JPS60162000A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:A compound (salt) expressed by formulas I -IV (R<4> is H or protecting group of mercapto). USE:An antigen for analyzing hepatitis B viruses and antigen for hepatitis B vaccines. PREPARATION:For example, amino acids having amino group protected with tert-butoxycarbonyl group are successively condensed with a chloromethylated styrene-divinylbenzene copolymer resin as a carrier according to the amino acid sequence of the aimed peptide, and protecting groups are removed. The above- mentioned operation is repeated successively to give a peptide resin, which is then treated with hydrogen fluoride in the presence of a solvent to separate the resultant peptide from the carrier. Mercuric acetate and acetic acid are if necessary added thereto to adjust pH to 4, and hydrogen sulfide gas is blown thereinto under agitation to reduce the peptide and form an intramolecular cystine type peptide. Thus, the aimed peptide expressed by formulas I -IV is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide, and more particularly, to a novel peptide and a pharmaceutical useful in the analysis of hepatitis B virus antigens, and as an antigen composition for hepatitis B vaccines. The salts thereof are permissible as

The novel peptides of this invention have the following formulas C11 to [X
Indicated by [].

[I] [II] [III] [IV] [Vl [■] [■] [Vl[] [IX] [X] [XI] (In each of the above formulas, R4 means hydrogen or a mercapto protecting group) This In the specification, regarding amino acids, protecting groups, active groups, solvents, etc., IUPAC-IUB commissio
Non Biological Nomenclatu
It may be indicated by an abbreviation based on re or a common abbreviation in the field, examples of which are as follows. Leu: leucine, (4s rainbow stein, Hi
s: histidine, Pro nibroline, He: isoleucine, Asp: aspartic acid, Asn: asparagine, Thr: threonine, Ala: alanine, Gln:
Glutamine, Gly nigericin, Lys: Lysine, Ph
e: Phenylalanine, Arg: Arginine, Glu:
Glutamic acid, Val: valine, Trp:) ribtophan, Set: serine, Boc: t-butoxycarbonyl, cl-z: 2-chlorobenzyloxycarbonyl, Bzl: benzyl, Acm: acetamidomethyl, Tos-p-)luenesulfonyl, Pac: phenacyl ertel, cHex dichlorohexyl, DMF: N
, N-dimethylformamide, TFA nitrifluoroacetic acid, WSCD: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, )IOBT: N-hydroxybenzoliazole, OSu: N-hydroxysuccinimide ester.

Compounds [I] to [X[] of this invention and pharmaceutically acceptable salts thereof can be produced by various methods, and these methods will be explained below.

(1) Method-1 (Peptide synthesis by solid phase method): 1) Method 1-1 R1-9er (R8)-resin [11a]-+2) Method 1
-2 R”-Crys(Ra)-resin [I[1a] →[l
lIb] 3) Method 1-3 R'-3et(R”)-Resin [IVa]-+ (2) Method 2 (Peptide synthesis by liquid phase method) (I-b) (3) Method 3 (Protecting group Elimination method) l) Method 3-1 [From] ↓ [I cl 2) Method 3-2 [11b] ↓ [11] 3) Method 3-3 [111b ↓ [mc] 4) Method 3-4 [ IVc] (4) Method 4 (elimination of mercapto protecting group and cyclization reaction): 1) Method 4-1 [■] [■] [■] 2) Method 4-2 [■] 3) Method 4- 3 [IVc] ↓ [IVcll 4) Method 4-4 [IVd] [X[] [IVd] In the above formula, R1 is an amine protecting group, R2 is a carboxy protecting group, R8 is a hydroxy protecting group, and Ra' is a mercapto protecting group. The group R means a guanidino protecting group and R6 means an imidazole protecting group, respectively. These definitions are explained below.

Regarding the amino protecting group in (+) R: Amine protecting groups include common amino protecting groups commonly used in the fields of amino acid and peptide chemistry, and preferred examples of such amine protecting groups include alkoxycarbonyl or cycloalkoxycarbonyl (e.g. t-butoxycarbonyl, t-pentoxycarbonyl, cyclohexylcarbonyl, etc.), substituted or unsubstituted phenyl lower alkoxycarbonyl (e.g. benzyloxycarbonyl,
Examples include aralkoxycarbonyl such as 2-chlorobenzyloxycarbonyl, substituted or unsubstituted allenesulfonyl (eg, benzenesulfonyl, P-)luenesulfonyl, etc.).

(2) Carboxy protecting group in R2: Carboxy protecting groups include common carboxy protecting groups commonly used in the fields of amino acid and peptide chemistry, and preferred examples of such protecting groups include lower alkyl (e.g. alkyl such as methyl, ethyl, etc.), cycloalkyl (e.g. cyclopentyl, cyclohexyl, etc.), heptano- or hacyphenyl lower alkyl (e.g. benzyl,
Examples include 7ralkyl such as diphenylmethyl, etc., and aroyl alkyl (eg, phenacyl, toluoyl ethyl, etc.).

(3) Regarding the hydroxy protecting group in Ra: Hydroxy protecting groups include common hydroxy protecting groups widely used in the fields of amino acid and peptide chemistry, and suitable examples of such hydroxy protecting groups include, for example, alkanoyl ( Examples include acyl such as acetyl, substituted or unsubstituted aralkyl (such as benzyl, 2,6-dichlorobenzyl, etc.), and the like.

(4) Regarding the mercapto protecting group in R4 and Ra': Mercapto protecting groups include protecting groups that do not leave off under the elimination conditions of amino protecting groups and hydroxy protecting groups, and preferred examples of such protecting groups include: , acylaminoalkyl (e.g., acetamidomethyl, benzamidomethyl, etc.), arylmercaptan (e.g., 3-nitropyridine-2-thiol, etc.), substituted or unsubstituted lower alkyl (e.g., t-butyl, etc.), substituted or Unsubstituted aralkyl (e.g. benzyl, p-methoxybenzyl, p-methylbenzyl, 3,4-dimethylbenzyl, diphenylbenzyl, trityl, etc.), substituted or unsubstituted lower alkyl mercapto (e.g. ethylmercapto, t-butylmercapto, etc.) ) etc. can also be used by appropriately selecting the removal conditions for each protecting group.

(5) Regarding the guanidino protecting group in R5 and the imidazole protecting group in R6: The guanidino protecting group and the imidazole protecting group include the usual guanidino protecting group and imidazole protecting group commonly used in the field of amino acid and peptide chemistry, Preferred examples of such protecting groups include, for example, nitro, substituted or unsubstituted arenesulfonyl (P-)
(eg, luenesulfonyl, etc.), substituted or unsubstituted phenyl lower alkoxycarbonyl (eg, benzyloxycarbonyl, etc.), and alkoxycarbonyl (eg, t-butoxycarbonyl, etc.).

(8) Regarding pharmaceutically acceptable salts: Compound [I]
Pharmaceutically acceptable salts in ~[X[] include, for example, alkali metal salts (e.g., sodium salts, potassium salts, etc.)
, alkaline earth metal salts (e.g. calcium salts, etc.), ammonium salts, organic amine salts (e.g. ethanolamine salts, triethylamine salts, dicyclohexylamine salts, etc.)
and addition salts of organic or inorganic acids such as trifluoroacetic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid. The above various methods will be explained below.

(1) About method 1: This method consists of compounds [IIa], [mal and [I
Va] according to the conventional method of peptide synthesis by solid phase method.
Each protected constituent amino acid is coupled sequentially,
This is a method for obtaining compounds [11bl, [mb] and [IVb].

The compounds [n a], [mal and [IV a] used here as starting materials are known substances (for example, Biopolymer Vol. 12, Vol. 251).
3, 1973) and a new substance, which can be prepared analogously to the method described in that document. Furthermore, as the resin used, any resin used in peptide synthesis by the solid phase method can be used, and examples of such resins include, for example, chloromethylated styrene-divinylbenzene copolymer. ,
Examples include polystyrene-type resins such as hydroxymethylated styrene-divinylbenzene copolymer and amine-methylated styrene-divinylbenzene copolymer, and polyamide-type resins such as polydimethylacrylamide resin. This method is generally carried out in one cycle of steps 1 to 11 as follows for each protected amino acid.

1) Step 1: This step is carried out to wash the starting material, the protected amino acid-resin, and to swell the resin. Suitable examples of solvents used for this purpose include methylene chloride, Examples include chloroform, dimethylformamide, or a mixed solvent thereof.

2) Step 2: This step is a step of removing the amine protecting group in the protected amino acid-resin, and the reaction in this step is carried out by a conventional method such as a method using an acid or a method using a base. be exposed.

Among the above-mentioned elimination methods, the method using an acid is most frequently used, so the addition method will be explained below.

This reaction is carried out in a solvent such as methylene chloride, chloroform, acetic acid, or water in the presence of an inorganic or organic acid such as trifluoroacetic acid, formic acid, P-toluenesulfonic acid, hydrochloric acid, or hydrobromic acid, preferably ethanedithiol or This is done by adding anisole.

Among the acids listed above, trifluoroacetic acid and formic acid are also used as solvents.

This reaction is usually carried out under cooling (eg -78°C) to room temperature.

3) Step 3: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as Step 1 above.

4) Step 4: This step is performed to shrink the resin and increase the cleaning effect, and the amino acid-resin is treated with alcohol (methanol, ethanol, propatool, 2-propanol, butanol, etc.), dioxane, etc. Preferably, it is treated.

5) Step 5: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as in Step 1 above.

8) Step 6: This step is carried out for desalting when the amino acid in the amino acid-resin obtained by the treatments in Steps 1 to 5 above exists as an acid addition salt at the α-7 amino group. , for example by treatment with a base such as triethylamine.

7) Step 7: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as Step 1 above.

8) Step 8: This step involves coupling each protected constituent amino acid and is carried out in a solvent such as methylene chloride, chloroform, dimethylformamide in the presence of a conventional condensing agent such as dicyclohexylcarbodiimide. It is also possible to convert the carboxy group of each protected amino acid into
It is also possible to activate it into an acid anhydride, active ester, etc. by a conventional method, and then carry out the reaction in the above-mentioned solvent.

9) Step 9: This step is carried out to remove impurities and swell the resin, and is carried out in substantially the same manner as in Step 1 above.

10) Step 10: This step is carried out to shrink the resin and improve the cleaning effect, and is carried out by substantially the same method as step 4) above.

11) Step 11: This step is carried out to wash and swell the resin, and is carried out in substantially the same manner as in Step 1 above.

Each of the above steps is generally performed at about room temperature, and each step (excluding step 8) is preferably repeated about 2 to 3 times.

(2) Method 2 (Peptide synthesis by liquid phase method) In this method, a compound (
This is a method of obtaining compound (Ib) or a salt thereof by reacting compound (I-2) or a salt thereof with I-1) or a salt thereof.

The compound (I-
1) and (I-2) are each new compounds, and these compounds can be produced by the Examples described later and methods similar to them.

The reaction of this method is carried out as follows.According to one method, first, the carboxyl group of compound (I-1) or a salt thereof is converted into an acid halide, acid azide, or acid anhydride by a conventional method. , mixed acid anhydride, active ester, etc., and react them with compound (I-2) to obtain compound (Ib). According to another method, compound (r-i) or a salt thereof and compound (I-2
) or its salts are reacted directly in the presence of a conventional condensing agent such as N,N-dicyclohexylcarbodiimide.

Among these activation methods, preferred methods and condensing agents are selected depending on the type of carboxy-protecting group of compound (I-1) and reaction conditions (eg, reaction solvent, reaction temperature, etc.).

This reaction is carried out in a solvent such as methylene chloride, chloroform, tetrahydrofuran, dioxane, ethyl acetate, methanol, ethanol, water, etc., with cooling (e.g., -20°C).
) to proceed smoothly at room temperature. Further, the reaction carried out in the presence of a condensing agent is generally carried out under anhydrous and mild conditions.

(3) Regarding method 3: This method is applicable to compounds [Ibl], [Ibl].

[Ibl and [IVb] are each an amine protecting group,
Upon elimination reaction of carboxy protecting group, hydroxy protecting group, guanidino protecting group and imidazole protecting group,
Compound [Ic], respectively.

This is a method to obtain [lIc1, [mc] and [IVc].

The methods for removing the amine protecting group, guanidino protecting group and imidazole protecting group include substantially the same method as Step 2 of the above method (I), the catalytic reduction method, the liquid ammonia-alkali metal method, the acid zinc method, and the hydrazine method. The carboxy protecting group and the hydroxy protecting group are removed by conventional methods such as hydrolysis and reduction.

■) Hydrolysis: Hydrolysis is preferably carried out in the presence of an acid or a base.

Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid,
Sulfuric acid, etc.) organic acids (eg, formic acid, acetic acid, trifluoroacetic acid, propionic acid, benzenesulfonic acid, P-)luenesulfonic acid, etc.), acidic ion exchange resins, etc.

Suitable bases also include alkali or alkaline earth metal hydroxides, carbonates or bicarbonates (eg hydroxides), potassium hydroxide, sodium carbonate,
(potassium carbonate, lithium carbonate, sodium hydrogen carbonate, calcium hydroxide, magnesium hydroxide, etc.), ammonium hydroxide, and other inorganic bases.

Hydrolysis is carried out under relatively mild conditions such as cooling or heating, and with solvents that do not adversely affect the reaction [e.g., water, wood-philic solvents such as alcohols (e.g., methanol, ethanol, propatool, etc.), acetone, N , N-dimethylformamide, tetrahydrofuran, dioxane, dimethyl sulfoxide, or a mixed solvent thereof.

2) Reduction: Reduction methods including chemical reduction and catalytic reduction methods are carried out by conventional methods.

Preferred reducing agents used in chemical reduction include, for example, metals (e.g. tin, zinc, iron, etc.) or metal compounds (e.g. chromium chloride, chromium acetate, etc.) and organic or inorganic acids (e.g. formic acid, acetic acid, propionic acid, trichloride, etc.). Fluoroacetic acid, P
-) combinations with luenesulfonic acid, hydrochloric acid, hydrobromic acid, etc.).

Preferred catalysts used for catalytic reduction include platinum catalysts (e.g. platinum plate, platinum sponge, platinum black, platinum colloid, platinum oxide, platinum wire, etc.), palladium catalysts (palladium sponge, palladium black, palladium oxide, palladium carbon, etc.). , palladium colloid, palladium-barium sulfate, palladium-barium carbonate, etc.), nickel catalyst (reduced nickel, nickel oxide, Raney nickel, etc.), cobalt carrier (e.g. reduced cobalt, Raney cobalt, etc.),
Examples include iron catalysts (for example, reduced iron, Raney iron, etc.), copper catalysts (for example, reduced copper, Raney copper, Ullmann copper, etc.). Reduction is usually carried out in a solvent. Preferred solvents include:
For example, water, alcohol (e.g. methanol, ethanol,
propatool, etc.), acetic acid and other common organic solvents or mixtures thereof. Furthermore, the above-mentioned liquid acid used in the chemical reduction can also be used as a solvent. In addition to the above-mentioned solvents, preferred solvents used in the catalytic reduction include diethyl ether, dioxane, tetrahydrofuran, etc., or mixtures thereof.

The reaction is rapidly carried out under relatively mild conditions such as cooling or heating.

Depending on the type of amino-protecting group, guanidino-protecting group, imitazole-protecting group, carboxy-protecting group, and hydroxy-protecting group, they may be removed at the same time by the above elimination reaction. Examples include a method of treating with acids such as hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, etc., and this reaction is carried out in the presence or absence of a normal solvent that does not affect these acids. be able to.

Good results are often obtained in this method when the reaction is carried out in the presence of thiols such as ethanesyl and thiophenol, thioethers such as methionine, dimethyl sulfide and thioanisole, anisole or cresol.

Further, the above acid can also be used as a solvent. This reaction is usually preferably carried out at around 0.degree. C. by cooling.

(4) Regarding method 4: This method uses compounds [Ic], [llIc] and [
IVc] is subjected to a mercapto-protecting group elimination reaction to produce compounds from which the corresponding mercapto-protecting group has been removed, as well as compounds from which an intramolecular S-5 bond and/or an intermolecular S-5 bond has been formed (i.e., Compound [Id], [IIId
l.

[rVdl, [VI, [VII, [VI[
], [VI].

[IX], [X] and [X[]).

As a method for obtaining compounds with these intramolecular and/or intermolecular S-8 bonds, compounds having a free mercapto group (i.e. compounds [Id] and [IIId
There are two methods: one that goes through l and [rVdl), and one that does not go through them. These will be explained below.

1) Method via a compound having a free mercapto group: 1)-1 (Compound [Ic] - Compound [Id], Compound [
lIc1 + compound [IId], compound [mc] - + compound [I [1dl compound [IVc] → compound [rVdl)
: This method is applicable to compound [Ic], [mc] or [IV
c] metal salts such as mercury acetate, mercury trifluoroacetate, and silver acetate, organic phosphorus compounds such as triphenylphosphine and tributylphosphine, mercaptoethanol, mercaptoacetic acid, ethanedithiol, and thiophenol.

By treating with reagents such as alkyl or arylthiols such as dithiothreitol and dithioerythritol, the corresponding compounds [Id] and [I[
Id] and compound [rVdl.

This reaction is usually carried out in a solvent, and preferred solvents include, for example, water, alcohol (eg, methanol, ethanol, propatool, etc.), dioxane, dimethylformamide, and mixed solvents thereof.

This reaction is rapidly carried out under relatively mild conditions such as cooling to heating.

Ri-2 (Compound [I dl → Compound [VII and (or) [■], Compound [md] → Compound Compound I []
, compound [IVd] → compound [IX] , [X] or [X[]): This method involves treating compound [1d], compound [md] or compound [IVd1] under relatively mild oxidative conditions. According to the method, compound [VI] corresponds to compound [I dl.

[VII and/or compound [■] to compound [md
This is a method for obtaining compound [IX], [X] or [XI] corresponding to compound [VII[] and compound [IV dl].

Preferred examples of the oxidizing agent used here include oxygen in the air, potassium ferricyanide, and the like.

2) Method not via a compound having a free mercapto group: (Compound [Ic]→Compound compound, [VII and (
or) [■], compound [llIc] → compound [■]
): This method is used for compound [I c] or compound [mc
] by treating with an electronic reagent such as iodine, dithiocyanogen, etc., a compound [I c] can be obtained.
VII and/or compound [■], also compound [m
This is a method to directly obtain compounds [■] corresponding to [c].

This reaction is usually carried out in a solvent, and examples of preferred solvents include water, alcohol (eg, methanol, ethanol, propatool, etc.), dioxane, dimethylformamide, and the like.

Further, this reaction is carried out under relatively mild conditions such as cooling to heating.

The compounds [I] to [XI] of this invention and the starting materials and intermediates for producing these compounds include one or more isomers due to asymmetric carbon atoms in the molecule,
All such isomers are included within the scope of this invention.

Peptides of this invention, namely compounds [I] to [X[]
and pharmaceutically acceptable salts thereof are useful in the analysis of hepatitis B virus antigens as described above, and are also useful as antigen compositions for hepatitis B vaccines and the like.

Next, examples of the present invention will be shown, and each amino acid in each example refers to the L-configuration.

Example 1-1 H
oe @Pro OSu (7.8 g) was added while cooling on ice. The reaction solution was stirred at 0-5°C for 2 hours, returned to room temperature, and further stirred for 6 hours. During this time, the pH of the reaction solution was
It was maintained at around 7.5 by adding triethylamine. After the reaction, acetonitrile was removed, and the precipitated oily precipitate was extracted with ethyl acetate (200 al). The extract was mixed with 2.5% hydrochloric acid (100+sl, twice) and water (100al
, 3 times), 2.5% sodium bicarbonate (100+
++1.2 times) and dried with magnesium sulfate. The solvent was distilled off and dried, and the residue was crystallized with n-hexane to give Boc e Pro-Glym 0Bz.
1 crystals (8.90 g) were obtained. Crystal is Example 1-
This is the raw material for step 2.

mp 68-89°C [α11. ) = -61,2" (C = 0.845, methanol) thin layer chromatography (silica gel: manufactured by Merck & Co.) Rf = 0.25 [benzene: ethyl acetate (3:1)
]Rf=0.47 [chloroform:ethyl acetate (2:
1) ] Example 1-2 Boc*Pro-Gly obtained in Example 1-1
* Add 0Bzl to T F, A (40+al),
The mixture was treated at room temperature for 30 minutes. After distilling off excess TFA,
The residue was dissolved in 50% acetonitrile in water (?Om+). This solution was cooled to 0°C, and triethylamine was added to neutralize it to 7.5%.
u*09u (3,28g) was added. The mixture was stirred at room temperature for 7 hours, during which time the pH of the reaction solution was maintained around 7.5 by adding triethylamine. After distilling off the acetonitrile, the concentrated solution was diluted with ethyl acetate (70%
m+) and 2.5% hydrochloric acid (4h+1).

Separate the organic solvent layer and add water (30+al, 2 times),
After washing with 2.5% sodium hydrogen carbonate (30+al, 2 times) and water (30+al, 3 times), it was dried over magnesium sulfate. When the solvent was distilled off, oily Boc a Leu -Pro -Gly * 0Bz
1 was obtained (4.30 g), and the crystal was used as the raw material for Example 1-3.

Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf = 0.28 [chloroform: ethyl acetate (2:1
] Example 1-3 Boc e Leu -Pro -G17 *0Bzl obtained in Example 1-2 in methanol (17+ol)
(1.71 g) was added thereto, and the mixture was washed with a 1N aqueous sodium hydroxide solution (3, full ml) at room temperature for 7 hours.

Methanol was distilled off and the concentrate was diluted with water (15+al). This solution was washed with ethyl acetate (20+al), adjusted to pH 2 with 5% hydrochloric acid, and extracted with ethyl acetate (20+al).
0+alX 2) Combine the extracts and wash with water.
0+slX 2) and dried over magnesium sulfate. 4
．． When the medium is distilled off and dried, powdered Boc *Leu
-Pro-Gly eOH (1.28 g) was obtained. Quartz is used as a raw material in Examples 1-11.

[α]D=-10,21" (C=0.594, methanol) A portion (40 ml) of this compound was dissolved in diisopropyl ether, and dicyclohexylamine (20 ml) was dissolved in diisopropyl ether.
mg) was added. When the precipitated crystals were collected by filtration, dicyclohexylammonium salt was obtained.

mp14? ~149°C [α] = -87, El" (C: = 1.0, methanol) Example 1-4 Boc reaction Thr (Bzl) OH (30.94 g) and triethylamine (10.1 g) were dissolved in ethyl acetate ( 3001
In addition to B+), this mixture also contained zenathyl bromide (19
, 9g) were added. The reaction mixture was stirred at room temperature for 15 hours. After removing triethylamine hydrochloride by filtration, the filtrate was mixed with 10% citric acid (150alX 2) and water (1
50+alX 3 ), 2.5% aqueous sodium hydrogen carbonate solution (15hlX2) and water (150alX 3
) and dried over magnesium sulfate. When the solvent was distilled off, an oily substance (42.7 g) was obtained, which was dissolved in TFA (230 al) for 5 minutes (0°C).
Then, each mixture was stirred for 30 minutes (at room temperature).

After evaporating excess TFA, the residue was dissolved in a solution of 3.6N hydrogen chloride in dioxane (4 hl), and the solvent was further evaporated to dryness. Recrystallization of the residue with ether yields HCI a Thr(Bzl)*0Pac
(35.4 g) was obtained.

mp 139-142°C [α]D=-12,80' (C=0.355, in methanol) Example 1-5 Boc 11Ser(Bzl)OH (4,43g) and N
- Methylmorpholine (1,52 g) was added to methylene chloride (9
Isobutyl chlorocarbonate (2.05 g) was added to the mixture while maintaining the mixture at -10 to 15°C, and the mixture was stirred at the same temperature for 25 minutes. Then add this solution to 14
Cooled to 0°C, HC:I-T obtained in Example 1-4
A mixed solution of hr(Bzl)OPac (5.48 g) and N-methylmorpholif (1.52 g) in methylene chloride-DMF (volume ratio 3:1.200 ml) was added to the cooling liquid. 1 hour at -30 to -40℃, then 1.5 hours at 0℃
After stirring for an hour, the mixture was concentrated under reduced pressure. Dissolve the concentrated solution in ethyl acetate (3001111) and add 2% hydrochloric acid (
10hlX2), water (10hlX3), 5% aqueous sodium bicarbonate solution (lohlX2) and water (100mlX
3) (After washing in the order of D, it was dried with magnesium sulfate. When the solvent was evaporated to dryness and the residual liquid was crystallized with petroleum ether, Boc 5et(Bzl)-Thr(Bzl)
e0Pac was obtained. The crystal is used as a raw material in Examples 1-6.

mp 58-60°C [α]D=-4,856 (C=o, 5o, in methanol) Thin layer chromatography (Silica gel: Merck & Co.) Rf=0.43 [Benzene: Ethyl acetate (3:1)
]IR) IC11-' (Nujol) 3320.1745.1700.1880.1880 Example 1-6 Boc*5et (Bzl) obtained in Example 1-5
-Thr(Bzr) Φ0Pac (6.1 g) was treated with TFA (80+ol) at room temperature for 30 minutes. After distilling off excess TFA, the residue was treated with acetonitrile (12
It was dissolved at 0m. The solution was cooled to 0°C and added with N
-Methylmorpholine (1.01 g) and Boc' e↑h
r(Bzl) Φ0Su (4.0 g) was added. The mixture was stirred at 0-5° C. for 10 hours, during which time N-methylmorpholine was added to maintain the pH at about 7.5. Thereafter, acetonitrile was distilled off, and the residue was dissolved in ethyl acetate (200 ml). The obtained solution was mixed with 10% citric acid (100 m
lX 2 ), water (100ml x 3), 5% sodium bicarbonate aqueous solution (100ml x 2), water (100ml
It was washed in the order of X 3 ) and dried over magnesium sulfate. The solvent was distilled off, and the residue was recrystallized from a mixture of ethyl acetate and n-hexane (volume ratio 1:10, IE 15 ml). When you collect the crystals, BoCΦThr(Bzl) -5e
t(Bzl) -Thr(Bzl) 1IOPac(8
, 4g) was obtained.

Proverb p 80-82℃ [α]D=+11.8@(C=0.814, in DMF)
Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf-0,35 (benzene: ethyl acetate (3:l))
IR10 layer-1 (Nujol) 3225.1750.11190.1845 Example 1-
7 Methylene chloride (45ml), acetic acid (5ml), DM
Bo obtained in Example 1-6 was added to the mixture consisting of F(51).
c * Thr(Bzl) -5er(Bzl) -T
Zinc dust (t-7g) was added to a solution prepared by adding hr(Bzl)*0Pac (1.88g) and treated at room temperature for 3 hours. After removing the zinc dust by filtration, the filtrate was concentrated to about 10+1 and dissolved in ethyl acetate (70 ml). This solution was mixed with 2.5% hydrochloric acid (50+++l×2) and water (50+ol
After washing in the order of X4) and drying with magnesium sulfate, the solvent was distilled off to dryness. The oily residue was dissolved in n-hexane (
Boc
ΦThr(Bzl)-Set(Bzl)-Thr(Bz
l) OH (1.30 g) was obtained. Crystal is Example 1-
9 raw materials.

+op 101-104°C [α]D=+22.5° (C=0.193, in methanol) Example 1-8 Boc* Thr (Bzl) obtained in Example 1-6
-5et(Bzl) -Thr(Bzl) -0Pac
(1.80g) was mixed with TFA (15ml) at room temperature.
Processed for 0 minutes. After distilling off excess TFA, 3.8
A solution of N hydrogen chloride in dioxane (1 ml) was combined and the dioxane was evaporated to dryness. The residue was recrystallized from n-hexane, collected and dried over potassium hydroxide pellets under reduced pressure to yield H(:l 11Thr(Bzl)-3er(Bz
l)-Thr(Bzl) 0Pac (1,43 g) was obtained.

mp 128~128℃ [(!], = -11,21° (C=0.193, meta/
Example 1-9 Boce Thr (Bzl) obtained in Example 1-7
-5er(Bzl)-Thr(Bzl)0)1 (74
5 mg), MCI II Thr obtained in Examples 1-8
(Bzl)-9et(Bzl)-Thr(Bzl) ・
0Pac (731+ig) and HOB T (149
+ mg) in DMF (61) was cooled in a water bath, and added to it was added W S CD (155 + mg) in DMF (61).
) was added. After stirring at the same temperature for 3.5 hours, the mixture was diluted with ethyl acetate (Bowl). This solution was mixed with 10% citric acid (5Q+alX2), water (50+alX2),
2.5% sodium bicarbonate aqueous solution, water (50+al
After washing in the order of X 3 ) and drying over magnesium sulfate, the solvent was evaporated to dryness. Crystallization of the residue with ether yields Boa 7hr(Bzl)-5er(B
zl) -Thr(Bzl) -Thr(Bzl) -
5er(Bzl) -Thr(Bzl) -0Pac
(1.08 g) was obtained.

mp 136-138℃ [α co. =-7,01@(C=0.23, in methanol) Thin layer chromatography (Silica gel: Merck & Co., Ltd.) Rf=0.34 [Chloroform: Ethyl acetate (2:1
] Example 1-10 Boc IIThr (Bzl) obtained in Example 1-9
-3er(Bzl) -Thr(Bzl) -Thr(
Bzl) -5et(Bzl)-Thr(Bzl) ・
0Pac (3.3 g) was treated in TFA for 30 minutes at room temperature. After distilling off the TFA, the oily residue was heated to 2.75N
- Hydrogen chloride dissolved in dioxane (2,2+al) solution. The solution was concentrated under reduced pressure, and the oily residue was washed with n-hexane (30 ml) and then recrystallized from ether to give HCI ・Thr(Bzl)-5et(Bzl)
-Thr(Bz I) -Thr(Bzl) -5et
(Bzl) -Thr(Bzl) ・0Pac(3,1
g) was obtained.

mp 118-118°C (decomposition) [a] n = -84-4''(C'''' 1, in methanol) Thin layer chromatography (silica gel: manufactured by Merck & Co.) Rf = 0.28 [Ethyl acetate: Acetic acid (10: 1)] Example 1-11 Boc a Leu-Pro- obtained in Example 1-3
Gly aO)1 (0.99 g), HCI φThr(Bzl) -Set obtained in Example 1-10, (B
zl) -Thr(Bzl) -Thr(Bzl)-3
er(Bzl) -Thr(Bzl) * 0Pac(
3.0 g) and HOBT (0.38 g)
WSCD (0.40 g) was added to the mixture while cooling in a water bath. This mixture was stirred at 0-5°C for 3 hours and then at room temperature for 15 hours, then mixed with water (15C1 ml) and ethyl acetate (150n).
+1). The ethyl acetate layer was separated and mixed with 2% hydrochloric acid (100ml), water (50ml
After washing in the order of alX 3) and drying with magnesium sulfate, the solvent was distilled off. Recrystallization of the residue from ether yields Boc*Lue-Pro-Gly-T
hr(Bzl) -5er(Bzl) -Thr(Bz
l) -Thr(Bzl)-3et(Bzl) -Th
r(Bzl)-0Pac (3.25 g) was obtained.

mp 172-175°C (decomposition) Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf = 0.42 [Chloroform: methanol (20:
l)] Example 1-12 Boc*Lue-Pro obtained in Example 1-11
-Gly-Thr(Bzl) -5et(Bzl) -
Thr(Bzl) -Thr(Bzl) -8er(B
zl) -Thr(Bzl) ・0Pac(3,4g)
While cooling a solution of DMF (40 ml) and acetic acid (20 ml) in a water bath, zinc powder (5 ml) was added to the solution.
, 0g) was added thereto, the temperature was returned to room temperature, and the mixture was stirred for 1 hour. After removing the zinc dust by filtration, the filtrate was concentrated under reduced pressure. The concentrated solution was dissolved in ethyl acetate (1001), washed with 1% hydrochloric acid (80 ml) and water (100 ml×2), dried over magnesium sulfate, and the solvent was evaporated to dryness. Recrystallization of the residue from isopropyl ether yields Boc e Lue
-Pro-Gly -Thr(Bzl) -5et(B
zl) -Thr(Bzl) -Thr(Bzl) -
3et(Bzl) -Thr(lzl) ・OH(2,
28g) was obtained. The crystal was the raw material of Example 1-22.

rrrp 173-176℃ (decomposition) [α]D =+
5' (C = 1. in DMF) thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf = 0.41 [R ao*rum: J )) /
-Lys (10:1)] Example 1-13 Boc-Lys (C:1.-z) @0HIIt-Bu
-NH2 (7.38 g) was partitioned between ethyl acetate and dilute hydrochloric acid, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with saturated aqueous sodium chloride solution, and dried over magnesium sulfate. After distilling off the solvent, HCI ・Thr(Bzl)
90Pac (5.0g) was dissolved in methylene chloride (100ml
) and DMF (70 ml) (7) was added to the residue obtained above. Then -50°C
To the solution, cooled Cinnabar WscD (2.34 g) was added. This was stirred for 3 hours, and the temperature was gradually raised to 0°C, and the mixture was stirred for an additional hour while maintaining this temperature. The mixture was concentrated and poured into a mixture of ethyl acetate and water. After separating the organic layer and extracting the remaining aqueous layer twice with ethyl acetate, the organic layers were combined and washed with dilute hydrochloric acid, water, aqueous sodium hydroxide solution, water, and saturated aqueous sodium chloride solution in this order. It was then dried over magnesium sulfate and concentrated. Crystallization of the residue from ether-diisopropyl ether yields Boc eLys(CI-z)-
Thr(Bzl) 0Pac (5.85g) was obtained.

mp 79-84.5°C [α]D = -9,33° (C = 0.5, in chloroform) Elemental analysis: C38H45N3 o,, CI calculated value C: 83.02 H: 8.40 N: 5 .80 Experimental value C: 81.99 H: 8.12 N: 6.43 Example 1-14 Boa * Lys (CI-
z)-Thr(Bzl)-0Pac(5.5g) to T
The mixture was treated with a mixture of F A (50 ml) and anisole (5 ml) at 0° C. for 30 minutes and then at room temperature for 30 minutes.

The mixture was concentrated and ether was added to the residue to obtain a crystallized product (5.19 g). This was dissolved in methylene chloride (100 ml), and Boce Cys (Ac
m) a 09u (2.88g) and triethylamine (
1.2 ml) was added. The mixture was stirred at room temperature for 15 h and 3-(N,N-dimethylamine)propylamine (0
, 1 ml) was added. The mixture was concentrated and extracted with ethyl acetate. The organic layer was washed sequentially with water, dilute hydrochloric acid, water, and saturated aqueous sodium chloride solution. After distilling off the solvent,
The residue was crystallized from a mixture of ethyl acetate and ether to give Boc ・Cys(Acm)-Lys(CI-z)-
Thr(Bzl)*0Pac (4.9 g) was obtained.

mp 93.5-87℃ [α]D=+3.41° (C:=O, 1329, in chloroform) Elemental analysis: C44H56N5011SC1 calculated value C:
58.04 H:8.20 N:? , 69 S: 3.5
2 Experimental value C: 57. E18 H:8.13 N77.
4BS: 3.88 Example 1-15 (1) Boc*Cys(
Acm)-Lys(CI-z)-Thr(Bzl)Pac (4.74 g) was treated with TFA (50 ml) and anisole (5 ml) at 0° C. for 30 minutes and at room temperature for 20 minutes, respectively. After concentrating the mixture, the residue was dissolved in dioxane (10 ml) and a further 1.75N solution of hydrogen chloride in dioxane (3 ml) was added. The resulting mixture was concentrated again and the residue was treated with ether to give a solid. This was dissolved in DMF (30+al) and further triethylamine (30++1) was added.

(2) Separately manufactured Boc * Gly-Pro *
NHNH2 (1,89g) in DMF (30ml)
Dissolve 2.75N hydrogen chloride in dioxane (4,2!
3ml) solution was added dropwise while cooling in a water bath. Next, tertiary butyl nitrous acid (808 mg) was added, and after stirring at the same temperature for 15 minutes, triethylamine (1.19 g) was added to neutralize the reaction solution, and the solution obtained in (1) was added. Further triethylamine (291 mg) was added, and after 3.5 hours while stirring under water bath cooling, further triethylamine (24On+
g) was added. During this time (4 hours), the temperature of the reaction solution was gradually raised to room temperature. The solution was concentrated and poured into a mixture of ethyl acetate and water. The aqueous layer was extracted again with ethyl acetate, the organic layers were combined, washed in this order with hydrochloric acid, water and a saturated aqueous sodium chloride solution, and then dried over magnesium sulfate.
Concentrated. The residue (5.42 g) was recrystallized from a mixture of cyclohexane and ethyl acetate (1:8), resulting in Boc
Fist Gly-Pro-Cys (Acm)-Lys (CI
-z)-Thr(Bzl) -0Pac (4,55g
)was gotten.

mp 115 ~ l18℃ [α]D=-35,8° (C=0.805, in DMF)
Elemental analysis: C5, H66N70.3SC1 calculated value C:
58.20 H:8.32 N:9.31 S:3.0
5 Experimental values C: 58.37 H: 6°48 N: 9.1
2S:3.25 Example 1-16 Boc*Gly-Pr obtained in Example 1-15 was added to a mixture of DMF (50 ml) and acetic acid (50 ml).
o-Cys(Acm)-Lys(C:I-z)-Thr
(Bzl)-0Pac (4.7 g) was dissolved, and zinc dust (7.1 g) was added to the solution while cooling it on ice, followed by stirring at room temperature for 1 hour. The reaction solution was filtered, the filtrate was concentrated, and the residue was
- After washing with hexane, water was added, and then hydrochloric acid was added to adjust the pH to 2, followed by extraction with ethyl acetate. The extract was washed with water and an aqueous sodium chloride solution, dried over magnesium sulfate, and then concentrated again. Treatment of the residue with ether yields Boa*Gly-Pro-Cys(Acm)-L
ys(CI-z)-Thr(Bzl) Φ0H(3,8
4g) was obtained. The crystal was the raw material of Examples 1-20.

Np 86-83℃ [α]D=-42,28" (C=1, in methanol)
Elemental analysis: C43H6oN70.2SC1 calculated value C:
55.27 H:(3,47N:10.48 S:3.
43C1:3. ? 9 Experimental values C: 53.69 H: Ei, 35 N: 10.
19 S:3.53C1:3.79 Example 1-17 Boa IIThr (Bzl) ・09u (5.3g)
and pTsOHe H++11e ll0Bzl(5,
0g) was dissolved in methylene chloride (30+al) and triethylamine (1.81ml) was added while cooling in a water bath. After a further 4 hours and 5.5 hours, 0.54+*l and 0.38 ml of triethylamine were added again. The reaction mixture was left in the freezer for 3 days and then concentrated. The residue was dissolved in ethyl acetate, washed successively with water, dilute hydrochloric acid, water and saturated aqueous sodium chloride solution, and dried over magnesium sulfate.

After distilling off the solvent, crystallization from petroleum ether yields Bo
a e↑hr(Bzl)-11e ・OBz+ (5,
95 g) was obtained.

mp 49-53.5℃ [α]D = + 9.38° (C-1, in chloroform) Elemental analysis: 029H4ON20G Total 1 o [α'i C: 87.95 H near, 88 N
: 5.4θ experimental value C: H37, 42H near, 81 N
:5.41] Example 1-18 Boc*Thr(Bzl
)-11e-0Bzl (5,5g) T F A (3
0 ml) and anisole (51) for 25 minutes at 0°C. The mixture was concentrated and dissolved in dioxane (51).

Add 2.75N hydrogen chloride in dioxane (5 ml) to this solution.
The solution was added and concentrated. The residue was washed several times with n-hexane, each time the n-hexane being decanted off. The precipitate was dissolved in methylene chloride (50i1) and Cy in Boc
s(Acm) e 03u (4.18 g) and triethylamine (1.E 17 ml) were added under water bath cooling. After returning to room temperature and stirring for 28 hours, 3-(N,N-dimethylamino)propylamine (0.18 ml) was added, concentrated, and extracted with ethyl acetate. The organic layer was washed with water, diluted hydrochloric acid, water, and a saturated aqueous sodium chloride solution in this order, dried over magnesium sulfate, and further concentrated. When a mixture of ether and diisopropyl ether is added to the residue and crystallized, Boc ・Gys(Acm)-Thr(
Bzl)-11s-0Bzl (4.70 g) was obtained.

mp 82-64.5℃ [α], = + 24.48° (C = 0.537, in chloroform) Elemental analysis: C3, H5oN408S Calculated value C: 81.20 H:? , 34 N: 8.18
S: 4.8? Experimental value C: 60.48 H: 8.99
N: 8.10 S: 4. ? 55 Example 1-19 Boc*Cys(Acm
)-Thr(Hzriile・0Bzl(4,Og) is treated with TFA in the same manner as in the first part of Example 1-18, and further treated with HCI dissolved in dioxane, )1G
1 ・H・Gys(Acm)-Thr(Bzl)-11
e*0Bzl (3.8 g) was obtained.

Example 1-20 Example 1-16-t' Obtained Boc e Gly-P
ro-Cys(Acm)-Lys(CI-z)-Thr
(Bzl) ・0H (3.5 g), HCI*He Cys(Acm)-Thr(
Bzl)-11e 0Bzl (2,12g) and l
-Hydroxybenzotriazole (0,51g) D
Add WSCD to the solution formed by adding M F (50 ml).
(0.58 g) was added dropwise at -30°C. The reaction temperature was raised to 0° C. in 30 minutes, stirred at the same temperature for an additional 3 hours, and then left overnight at room temperature.

Ethyl acetate (150+ol) and water (300+ ol) were added to the reaction mixture.
ml) was added to separate the organic layer and the aqueous layer. The aqueous layer was extracted again with ethyl acetate, the organic layers were combined, and then 2% hydrochloric acid and 2%
It was washed sequentially with an aqueous sodium hydrogen carbonate solution and water, and concentrated. The residue was developed on a column of silica gel (40 g) and eluted with a mixture of chloroform and methanol (98:2) to give Boc*Gly-Pro-Cys (Ac
m)-Lys(C:l-z)-Thr(Bzl)-Cy
s(Acm)-Thr(Bzl)-11e 110Bz
1 (2.87 g) was obtained.

mp 183-188℃ [α] = -18,58" (C = 1, in DMF) Elemental analysis" 78 HlooNII 017 s2C1 calculated value C: 58.33 H: fl, 70 N: 10.25
S: 4.27 experimental value C: 5? ,87H:8.55
N: 10.11 S: 4.45 Example 1-21 Boc-Gly-Pro- obtained in Example 1-20
Cy s (Ac+m)-Lys(C1z)-Th r
(Bz l )-Cys (Acm) -Th r
(Bz l )-Ile eOBzl (2,5 g) to T
F A (25ml) and anisole (2.5ml)
and treated at room temperature for 20 minutes. After removing TFA,
The residue was dissolved in a solution of 2.75N hydrogen chloride in dioxane (2 ml) and diluted with dioxane (20 ml). After distilling off dioxane, ether is added to the residue to crystallize it, resulting in HCI ・Gly-Pro-Cys (
Acm)-Lys(CI-z)-Thr(Bzl)-C
:ys(8cm)-Thr(Bzl)-11e ・0B
zl (2.34 g) was obtained.

mp 178-180°C (decomposition) [α: +D = -20,5' (C = 1, in DMF) Thin layer chromatography (silica gel: manufactured by Merck & Co.) RfI = 0.23 (n-butanol: acetic acid: water: =4
=l:5) Example 1-22 Boc @Leu-Pro- obtained in Example 1-16
Gly-Th r (Bz I )-5et (Bzl)
-Thr(Bzl)-Thr(Bzl)-Set (
Bz])-Thr(Bzl) ・OH (0.40g)
and HCI e Gly-Pr obtained in Example 1-21
o-Cys(Ac+++)-Lys(fl;lz)-T
hr(Bzl)-Cys(Acm)-Thr(Bzl)
-IIe 〇Bzl (0,3'5g) HOB T
(35,9 g) in DMF (7 ml) at 0 °C.
After cooling to , W S CD (41.2 mg) was added. After stirring at the same temperature for 15 hours, ether (80+o
diluted with l). The precipitated crystalline solid was collected by filtration and mixed with water (
Fowri, 50% Improper Tools water (10ml
) solution, Boc e Leu-Pro
-Gly-Th r (Bz 1)-5e t(Bz
1)-Thr(Bzl)-Thr(Bzl)-9e t
(Bz I)-Th r (Bz l)-G ly
-Pro-Cys(Acm)-Lys(C1z)-Th
r(BzI)-Cys(Acm)-Thr(Bz
l)-11eOBzl (0.82 g) was obtained.

MP20? ~210°C (decomposition) [α]D = -9,3@ (c = i, in DMF) Thin layer chromatography (silica gel: manufactured by Merck & Co.) Rf = 0.48 (Chloroform: methanol = 10 =
1) Example 1-23 Boc a Leu-Pro obtained in Example 1-22
-Gly-Thr(Bz 1)-8et(Bz 1)
-Thr(Bz 1)-Thr(Bz 1)-9et
(Bz 1)-Th r (Bz I)-G I
y-Pro-Cys(Ac+w)-Lys (fll:
lz) -Thr(Bzl)-Cys(Ac
m)-Thr(Bzl)-11e110Bzl (0,
59 g) with hydrofluoric acid (6 ml) and anisole (0.8
m1) in a water bath for 1 hour. After removing the hydrofluoric acid, the residue was extracted with water (20!II). After washing the extract with ether (20 ml), a column of Dowex I
150 + al), and the ninhydrin positive fraction (15
0 ml) and concentrated to about 30 ml. When this was freeze-dried, 3.6 g of crude powder was obtained, and CM-
52 (2,8X 45cm) column and 0.05
After developing with a linear concentration gradient prepared from 4001 M pyridine-acetate buffer (pH 4,8) and 0.2M pyridine-acetate buffer (pH 4,8), 4001 was added using the TNBS method.
Resolution of each fraction (8+al) was performed at a wavelength of 20 nm. The main peak fractions (tube no. 33-45) were combined, concentrated and lyophilized to yield 0.30 g of powder. Sephadex G-15 (3, IXEi)
2c+m) column and developed with 0.1% acetic acid.

The eluate was analyzed by UV absorption at 230 nrtr, the main peak fraction was collected, concentrated again and lyophilized to give 0.28 g.
of powder was obtained. Finally, add this powder to Sephadex G.
-25[3,I Each fraction of the eluate was analyzed according to the TNBS method, and the main peak fraction (tube number, 109
~133) are combined, concentrated, and lyophilized to yield H-Leu-
Pro-Gly-Thr-Ser-Thr-Thr-S
er-Thr-Gly-Pro-Cys (Acm
) -Lys-Th r-Cys (Acm)-Thr
-Ile-0H (0.18 g) was obtained.

[α]D=-82.7° (c=i, in water) Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf'=0.31 (n-butanol:acetic acid:water:pyridine=3:l:2: 1) High performance liquid chromatography retention time = 4.4 minutes Column: Nucleosil 10C (4X250mm) Eluent: 0.1M phosphate buffer (pH 4,5) Niacetonitrile (82:18) Flow rate: 1. Oml/min Detection: UV21On+11 Amino acid analysis of acid hydrolysis Leu: 1.00 Pro: 2.53 (2) Gly: 2
．． 15(2) Lys:1.00Ser:2.5&(2
) Thr:5.8? (61) 11e: 0.99 Example 2 Nt-butoxycarbonyl-〇-benzylserine resin [4 g, serine content 0.545 mmol/g, styrene-1% divinylbenzene copolymer] as starting material year,
Phe, Arg, Va according to Example 5-1 described below
l,Ser,Ala,Gly,Glu,Tr
p, Leu, Phe, Arg.

Solid phase synthesis reactions were performed in the order of Ala, Phe, Ala, and Trp. In each step, θo1 solvent was used.

After reacting the 8th amino acid (first Trp residue), in step 2, 5% ethanedithiol was added to a solution of 50% TFA in methylene chloride to perform an elimination reaction of the Boc group. Ta. Finally Boc-Tr p-A
l a-Phe-A 1a-A rg-Phe-Leu
-Trp-Glu-Gly-Ala-7.35 g of fat was obtained.

A portion (0.70 g) was treated with hydrogen fluoride (10 ml) at 0° C. for 1 hour in the presence of anisole (1 ml) and ethanedithiol (0.5+al). After distilling off the hydrogen fluoride, the residue was washed with ether (10ml x 2) and extracted with TFA (20ml). The extract was distilled off, dried under reduced pressure on potassium hydroxide pellets for 5 hours, and then treated with ether to powder, yielding a crude product (420 mg) from which the resin moiety and protecting group had been removed.

This crude product (210+mg) was developed on a LH-20 column chromatograph (3X 82cm) and eluted with 50% acetic acid. Each elution fraction (7 g) was subjected to absorbance measurement at 280 nm and the main peak fraction (tube no. 20-3
0) were collected. This was concentrated to 301 and freeze-dried to obtain 120 mg of powder. This powder was mixed with n-
It was developed on an LH-20 reverse phase fractionation chromatograph using a solvent system of butanol/acetic acid/water (2:1:10 by volume). The column (3.2 x 8 Bcm) was equipped with the above-mentioned LH-2.
0 is swollen and packed in advance in the upper layer of the above solvent system, and the lower layer (
51) and 50% acetic acid (31).
) was dissolved and added to the column. The column was developed using the lower layer of the above solvent system. The eluate was equilibrated by adding ethanol (1 ml) to each fraction at 280 nm.
The main peak fraction (tube No.
, 24-34) were combined and concentrated under reduced pressure. Residue 50
When dissolved in % acetic acid (20+al) and lyophilized, H-
Trp-A 1a-Phe-A Ia-Arg-Phe
-Leu-T rp-G 1u-G 1y-A 1a-
Ser-Va I-A rg-Phe-3er-OH
c7) Powder (35 mg) was obtained.

Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf = O, E18 (n-butanol: acetic acid: water = 4:
1:5) Detected by ninhydrin test Amino acid analysis of amino acid decomposition product with 3N methanesulfonic acid containing 2% tryptamine Glu: 1.0 (1) Ala: 3.18 (3) Ph
a: 3.26 (3) Arg: 2.19 (2) Leu:
1.02 (1) Gly: 1.18 (1) Ser: 1.
91 (2) Val: 1.13 (1) Trp: 1.83
(2) High speed chromatography retention time: 1i3.2 min Column: Nucleozil I OCl8 (4X250++w
) Eluate: 0. IM・ni2HPO-H3PO4 (pH 4
, 5) Gradient concentration solvent from 20% acetonitrile to 80% acetonitrile (30 minutes) Flow rate = 117 minutes Detection: UV21Or++w, 280 nm Example 3 Example 1-23'l? The obtained H-Leu-Pro-G
ly-Thr-8e r-Th r-Thr-5e r
-Thr-Gly-Pro-Cys (Acm)-Ly
s-Thr-Cys(AcIl)-Thr-11e-
0)! (100 mg) in water (30 ml) was added mercuric acetate (34.9 mg), and then acetic acid was added to adjust the pH.
After adjusting the temperature to 4, the mixture was stirred at room temperature for 1 hour. Next, hydrogen sulfide gas was blown in for 5 minutes, and the precipitated forge was filtered off. The filtrate was diluted with water (22 hl) and diluted with dilute aqueous ammonia.
After adjusting to H8.0, the mixture was stirred overnight. This solution was concentrated under reduced pressure, and the concentrated liquid (5 ml) was obtained from Cef7dex G-25 (
It was passed through a column of 3,2×11 ecm) and developed with 0.1% acetic acid. The eluate was analyzed by UV absorption of 23Or+++++, all fractions were collected, concentrated, and lyophilized to obtain the following intramolecular cystine-type peptide (85 mg).

[α]D=-38,9' (C=1, in water) Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf=0.32 (n-butanol:acetic acid:water:pyridine=3:1:2:1 ) High performance liquid chromatography Retention time: 4.8 minutes Column: Nucleosil 10 CIB (4X 250m
m+) Eluate: 0. I M -K2HPO4-H3P
04 buffer (pH 4, 5): CH3CN (82:18
)Flow rate: 1. Om17 min Detection: UV210 nm Amino acid analysis of hydrolyzate by alcohol Leu: 1.00 Pro: 2.07 (2) Gly: 2
．． 00(2) Lys:1.01(1)11e: 1.
02(1) Thr + Ser: 8.05(8) (Cys) 2: 0.81(1) Example 4 H-Leu-Pro-Gly obtained in Example 1-23
-Thr-9a r-Th r-Th r-3e r-
Thr-GI y-Pro-Cys (Acm)-
Lys-Thr-C: ys(Acm)-Thr-11
Dissolve e-OH (20hg) in water (21) and add vinegar f! #
After adding mercuric (76.5+wg), more acetic acid was added to bring the pH to 2. After stirring at room temperature for 1 hour, hydrogen sulfide was bubbled in for 5 minutes. Excess hydrogen sulfide was then removed by bubbling nitrogen for 1°. The precipitated mercury sulfide was removed by filtration, and 5% ammonia water was added to the filtrate to adjust the pH to 8 to 9, followed by stirring overnight at room temperature. Concentrate under reduced pressure, dissolve the residue in a small amount of 0.1% acetic acid, and apply it to a Sephadex G-25 column (3.2X
80 cm) and eluted with 0.1% acetic acid. 30
Elution fractions from 0 ml to 400 ml were collected and lyophilized. The obtained powder (100 mg) was subjected to partition chromatography using a Sephadex G-25 column (3.2×85 cm) using an n-butanol:pyridine=1% 11F (5:3:1) system. 1.18i to 1.45
The eluted fractions up to JJ were collected and concentrated under reduced pressure. Dissolve the residue in a small amount of water and apply it to a Sephadex G-25 column (3
, 2×85 cm) and eluted with 0.1% acetic acid. The eluted fractions from 290 ml to 320 ml were collected and lyophilized to obtain the following mixture of intermolecular cystine-type peptides (30.0 mg).

Thin layer chromatography (silica gel: manufactured by Merck & Co., Ltd.) Rf = 0.24 (n-butanol: acetic acid: water: pyridine = 3:1:2:1) High performance liquid chromatography retention time: 5.0 minutes, 7.3 minutes column : Nucleozil OC18C13 (4X250
Eluent: 0.1Ml K2HPO4-H3P04 buffer (pH 2,5) Niacetonitrile (8:2) Flow rate:
1. Om17 min detection: UV210 nm Example 5-1 Nt-butoxycarbonyl-5-acetamidomethyl cysteine resin [4.0 g, cysteine content i0.48
mmol/g, styrene-1% divinylbenzene copolymer] was used as a starting material, and a peptide bond-forming reaction was carried out according to the solid phase synthesis schedule shown in the table below. The reaction order of the amino acids used in the reaction is Set, Thr, Pr.
o, Ser, His, Asn, Ser, Th
r, Pro.

Ser, Gln, Ser, Asn, Gln,
Gly, Pro, Cys, Thr, Pro, A
A peptide having 21 amino acid residues was synthesized by repeating the schedule shown in the table for 20 cycles. Note that the α-ami7 group of these amino acids is t-
Regarding the butoxycarbonyl group and other side chain functional groups, Set and Thr were protected with a benzyl group, His with a p-)luenesphonyl group, and Cys with an acetamidomethyl group.

That is, starting materials (
Alternatively, the peptide chain can be extended by sequentially reacting the solvents and reagents listed in the same table on an intermediate substance obtained by extending the peptide chain by coupling an amino acid to the N-terminal amine group of the starting material. Ta. The coupling reaction corresponding to step 8 of the inner cylinder in the same table was generally carried out in a methylene chloride solution, but only when reacting N-t-butoxycarbonyl Cys protected with an S-acetamidomethyl group. A (a:X) mixture of methylene chloride and dimethylformamide was used. In addition, when coupling Asn and Gin, each N-t
The -butoxycarbonyl protected amino acid was added to a dimethylformamide-methylene chloride (171) mixture of N-hydroxybenzotriazole, activated, and used in the 8th step. Also, the 7th (Ser) is changed to the 8th (Thr).
).

For each coupling reaction of the 15th (Gly), a mixture of dichloromethane and dimethylformamide (X+
1), especially the 8th (Thr) and 15th (Thr)
In the coupling reaction of Gly), N-hydroxybenzotriazole was added.

The completion of each coupling reaction was determined by Kaiser's ninhydrin test and for proline by the isatin test. When it was found that the coupling was insufficient (in the experiment, the coupling reaction was between the 16th Pro and the 18th Thr), steps 8 to 11 in the table were repeated. At this time, the second coupling reaction was carried out by adding an equivalent amount of N-hydroxybenzotriazole to a methylene chloride-dimethylformamide mixture (3+1).

After coupling the last Ala, the resulting resinous material was washed with methanol and dried under reduced pressure, resulting in a peptide resin (s, 4gg) represented by the following formula:
I was able to get

Boc-Ala-Pro-Thr(Bzl)-Cys(
Acm)-Pro-Gly-Gln-Asn-Ser(
Bzl)-Gin-5er(Bzl)-Pro-Thr
(Bzl)-8er(Bzl)-Asn-H35(
Tos)-8er (Bzl) Example 5-2 The peptide resin (4.20 g) obtained in Example 5-1 was dissolved in liquid hydrogen fluoride (50TR1) in the presence of anisole (5Tnl).
) for 1 hour at 0°C. Next, hydrogen fluoride was distilled off, and the residue was left with water (1'OOmA') and ether (50
d) was added. After removing the resinous material from the furnace, separate the aqueous layer and add it to Dowex 1×2 (acetate type + 3QQ + rt).
1) was developed and eluted with water. The fractions positive for the ninhydrin test were collected and lyophilized to produce crude H-Al
a-Pro-Thr-Cys(Acm)-Pro-Gl
y-G, In-AsnIn-Asn-5er-Gln-
8er-Pro-Thr-5er-Asn((is-3
er-Pro-Thr-8er-Cys(A (1,1
gg) was obtained. This crude product was prepared from carboxymethylcellulose equilibrated with 0.01M pyridine-acetate buffer (pH 5,4). '7/Fum (3.2x60cm)
and eluted with a linear gradient prepared from 1.21 m each of 0.01M pyridine-acetate buffer and 0.1M pyridine-acetate buffer. Both 10g portions were tested for torinide using the benzenesulfonic acid method (wavelength: 420nm), and fractions 172 to 185 were collected as the main fraction and freeze-dried to obtain a slightly purified peptide (349■). Ta. The crystal was passed through a high-performance liquid column using a preparative μm Bonderback CI8 column (trademark manufactured by Waters Co., Ltd.) at a flow rate of 3. Purification was performed by OTIL 6/min using 0.1 TFA-acetonitrile (85:15) as eluent. For purification, a 60fng sample was divided into three 20μ portions, and the main fraction was collected and concentrated. The residue was dissolved in 0.1 M heptammonium acetate solution and purified with Sephadex G-15 (2,3X4
After passing through a column of 9Cm), it was eluted with 0.1 thiacetic acid. Fractions of 3 g each were collected and lyophilized to obtain the purified peptide (55■).
was gotten.

Amino acid analysis of hydrolyzate with 6N hydrochloric acid Ala +
1.00 Pro 70.88X4Thr s O,7
9X3 Gly s 1.04Gin: 1.18X
2 Asp + 1.02X2Ser: 0.81X
5 Hls s 1.18 Amino acid analysis of enzymatic decomposition product Analysis conditions! 011M phosphate buffer, pH 7 (400μ)
The substrate (2,25 µ) was dissolved in the solution and treated with proline-specific endopeptidase (enzyme number 3.4.21.26: 1 mg/m! 20 μl of solution) at 37°C for 2 hours. 0.1 M phosphate buffer (600 μl), pH 8, was added, followed by aminobeptidase M (2 units), and the mixture was incubated at 37° C. for 20.5 hours.

A1a+ 1.00 Pro: 0.94X4Thr+
Gln: 4.05 Cys(Acm) + 0.96
x2Gly + 0.9 9 Ser 1 Asn: 5
．． 74 His could not be quantified because the ammonia peaks overlapped.

[α] D knee 132.76° (C = 0.25 in water) thin layer chromatography (Merck Cell 4th) Rf = 0
．． 65 (n-butanol: acetic acid; water: pyridine = 2
: 2 : 2 : 1) Positive with ninhydrin and Pauli reagent High performance liquid chromatography Retention time 24.2 minutes Column: Nucleosil 10C1B (4X250mm)
Eluate: 0. IM-H, PO4- to 2HPO, buffer (PH4,8) Niacetonitrile (88712) flow rate: x, oml/min detection'UV 210nm Example 6-1 H-Ala obtained in Example 5-2 -Pro-Thr
-Cys(Acm)-Pro-Gly-Gin-Asn
-5er-Gln-8er-Pro-Thr-3er-
Asn-His-8er-Pro-Thr-Ser-C
ys(Acm)-0H (147fn9) in water (100
TLe) and mercury acetate (46r+v) was added.

Further, 1% acetic acid was added to adjust the pH to 4, and the mixture was stirred for 1.5 hours.

Hydrogen sulfide was bubbled into the reaction mixture for 15 minutes, followed by nitrogen gas for 15 minutes to dissipate residual hydrogen sulfide.

The mixture was filtered through a cellulose pad, washed with a small amount of water, and the filtrate and washings were combined (152 mg).

Add 0.28% ammonia water to this to adjust the pH to 8, and
After stirring for 16 hours, it was concentrated. The residue was developed on a Sephadex G-75 column (3,2X71CII+), eluted with O1 acetic acid, and divided into fractions of 6 g each. The main fraction (ltln27-36) was collected and lyophilized to produce a crude intramolecular cystine-type peptide (88,
2■) was obtained.

Example 6-2 The crude peptide (80~) obtained in Example 6-1 was purified using a preparative μ
m bonder pack 0□8 columns (7,8X300mm) (
Waters Co., Ltd.) was subjected to high-performance liquid pomatography using o, t% TFA-acetonitrile (85
715) was purified as an eluate.

Purification was carried out by dividing 80 μl of sample into 4 portions of 20 mfl each, and the main fraction was collected and concentrated. The residue was dissolved in 0.1 M aqueous ammonium bicarbonate solution, dissolved in a Sephadex G-15 column (z, 3 x 49 cm), and eluted with 0.1% acetic acid. Then, 3 g of each fraction was collected, and the main fraction (fraction 13 to 21) was collected and lyophilized. Example 5-1
An intramolecular cystine-type peptide with the structure described above could be obtained as a purified product (49.2■).

Amino acid analysis of hydrolyzate with 6N hydrochloric acidAla:
1.00 Pro: 0.86X4Thr: 0.
87X3 Gly: 1.02Gin: 1.03
X2 Asp: 0.98X2Ser: 0.8X
5 His: 1.17C5rstine: 0.51 Amino acid analysis of enzymatic decomposition product Decomposition conditions + 0.1M phosphate buffer (500d) at pH 8
The substrate (1,64f#) was dissolved in the solution, and the conditions described in Example 4-2 were then followed (however, the incubation period was 20 hours).

Aha + 1.00 Pro j 0.88X4Th
r-I-Gln: 3.77 Cystine+1.01
Gly: 1,18 Ser+Asn: 4.9
5 [α) D = -131,04° (C = 0.50, H
2O) Thin layer chromatography (Merck cellulose)
Rf = 0.56 (n-butanol:acetic acid:water:pyridine = 2:2:221) Positive with ninhydrin and Pauli reagent High performance liquid chromatography, retention time: 6.1 min Kazim: Nucleozil 10C1 [1 (4x250mm)
Eluent; 0.1% TFA-acetonitrile (88:12
) Flow rate: 1.0g/min detection + UV210nm Example 7-1 N-t-7') oxycarbonyl-〇-benzylserine resin (2.15g) [Serine content; 0.82mmol/
1% styrene-1% divinylbenzene copolymer] was used as a starting material and subjected to a peptide bond-forming reaction according to the solid phase synthesis schedule shown in the table of Example 4-1. However, the third
The process was carried out by treating with an ice-cooled mixture of triethylamine and methylene chloride for 1.5 minutes. The reaction order of amino acids used in the reaction is Ser + Pro.
+11e+Pro+11eyCys+Thr+Cyss
Asn+G1ytAsptSer*Pro+Lys+T
At hrjCysjCys, the table schedule is 15
By repeating the cycle, a peptide having 16 amino acid residues was synthesized. In addition, α of these amino acids
-The amine group is a t-butoxycarbonyl group, and the other side chain functional groups are: Set+Thr is a benzyl group, Cys is an acetamidomethyl group, Asp is a cyclohexyl ester, and Lys is a 2-chlorobenzyloxycarbonyl group. Each was protected.

That is, starting materials (
Alternatively, the peptide chain can be extended by sequentially reacting the solvents and reagents listed in the same table on an intermediate substance obtained by extending the peptide chain by coupling an amino acid to the N-terminal amine group of the starting material. Ta. The coupling reaction, which is the eighth step in the same procedure, was generally carried out in a methylene chloride solution, but only when reacting Cys protected with an S-acetamidomethyl group, a mixture of methylene chloride and dimethylformamide was used. A (2:X) mixed solution was used. In addition, in the Asn coupling reaction, N
-t-Butoxycarbonylasparagine was added to a dimethylformamide-methylene chloride (1:1) mixture of N-hydroxybenzotriacyl/l/(1,1 eq.) and dicyclohexylcarbodiimide (1 eq.), and the mixture was heated at 0°C. 1
The mixture was activated by stirring for 0 minutes and used in the 8th step. Introducing the seventh and subsequent amino acids (Cy
After completion of the first reaction using s, the second step shown in the table was carried out by adding 5 volumes of ethanedithiol to the methylene chloride-trifluoroacetic acid mixture. Furthermore, when introducing the 12th and subsequent amino acids after coupling Asp, the neutralization in the 6th step was carried out using a water-cooled solution of triethylamine, and the treatment time was reduced to 1.5 minutes. Shortened.

The completion of each coupling reaction was determined by Kaiser's ninhydrin test. When it was detected that the coupling was insufficient, steps 8, 9, 11 and 1 in the table were repeated in the order listed.

In the experiment, Cys (6th), G1n+Asp, Se
The above-mentioned process was repeated for each coupling reaction of r. After coupling the last Thr, the resulting resinous material was washed with methanol and further dried under reduced pressure, resulting in Vepti represented by the following formula,
A resin (s, 7ssg) was obtained.

Boc-Cys(Acm)-Cys(Acm)-Thr
(Bzl)-Lys(CI-z')-Pro-5et(
Bz 1 )-Asp(OHex)-Gly-Asn-
Cys (Acm)-Thr (Bz1)-Cys
(Acm)-I 1e-Pro-I 1e-Pro-
8er (Bzl) - 8er (Bzl) - Resin implementation M7
-2 The peptide resin (1,451F) obtained in Example 7-1 was treated with hydrogen fluoride (25 mg) in the presence of anisole (2,0-).
The mixture was stirred for 1 hour at -7 to -5°C. Then at the same temperature 2
Hydrogen fluoride was distilled off under reduced pressure at room temperature for 1 hour. The residue was treated with IN acetic acid (60 m) and ether (40 m).
The resin was filtered off and washed with IN acetic acid. The filtrate and washing liquid were combined, the ether layer was separated, and the aqueous layer was washed with DOWEX IX2 (acetic acid type, 1 s
o-) column and lyophilized to produce crude H-C
ys(Acm)-Cys(Aam)-Thr-Ly
s-P r o-8or-A gp-G ly-A
s n-Cys (A m)-Thr-Cys (
Aam)-11@-Pro ro-11e-Pro 8a
r-Be r-OH (66719) was obtained.

This crude fR product (6671q) was converted into carboxymethyl cellulase [Watt 720M-52,2,8X56, 0.1
Elute with the same buffer (each 1000 m) whose concentration was changed linearly between 0.1 M and 0.4 M. When the fractions of 419-30 were collected and lyophilized, 1
1111i product (448 cape) was obtained.

Thin layered helmet Madgraye (Merck's Cell 4th) at-
o, ao (n-b/-l: acetic acid: water: hyridine-3:1:2:1) Detected by ninhydrin test High performance liquid chromatography Retention time: 5.2 minutes Column: Nucleodi A15 G, ( 4X150m11
) Eluate: 0. I M IH, PO, -HK! PO,
Buffer solution (PH4,8)-acetonitrile (82:18) Flow rate: 0.7 m/min Detection: UV210 nm Example 7-3 H-Cys (human om)-Cys obtained in Example 7-2
(Aom)-Thr-Lys-Pro-8e r
-A up-G 1y-A an-Cys (A on
) Ihr-Cyg (Aom)-11e-Pro-
11e-Pr o-8or-8@r-OH(29,6q
) in water (20,0 ad) and mercuric acetate (1
After adding a solution of 9,7ki)- in water (2,0m), 0
．． 14ifl: 1- was added with acid to adjust the pH to 4. After stirring at room temperature for 2 hours, hydrogen sulfide was blown in for 5 minutes, and then nitrogen gas was added for 5 minutes! The remaining hydrogen sulfide was dissipated by blowing. The reaction solution was filtered with a membrane filter (0.45 μm), and the filtrate and washing solution were combined (aX, S*). Of these, 0.
Take out 20m and add 0.1M phosphate buffer (pH 8,0)
and 2 drops of a 0.01M (0.1M phosphate buffer, pH 7.0) solution of 5,5-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) were added to make exactly 5 tlt. After allowing this to stand for 1 hour, the absorbance at 412 nm was measured and found to be 0.893. This means that 12.94 μmol (92.4% of the theoretical value) of the peptide represented by the following formula is present in the original solution.

H-C75-Cy a-Thr-L78-P r O-
3e r-A a p-G 1y-As m-CY 5
-Thr-Cys-IIs-Pro-IIs-Pro
-8ir-8sr-OHExample 8 H-Cys(Aom)-Cy obtained in Example 7-2
s(Aam)-Thr-Lys-Pro-8e r-A
sp-Gly-AII n-Cys (Aam)-Th
r-Cys(-Aom)-1ie-Pro-1ie-P
ro-8er-8ir-OH (7,0119) to 0.1
% acetic acid (2g) and added to a 0.1% acetic acid (l-) solution of mercuric acetate (8.5W) at room temperature.
After stirring for an hour, 2-mercaptoethanol (0,38
m) was added and left overnight at room temperature. The next day, the precipitated insoluble matter was removed by filtration, and the filtrate was filtered with 7 Fadex G = 15 (2, I3
After applying it to a column of X3551), it was eluted with 0.1% vinegar. Obtain 3 g of each portion, measure the absorbance at 280 nm of each portion with a meter every minute, and collect the 15th to 19th fractions. The whole was diluted to 23 mg, of which 0.
When 40 dt was taken and the amount of thiol group was determined by Ellman's method, 65.0% of thiol was found based on the theoretical value. Another high-speed liquid bear! According to Tography, a peptide with a nearly single peak was observed at a retention time of 21 minutes. The crystals were identical to the peptides obtained in Examples 7-8. The column used is Nucleozil sc, (4X 2
The eluent was o, i and trifluoroacetic acid, the acetonitrile linear concentration gradient was 1 to 50% (30 minutes), the flow rate was 1.0 m/min, and the detection was performed at 210 ml.

To the above solution, add concentrated ammonia water diluted 100 times (3
, 5m) was added to adjust the pH to 8, the mixture was stirred at room temperature with the cap open for 24 hours, and then lyophilized to obtain a mixture of peptides (5,8m) represented by the linear formula.

High performance liquid chromatography retention time = 20 minutes Cod: Nuclei μm 0 Cl8 (4X250 cod)
Eluent 20, 1%, trifluoroacetic acid ace)-4! J/L' Linear concentration gradient I9b → 50% (80 minutes) Flow rate: 1.0g/min Detection: UV210nm Applicant: Foundation for Chemotherapy and Serum Therapy Research Institute Fujisawa Pharmaceutical Co., Ltd.

Claims (1)

[Scope of Claims] (However, in each of the above formulas, R4 means hydrogen or a mercapto protecting group) or a pharmaceutically acceptable salt thereof.