried over sodium hydroxide under reduced pressure in a desiccator, and then dissolved in 10 ml of DMF. After cooling, the solution was neutralized with 0.84 mL of triethylamine, and then 0.69 g of Z-β-Ala-ONB was added thereto. The mixture was stirred overnight, the solvent was distilled off under reduced pressure, and 50 ml of water was added thereto. The resulting powder was dried, washed with ethyl acetate and reprecipitated with water-dioxane-ethyl acetate. 0.98 g (83.3%) Melting Point 161-163 deg.

f) Preparation of β-Ala-Arg-Gly-Phe-Phe-NH ₂

In 40 ml of acetic acid, Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved with palladium as a catalyst and subjected to catalytic reduction for 14 hours. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl-sefadex (2.5 cm x 9 cm) and eluted from a 0.01 N aqueous ammonium acetate solution (700 mL) through a 1 N aqueous ammonium acetate solution (700 mL) by linear gradient gradient method. . The fractions were collected in 780-990 ml and lyophilized. Yield 500 mg (66%),

[Numbers in parentheses indicate theoretical values, the same applies hereafter]. (Peptide Content: 76%)

Example 35

Preparation of γ-Abu-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-7-Abu-ONB

After dissolving 4.75 g of Z-γ-Abu-OH and 3.94 g of HONB in 40 ml of tetrahydrofuran, and after ice-cooling, after dissolving DCC 3.94 g and ice-cooling thereto. To this was added 4.54 g of DCC. The mixture was stirred for 6 hours to filter out dicyclohexylurea formed. The solvent was distilled off under reduced pressure, and then 30 ml of ethyl ether was added to the residue, followed by addition of a small amount of water. The resulting crystals were collected by filtration. Quantity 6.0 g (75.3%), melting point 66-69 degrees Celsius

b) Preparation of Z-γ-Abu-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

0.53 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe- NH ₂ was treated with HBr-acetic acid and dissolved in 15 ml of DMF according to the same method as described in Example 34-e). After neutralizing this solution with triethylamine, 0.44 g of Z-γ-Abu-ONB was added. The mixture was stirred overnight and the solvent was distilled off under reduced pressure and water was added. The resulting powder was collected by filtration and dried sufficiently.

Washed with ethyl acetate and reprecipitated from ethanol-water. 0.38 g (65%), Melting point 167-170 degreeC,

c) Preparation of γ-Abu-Arg-Gly-Phe-Phe-NH ₂

Z-γ-Abu-Arg (NO ₂ ) -Gly-Phe-Phe-300 mg was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 6 hours using Pd as a catalyst. After the catalyst was filtered off, the solvent was distilled off under reduced pressure and the residue was passed through a column of carboxymethyl-sefadex (1.8 cm x 10 cm). Then, eluting was carried out by passing 1 N ammonium acetate solution (350 mL) from 0.005 N ammonium acetate solution (350 mL) by linear background method, and 290-340 mL fractions were collected and frozen. 180 mg (63%)

Example 36

Preparation of δ-AvaI-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-δ-Aval-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

In the same manner as in Example 34-e), 0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was treated with HBr-acetic acid and dissolved in 10 ml of DMF. This solution was neutralized with triethylamine. On the other hand, 0.30 g of Z-δ-AvaI-OH and 0.24 g of HONB were dissolved in 5 ml of tetrahydrofuran. While cooling with ice, 0.27 g of DCC was added thereto, and the mixture was stirred for 30 minutes. The dicyclohexyl urea was removed and the filtrate was added to the amine component [H-δ-Aval-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ ], followed by stirring overnight. The solvent was distilled off under reduced pressure and water was added. The resulting powder was collected by filtration and dried sufficiently. Washed with ethyl acetate and reprecipitated with DMF-ethyl acetate. Yield 0.62 g (70%), melting point 154-157 degreeC.

b) Preparation of δ-Aval-Arg-Gly-Phe-Phe-NH ₂

500 mg of Z-Aval-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 5 hours using Pd as a catalyst. After filtering off the catalyst, the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl-sepadex (1.8 cm x 9 cm) and eluted by passing a 1 N ammonium acetate solution (500 ml) from a 0.1 N ammonium acetate solution (500 ml) by a linear gradient gradient method. . Fractions of 290-370 ml were collected and dried in Tokyo. Quantity 290 mg (61%,

(2), peptide content: 81.4%

Example 37

Preparation of ε-Acap-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-ε-Acap-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

In the same manner as in Example 34-e), 0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was treated with HBr-acetic acid and dissolved in 10 ml of DMF. This solution was neutralized with triethylamine. On the other hand, 0.32 g of Z-ε-Acap-OH and 0.24 g of HONB were dissolved in 5 ml of tetrahydrofuran, and 0.27 g of DCC was added under ice cooling. The mixture was stirred for 3 hours to distill off the dicyclo hexylurea formed and add this filtrate to the amine component [H-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ ]. The mixture was stirred overnight. The solvent was distilled off under reduced pressure and water was added. The resulting powder was filtered off and dried sufficiently. Then washed with ethyl acetate and recrystallized with DMF-ethyl acetate. 0.49 g (55%), melting point 102-104 ° C

b) Preparation of ε-Acap-Arg-Gly-Phe-Phe-NH ₂

400 mg of Z-ε-Acap-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 7 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. This residue was passed through a column of carboxymethyl-sefadex (1.8 cm × 10 cm), and then eluted by passing a 1 N ammonium acetate solution (500 mL) from a 0.1 N ammonium acetate solution (500 mL) by a linear gradient gradient method. Done. 250-330 ml of fractions were combined and lyophilized. 200 mg (51%)

Example 38

Preparation of Lys-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Di-Z-Lys-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

In the same manner as in Example 34-e), 0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was treated with HBr-acetic acid and dissolved in 10 ml of DMF. This solution was neutralized with triethylamine.

On the other hand, 0.54 g of di-Z-Lys-OH and 0.26 g of HONB were dissolved in 5 ml of tetrahydrofuran, and 0.30 g of DCC was added while cooling with ice. The mixture was stirred for 4 hours and the resulting dicyclohexylurea was filtered off. The filtrate was added to the amine component [H-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ ] and then stirred overnight. The solvent was distilled off under reduced pressure, and water was added to the residue. The resulting powder was collected by filtration and dried sufficiently. It was then washed with ethyl acetate and then reprecipitated with DMF-ethyl acetate. Yield 0.82 g (77%), melting point 200-204 ° C (decomposition),

b) Preparation of Lys-Arg-Gly-Phe-Phe-NH ₂

600 mg of di-Z-Lys-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 6 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl sefadex (1.5 cm x 11 cm) and eluted by passing a 1 N ammonium acetate solution (700 mL) from a 0.1 N ammonium acetate solution (700 mL) by a linear gradient gradient method. 650-760 mL of fractions were combined and lyophilized. Yield 280 mg (52%),

Example 39

Preparation of α, γ-Dab-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of di-A-Dab-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

In the same manner as in Example 34-e), 0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was treated with HBr-dissolved to dissolve in DMF10 mg. This solution was neutralized with triethylamine. On the other hand, 0.50 g of di-Z-α, γ-Dab-OH and 0.26 g of HONB were dissolved in 5 mg of tetrahydrobutane, and 0.30 g of DCC was added while ice-cooling. The mixture was stirred for 4 hours and the dicyclohexylurea formed was filtered off.

The filtrate was added to the amine component H-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ , and the mixture was stirred overnight. The solvent was distilled off under reduced pressure and water was added to the residue. The resulting powder was collected by filtration and dried sufficiently. The powder was washed with ethyl acetate and reprecipitated with DMF-ethyl acetate. Yield 0.82 g (78%), melting point 184-188 degreeC.

b) Preparation of α, γ-Dab-Arg-Gly-Phe-Phe-NH ₂

600 mg of Di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 6 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl-sefadex (1.5 cm x 13 cm) and eluted by passing 1 N ammonium acetate solution (700 mL) from 0.1 N ammonium acetate solution (700 mL) by linear gradient gradient method. Done. 610-680 mL of fractions were combined and lyophilized. Yield 255 mg (46%),

Example 40

Preparation of Arg-Arg-Gly-Phe-Phe-NH ₂

a) Preparation of Z-Arg (NO ₂ ) -Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂

In the same manner as in Example 34-e), 0.80 g of Z-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was treated with HBr-acetic acid and dissolved in 10 ml of DMF. This solution was neutralized with triethylamine. This solution was neutralized with triethylamine. On the other hand, 0.39 g of Z-Arg (NO ₂ ) -OH and 0.22 g of 2,4-dinitrophenol were dissolved in 5 ml of tetrahydrofuran, and 0.25 g of DCC was added while ice-cooling. The mixture was stirred for 2 hours and the dicyclohexylurea formed was filtered off. This filtrate was added to the amine component [H-Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ ], followed by stirring overnight. The solvent was distilled off under reduced pressure and water was added to the residue. Washed sufficiently with oily product and reprecipitated with DMF-ethyl acetate. 0.90 g (89%), melting point 89-92 deg.

b) Preparation of Arg-Arg-Gly-Phe-Phe-NH ₂

700 mg of Z-Arg (NO ₂ ) -Arg (NO ₂ ) -Gly-Phe-Phe-NH ₂ was dissolved in 40 ml of acetic acid, and catalytic reduction was performed for 9 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl-cellulose (3 cm x 18 cm) and eluted by passing 0.3 N ammonium acetate aqueous solution (1 L) from 0.005 N aqueous ammonium acetate solution (1 L). Elution fractions of 890-1,410 ml were collected and lyophilized. Yield 380 mg (45%),

Example 41

Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-β-Ala-Arg (NO ₂ ) -Gly-OEt

130 ml of 25% HBr-acetic acid was added to 26.3 g of Z-Arg (NO ₂ ) -Gly-OEt, and stirred at room temperature for 60 minutes, followed by ethyl ether. This precipitate was collected by filtration and dried over sodium hydroxide. The dried precipitate was then dissolved in 150 ml of DMF and ice cooled before neutralizing the solution with triethylamine. Thereafter, 23.1 g of Z-β-Ala-ONB was added, and the mixture was stirred overnight. The solvent was distilled off under reduced pressure and the residue was dissolved in 300 ml of ethyl acetate containing a small amount of n-butanol. The solution was washed with water, a saturated aqueous solution of sodium bicarbonate (100 mL x 3) and a saturated aqueous solution of sodium chloride (100 mL x 3), and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure and ethyl ether was added. The resulting powder was recovered by filtration and recrystallized from ethanol-ethyl acetate. Yield 22.9 g (75%), melting point 97-99 ℃

b) Preparation of Z-β-Ala-Arg (NO ₂ ) -Gly-OH

12.1 g of Z-β-Ala-Arg (NO ₂ ) -Gly-OEt was dissolved in a mixture of 50 ml of acetone and 25 ml of water, and 27.8 ml of an aqueous sodium chloride 1N sodium hydroxide solution was added dropwise over 30 minutes under ice-cooling. The mixture was stirred under the same conditions for 1 hour and further stirred at room temperature for 2 hours, and then 3.8 ml of 1N hydrochloric acid was added, and acetone was distilled off under reduced pressure. 50 ml of water was added to the residue, and the mixture was acidified with 6N hydrochloric acid to obtain an oily substance which immediately solidified.

The solid material thus obtained was collected by filtration. 10.3 g (86.0%) of water, melting point 101-103 deg.

c) preparation of Z-Ph-Tyr-OMe

After suspension of ice-cooled by suspending 5.10 g of Tyr-OMe HCl in 100 mL of tetrahydrofuran, the suspension was neutralized with 3.08 mL of triethylamine. 5.99 g of Z-Phe-OH, 3.94 g of HONB, and 4.54 g of DCC were added thereto, and the mixture was stirred overnight. The formed dicyclohexyl urea was filtered off and the solvent was distilled off by evaporation under reduced pressure. The residue was dissolved in 200 ml of ethyl acetate, and the solution was washed with a saturated aqueous solution of sodium hydrogen carbonate (100 ml x 3), 1N hydrochloric acid (100 ml x 3) and water, and then dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure, petroleum benzine was added to obtain a gelatinous residue, which was recovered by filtration and reprecipitated from ethyl acetate-petroleum benzene. Quantity 6.00 g (63%), Melting point 132-133 ° C,

d) preparation of Z-Phe-Tyr-NH ₂

Z-Phe-Tyr-OMe 3.0g was dissolved in 40 ml of methanol, saturated with ammonia, and the solution was stored in a closed container for 1 week to separate needle-like material. After evaporation of the solvent under reduced pressure, ethyl ether was added and the resulting crystals were collected by filtration and recrystallized from ethanol-water. Yield 2.61 g (90%), melting point 217-218 ° C,

e) preparation of Z-Phe-Phe-Tyr-NH ₂

A small amount of anisol was added to 1.15 g of Z-Phe-Tyr-NH ₂ followed by 11 ml of 25% HBr-acetic acid. After stirring for 40 minutes at room temperature, ethyl ether was added. The resulting precipitate was filtered off and dried over sodium hydroxide. On the other hand, DCC 0.52g was added dissolving 0.67 g of Z-Phe-OH and 0.45 g of HONB in 5 ml of tetrahydrofuran. The mixture was stirred for 4 hours and the resulting dicyclohexylurea was filtered off.

The amine component [H-Phe-Tyr-NH ₂ ] was dissolved in 10 mL of DMF and the solution was neutralized with triethylamine.

The ester solution was added to this solution, and the mixture was stirred overnight. The solvent was distilled off under reduced pressure and then water was added thereto. The resulting crystals were collected by filtration, extracted with ethyl acetate and recrystallized with DMF-ethyl acetate. Yield 1.01 g (74%), melting point 218-221 ° C (decomposition),

f) Preparation of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

0.91 g of Z-Phe-Phe-Tyr-NH ₂ was suspended in a mixture of 30 ml of methanol and 20 ml of DMF, followed by catalytic reduction for 7 hours. Methanol was distilled off under reduced pressure. On the other hand, 0.72 g of Z-β-Ala-Arg- (NO ₂ ) -Gly-OH and 0.32 g of HONB were dissolved in 5 ml of DMF, and 0.37 g of DCC was added while ice-cooling. The mixture was stirred for 6 hours to remove vivid dicyclohexylurea. This filtrate was added to the amine component [H-Phe-Phe-Tyr-NH ₂ ], followed by stirring overnight. The solvent was distilled off under reduced pressure to obtain a solid material which was titrated with water. The resulting powder was collected by filtration and reprecipitated from DMF-ethyl acetate.

Yield 0.88 g (62%), melting point 174-177 degreeC,

g) Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-NH ₂

740 mg of Z-β-Ala-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 7 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl-sepadex (1.6 cm x 15 cm) and eluted by passing through an aqueous solution of 1 N ammonium acetate (700 mL) from an aqueous solution of 0.005 N ammonium acetate (700 mL) by linear gradient gradient method. Done. Elution fractions of 580-790 mL were collected and lyophilized. 540 mg (75%),

h) Preparation of β-Ala-Arg-Gly-Phe-Phe-Tyr-NH ₂

β-Ala-Arg-Gly- Phe-Phe-Tyr-NH 2 · 2 was dissolved nitrate trihydrate (9.33g) in water 20㎖, was added with stirring to 2.2g of citric acid monohydrate to the solution. The resulting crystalline precipitate was collected by filtration and washed with a small amount of cold water to obtain 10.4 g of the corresponding dicitramate.

Example 42

Preparation of γ-Abu-Arg-Gly-Phe-Phe-Tyr-NH ₂

a) Preparation of Z-γ-Abu-Arg (NO ₂ ) -Gly-OEt

40 ml of 25% HBr-acetic acid was added to 4.38 g of Z-Arg (NO ₂ ) -Gly-OEt, and stirred at room temperature for 30 minutes, followed by ethyl ether. The resulting precipitate was filtered off and dried over sodium hydroxide.

Then, 30 ml of DMF was added thereto, followed by ice cooling, and the solution was neutralized with triethylamine.

4.16 g of Z-γ-Abu-ONB was added to this solution, and the mixture was stirred overnight. The solvent was fed under reduced pressure, and water was added to this residue. Water was removed by decantation and ethyl ether was added to the residue to solidify the residue.

This was then filtered off and reprecipitated from acetonitrile.

Yield 2.5 g (48%), melting point 60-62 ° C,

b) Preparation of Z-γ-Abu-Arg (NO ₂ ) -Gly-OH

2.0 g of Z-γ-Abu-Arg (NO ₂ ) -Gly-OEt was dissolved in 20 ml of acetone, and 5.7 ml of an aqueous solution of 1N sodium hydroxide was added while cooling with ice. The mixture was stirred at room temperature for 2 hours, then 6 ml of 1N hydrochloric acid was added, and then the acetone was distilled off under reduced pressure. The resulting crystals were filtered off and recrystallized from ethanol-water.

Yield 1.86 g (98%), melting point 113-115 ° C,

c) Preparation of Z-γ-Abu-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

A small amount of anisol was added to 1.34 g of Z-Phe-Phe-Tyr-NH ₂ followed by 13 ml of 25% HBr-acetic acid.

After shaking for 45 minutes at room temperature, ethyl ether was added and the resulting precipitate was filtered off and dried over sodium hydroxide. On the other hand, 0.99 g of Z-γ-Abu-Arg (NO ₂ ) -Gly-OH and 0.43 g of HONB were dissolved in 5 mL of DMF, and 0.50 g of DCC was added under ice-cooling. The mixture was stirred for 6 hours and the dicyclohexyl urea was filtered off. The amine component [H-Phe-Phe-Tyr-NH ₂ ] was dissolved in 10 mL of DMF and the solution was neutralized with triethylamine.

The above active ester solution was added to this solution to remove this mixture. The resulting powder was collected by filtration, washed with ethyl acetate and reprecipitated with DMF-ethyl acetate.

Yield 1.12 g (58%), melting point 139.5-141 ° C,

d) preparation of γ-Abu-Arg-Gly-Phe-Phe-Tyr-NH ₂

800 mg of Z-γ-Abu-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 14 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The solution was passed through a column of carboxymethyl-sefadex (1.5 cm × 10 cm) and passed through an aqueous solution of 1 N ammonium acetate (700 ml) from an aqueous solution of 0.1 N ammonium acetate (700 ml) by a gradient elution method. Done. Elution fractions of 410-600 ml were collected and freeze-dried. Water volume 412ml (52%),

Example 43

Preparation of ε-Acap-Arg-Gly-Phe-Phe-Tyr-NH ₂

a) Z-ε-Acap-Arg (NO ₂ ) -Gly-OEt

To 2.41 g of 2-Arg (NO ₂ ) -GIX-OEt, 12 ml of 25% HBr-acetic acid was added and shaken at room temperature for 40 minutes, followed by ethyl ether. The resulting precipitate was filtered off and dried over sodium hydroxide.

Meanwhile, 1.33 g of Z-ε-Acap-OH and 0.99 g of HONB were dissolved in 5 ml of tetrahydrofuran, and then 1.14 g of DCC was added while cooling with ice.

The mixture was stirred for 4 hours and the dicyclo hexylurea formed was filtered off.

The amine component was dissolved in 10 ml of DMF, the solution was neutralized with triethylamine, and then the active ester solution was added. The mixture was stirred overnight and the solvent was distilled off under reduced pressure. The residue was dissolved in 50 ml of ethyl acetate and the solution was washed with water, a saturated aqueous solution of sodium bicarbonate (50 ml x 2) and water, and then dried over anhydrous sodium carbonate. The solvent was distilled off under reduced pressure and petroleum benzine was added thereto. The resulting crystals were recrystallized from water-ethanol-ethyl acetate.

b) Preparation of Z-ε-Acap-Arg (NO ₂ ) -Gly-OH

1.8 g of Z-ε-Acap-Arg (NO ₂ ) -Gly-OEt was dissolved in 10 ml of acetone, and 2.9 ml of an aqueous solution of 2N sodium hydroxide was added with ice cooling. The mixture was stirred at rt for 2 h.

2.5 ml of 1N hydrochloric acid was added, and then acetone was distilled off under reduced pressure. 10 ml of water was added to the residue, and the insoluble matter was filtered off. The filtrate was acidified with 1N hydrochloric acid to remove crystals formed. These crystals were recovered by filtration.

c) Preparation of Z-ε-Acap-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

A small amount of anisole was added to 1.22 g of Z-Phe-Phe-Tyr-NH ₂ followed by 10 ml of 25% HBr-acetic acid. The mixture was shaken for 40 minutes at room temperature, and ethyl ether was added thereto. The precipitate was collected by filtration and dried over sodium hydroxide. On the other hand, 1.05 g of Z-ε-Acap-Arg (NO ₂ ) -Gly-OH and 0.36 g of HONB were dissolved in 10 ml of DMF, and 0.42 g of DCC was added thereto while cooling with water. The mixture was stirred for 6 hours to filter off byproduct dicyclohexylurea.

The amine component was dissolved in 10 ml of DMF to neutralize this solution with triethylamine.

The active ester solution was added to this solution, and the mixture was stirred overnight. The solvent was distilled off under reduced pressure and water was added. The resulting powder was collected by filtration, washed with ethyl acetate and reprecipitated with DMF-ethyl acetate.

d) Preparation of ε-Acap-Arg-Gly-Phe-Phe-Tyr-NH ₂

700 ml of Z-ε-Acap-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 40 ml of acetic acid, and catalytic reduction was performed for 14 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a column of carboxymethyl-sepadex (1.8 cm x 12 cm) and eluted by passing through an aqueous solution of ammonium acetate (700 mL) from an aqueous solution of 0.1 N ammonium acetate (700 mL) by linear gradient gradient method. 430-550 ml of the elution fractions were collected and lyophilized. 430 mg (62%),

Example 44

Preparation of Lys-Arg-Gly-Phe-Phe-Tyr-NH ₂

a) Preparation of di-Z-Lys-Arg (NO ₂ ) -Gly-OEt

Using 2.07 g of di-Z-Lys-OH, the same procedure as in Example 43-a) was carried out to obtain a gel target product. Reprecipitation with water-ethanol-ethyl acetate gave 2.57 g (73%) of the title compound.

b) Preparation of di-Z-Lys-Arg (NO ₂ ) -Gly-OH

2.0 g of di-Z-Lys-Arg (NO ₂ ) -Gly-OEt was dissolved in 10 ml of acetone, and 2.1 ml of an aqueous solution of 2N sodium hydroxide was added while cooling with ice. The mixture was stirred at rt for 2 h. 1.4 ml of 1N hydrochloric acid was added, and then acetone was distilled off under reduced pressure. The residue was diluted with 10 mL of water and the insolubles were distilled off. Then, the solution was made acidic with 1N hydrochloric acid, and the resulting gelatinous precipitate was collected by filtration.

c) Preparation of di-Z-Lys-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

Using the same method as Example 43-c), using 1.21 g of di-Z-Lys-Arg (NO ₂ ) -Gly-OH, the gelled target product was obtained. Yield 1.21 g (58%), Melting Point 199-203 ° C,

d) preparation of Lys-Arg-Gly-Phe-Phe-Tyr-NH ₂

1.00 g of di-Z-Lys-Arg (NO) ₂ -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 20 hours using Pd as a catalyst. The catalyst was filtered off and the solvent was distilled off under reduced pressure. The residue was passed through a carboxymethyl Sephadex column (1.8 cm x 10 cm). Elution was carried out by passing an aqueous solution of 1N ammonium acetate (500 mL) from an aqueous solution of 0.1 N ammonium acetate (500 mL) by a linear gradient gradient method, and the elution fractions of 650-890 mL were collected and freeze-dried. 468 mg (50%),

Example 45

Preparation of α, γ-Dab-Arg-Gly-Phe-Phe-Tyr-NH ₂

a) Preparation of di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-OEt

In 40 ml of ethyl ether, di-Z-α, γ-Dab-OH. ("DCHA" is short for dicyclohexylamine) was suspended. The suspension was then shaken with 0.5 N sulfuric acid. The ether layer was washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off, and the gel target product was obtained using the exact same method as in Example 43-a). This was reprecipitated from water-ethanol-ethyl acetate.

Yield 2.36g (70%), Melting Point 153 ~ 156 ℃,

b) Preparation of di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-OH

2.0 g of di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-OEt was dissolved in 30 ml of acetone, and 2.97 ml of an aqueous solution of 2N sodium hydroxide was added dropwise thereto under ice cooling. The mixture was stirred for 1 hour under the same conditions, and 3 ml of 1N hydrochloric acid was added. Acetone was distilled off under reduced pressure, and 10 ml of water was added thereto. The insolubles were filtered off and the filtrate was acidified with 1N hydrochloric acid. The resulting gelatinous precipitate was filtered off.

c) Preparation of di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂

Using 1.12 g of di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-OH, a gelled target product was obtained using the exact same method as in Example 43-c). Yield 1.02g (51%), Melting Point 191-195 ℃,

d) Preparation of α, γ-Dab-Arg-Gly-Phe-Phe-Tyr-NH ₂

600 mg of di-Z-α, γ-Dab-Arg (NO ₂ ) -Gly-Phe-Phe-Tyr-NH ₂ was dissolved in 30 ml of acetic acid, and catalytic reduction was performed for 12 hours using Pd as a catalyst. The catalyst was quenched and the solvent was removed by evaporation under reduced pressure. The residue was passed through a column of carboxymethyl-sepadex (1.8 cm × 10 cm), and then passed through an aqueous solution of 0.1 N ammonium acetate (500 mL) by a linear background method (500 mL). Elution was performed. Fractions of 490-620 ml were collected and lyophilized.

Amino acid analysis: Arg, 1.00 (1), Dab, 1.04 (1), Gly, 1.09 (1), Tyr, 1.02 (1), Phe, 2.13 (2): Peptide content: 79%.

In the following Examples of the present invention, substituents and amino acid or peptide structural formulas of the polypeptides of the present invention are described.

[List of Examples]

R ₁ -Arg-R ₂ -Phe-Phe-R ₃

Claims (1)

To this in preparing a polypeptide of formula (Ⅰ), the amino acid corresponding to R ₁ itself, or R ₁ is a hydrogen arginine, or N- terminal amino acid or amino acid residue R ₁ itself or as N- terminal amino acid, R ₁ A fragment of the polypeptide (I) having an arginyl group which is hydrogen is peptide condensed with the remaining fragment of the polypeptide (I), and when the condensation product has a protecting group, it is represented by the following general formula (I) Preparation of Polypeptides.

In the above formula, R ₁ is hydrogen or an amino acid residue that is basic or neutral, R ₂ is a neutral amino acid residue, R ₃ represents NH ₂ · Tyr-NH ₂ or a tyrosyl peptide residue consisting of 1 to 5 amino acid residues or an amide residue of a tyrosyl peptide thereof, wherein when R ₁ is hydrogen, R ₂ is Pro.