JPS60248700A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (R is H or lower alkyl; X1 and X2 are thiol-protecting group or X1 and X2 together form a single bond). USE:Preventive and remedy for hypercalcemia, diaclasis, osteo-malacia, rachitis, etc. PREPARATION:The compound of formula I can be prepared e.g. by reacting the peptide of formula II (A is Asn-Thr-Gly-Ser or Asp-Val-Gly-Ala) or its functional group-protected derivative in a medium containing a buffer solution of 4- 10pH, in the presence of trypsin or trypsin-like proteinase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel polypeptide, more specifically the following formula % formula % () where R represents a hydrogen atom or a lower alkyl group, and xl and or
Alternatively, the present invention relates to a peptide or a functional group-protected derivative thereof, wherein xl and X together represent a single bond.

The peptide of formula (1) or its functional group-protected derivative provided by the present invention has the following formula H-Cys-5er-Asn-Leu-5er-Thr.
-Cyg-1234567 Val-Leu-Gly-Lys-Len-5er-8
910111213 Gln-Glu-Leu-H4a-Lys-Leu-1
41516171819 Gln-Thr-Tyr-pro-Arg-Thr-2
02122232425 Asn-Thr-Gly-5ttr-Gly-Thr-
262728293031 pro -MHI (Summer-α) 2 and H-Cys-5er-Asn-Leu-5er-Thr
-Cys-1234567 Val-Lew-Gly-Llls-Leu-5er-
8910111213 Gln-Gls-Leu-H4a-Lys-Lew-1
41516171819 Gln-Thr-Tyr-pro-Arg-Thr-2
02122232425 Asp-VtXl-Gly-Ala-Gly-Thr-
262728293031 pro -NH, (B) 2 polypeptide, or a derivative of the polypeptide in which the disulfide bond between the 1st and 7th positions is ring-opened, or a functional group-protected derivative of these polypeptide compounds is useful as an intermediate for the production of

The polypeptides represented by the above formulas (I-α) and (1-b) have strong serum calcium and phosphorus lowering activity,
Excellent pharmacological effects such as promoting bone formation, suppressing bone resorption, promoting urinary phosphorus excretion, etc., for example, preventing hypercalcemia, bone fractures, osteomalacia, rickets, etc. caused by endogenous thyroid calcitonin deficiency. This compound is already known as a therapeutic agent.

Many methods have been proposed for producing the above-mentioned polypeptides. For example, in-phase synthesis using a resin support containing a removable amine group (for example, (Refer to Japanese Patent Publication No. 1, No. 1, No. 1, No. 1, No. 1, No. 1)), or a liquid phase synthesis method in which seven peptide fragments having a partial sequence corresponding to the above-mentioned polypeptide are formed in a liquid phase, and each of the seven fragments is further coupled in the liquid phase (for example, Japanese Patent Publication No. 54).
-29513) etc. are known. However, in these conventionally known methods, for example, in the case of in-phase synthesis, the purity of the final target product decreases, especially in the case of long-chain peptides, as the reaction at each stage does not proceed completely, making it extremely difficult to purify the product. The amino acid derivatives used must fully protect all side chain functional groups, all of which must also be completely removed in the final step, but there is always a risk of side reactions. In addition, there are disadvantages such as being uneconomical because a large excess of solvents and reagents are used, and making it difficult to scale up. On the other hand, in liquid phase synthesis, as the number of amino acids increases, their solubility changes slightly, making it increasingly difficult to find a suitable solvent, and the difficulty of separating them from unreacted substances and by-products increases accordingly. However, as the molecular weight increases, the molar concentration in the solution decreases, which makes the peptide chain extension reaction more difficult, and it is almost impossible to recover the raw materials.
This cannot be said to be a fully industrially satisfactory method.

The present inventors have proposed the above formula (1-α) which has fewer drawbacks as described above.
As a result of conducting research on the amount of lead for the purpose of providing a method for producing the polypeptide or its derivative (1-6), it was found that the purpose could be achieved by the method described below.

It has also been found that the peptide of formula (1) or its functional group-protected derivative is a novel compound that has not been described in conventional literature, and that it can be an extremely useful intermediate in carrying out these methods.

That is, a peptide or a functional group-protected derivative thereof represented by the formula % formula % (1) in which RSxi and A is Aan-Fhr-Gly-5ar or A'a
The peptide represented by p-Val-Gly-Ala or its functional group-protected derivative is reacted with trypsin or a trypsin-like protease in a medium containing a buffer solution of pH 4 to 10. It is possible to advantageously produce a polypeptide represented by the formula % formula % (1) in which A, X, and X have the above-mentioned meanings or a functional group-protected derivative thereof.

In addition, each abbreviation used to represent an amino acid unit in this specification has the following meaning.

Ala- = alanil mercenary k Based on Arg-argini, Asn- = Asuno group two groups, Asp-=based on asparti, Cys-=cysteine optic group, GLn-=Glutamininoki W1 GI B-x = Glutamino et al. c, ty-=Glycinosaurus; H-8-=histiso based, Tal- = Roy 1in Jibi- Lye-=lysyl group, pro-=prolyl group, Se de = seryl group, Thr-=threonyl group, Tyr-=tyrosyl group, Val-=valyl group. S- Used to represent.

This method is characterized by the above formulas (I-α) and (1-α).
When synthesizing peptides as shown in 6), a peptide fragment having the amino acid sequence from position 1 to position 24 and a peptide fragment having the amino acid sequence from position 25 to position 32 of the remainder are each synthesized in advance. First, the free carboxyl group or esterified carboxyl group of arginine at position 24 and the amino group of threonine at position 25 are coupled (amidated) using trypsin or a trypsin-like protease.

Conventionally, proteolytic enzymes such as trypsin have been mainly used to cleave peptide bonds, but in this method, they are extremely effective as condensing agents for the coupling reaction between the peptide fragment of formula (1) and the peptide fragment of formula (■). Surprisingly, it was discovered that it was excellent.

Trypsin used in this method has been approved as a proteolytic enzyme by the Enzyme Committee of the International Union of Biochemistry (J.U.B.).
It is a well-known enzyme registered with the prime number EC3, 4, 2LA, such as trypsin derived from Ushiyama, pig, etc., or tosyl-L-phenylalanyl chloromethyl ketone (
Trypsin treated with TpCK) or the like is commercially available, and any of these can be used in the present method.

In this method, not only trypsin but also trypsin-like proteolytic enzymes can be used. Examples of trypsin-like proteolytic enzymes that can be used include, for example, Streptomyces griseus (Streptomyces griseus).
grisss), Streptomyces flagi x (S
It is known that proteases are produced by proteases such as T. treptomllatts fradtae).
ZYMES w, Vo 1. I, 746 f,,
(see ACADEMIC PRESS publication in 1971),
An enzyme product containing a trypsin-like protease produced by Streptomyces griseus-1 is commercially available under the trade name "Pronase" (manufactured by Kaken Kagaku Co., Ltd.).

The formula (1) in the presence of such a specific proteolytic enzyme
) or a functional group-protected derivative thereof (hereinafter sometimes referred to as "component C") and the formula (It)
The coupling reaction with the peptide represented by or its functional group-protected derivative (hereinafter sometimes referred to as "component F") is carried out in a medium containing a buffer solution with a pH of 4 to 10, preferably 7 to 10, or a pH of 5 to 8. It is done.

The type of buffer to be used is not particularly limited as long as the pH value is within the above range, and various types can be used.For example, Tris-HCl buffer, Matsukuilpein's buffer, Phosphate buffer, ammonium acetate buffer, Adkins & Punchin buffer, veronal buffer, etc. are included. Among these, a Tris-HCl buffer with a pH of 8) is suitable.

When these buffers are used as reaction media, they are usually mixed with a water-miscible organic solvent, in particular a water-miscible organic solvent that at least partially dissolves at least one of the C component and the N component. be done. Examples of the water-miscible organic solvent 7 that can be used as part of the reaction medium include dimethylformamide (DMF), dimethyl sulfoxide (
DMSO), tylphosphoryltriamide (gxpA), and the like, among which dimethylformamide is preferred. These organic solvents may be used alone or in combination of two or more.

Advantageously, the mixing ratio of the buffer solution and the water-miscible organic solvent is generally in the range of 228 to 8:2, preferably 5 to 7:5 in volume ratio of buffer solution to organic solvent. .

The reaction between the C component and the N component in such a reaction medium is carried out at a temperature range in which the protease acts, generally from about 20 to
It can be carried out at a temperature within the range of about 50<0>C, preferably about 25<0>C to about 40<0>C.

On the other hand, the usage ratio of C component and N component is not strictly limited and can be changed depending on other reaction conditions used, but generally the molar ratio of C component/N component is 17
Advantageously, it is 1 to 1/l OO1, preferably 1/10 to 1/20.

However, the amount of the protease used is not critical and can be changed depending on the reaction conditions, but 0.003 to 0.2 g per 1 g of component C, preferably 0.05 to 0.
．． It is suitable to use an amount of 1 g.

Furthermore, when carrying out this method, a trace amount of calcium ions or the like may be present in the reaction system in order to stabilize the activity of proteolytic enzymes such as trypsin.

Under such reaction conditions, the coupling reaction can usually be completed in about 1 to about 20 hours.

In the above reaction, the formula (1
), the functional groups (-OH, -COOH, -NH,) present in the N-terminus and side chain are unprotected,
That is, it may be in a free form, or some or all of the functional groups present are protected by protecting groups known in the field of peptide chemistry.

It may be in the form of a functional group-protected derivative.

Similarly, the peptide of formula (1) used as the N component may be either a free type or a functionally protected derivative type.

Retaining groups that can be used to protect the functional groups present in the peptides of formula (1) and/or (1) are stable under the reaction conditions of the present invention and are easily removed from the raw acid after the reaction. It is desirable that the protective group can be removed as follows, and that no side reactions occur during the removal. Examples of the protecting group include the following.

(α) Protecting groups for amino groups include, for example, 1-butyloxycardenyl group, benzyloxycarbonyl group, p
-methoxybenzyloxycarbonyl group, trityl group, etc.

(b) As a protecting group for a carboxyl group, for example, t-
Butyl group, benzyl group, etc.

(C) Examples of the protecting group for hydroxyl group include benzyl group and t-butyl group.

(d) Examples of thiol-protecting groups include benzyl group, acetamidomethyl group, and trityl group.

In addition, the formula (1) used as a starting material in this method
) and/or the peptide of formula (1) can also be used in the form of salts, and therefore the terms "peptide" and "polypeptide" as used herein include the acidic and basic functionalities present. All of the groups are in the free state (
The term is used to include not only poly)peptides but also (poly)peptides in which at least some of these acidic and basic functional groups are in the form of salts. Examples of such salts include:
Examples include hydrochloride, hydrobromide, trifluoroacetate, 7'-toluenesulfonate, acetate, triethylamsonium salt, and the like.

By the present method described above, a polypeptide or a functional group-protected derivative thereof represented by the following formula % formula % (1) where A, X, and X have the above-mentioned meanings can be obtained.
It can be synthesized with high purity and high yield without side reactions.

In the above formula (II), the compound in which both Xl and The corresponding formula (1) when representing a single bond is
The compound can be produced.

The oxidation can be carried out using an oxidizing agent and means commonly used in the field of peptide chemistry to form a disulfide bond between two cysteine units in a polypeptide. , for example H
e l v, Ch in, Act a.

51, pp. 2061-2064 (1968), J.A.
mtr, Chem, Soe, vol. 94, 5456-54
Reference should be made to guest contributions such as p. 61 (1972).

In addition, when the compound of the above formula (2)) has a protecting group, methods known in the field of peptide chemistry, such as E, 5
chroder and KsL/mhlce4”T5-pm
ptsde8Vo1.1.3-75, 1965 Ac
The protecting group can be removed by the method described in, published by Academic Press. When the compound of formula (II) is subjected to the above-mentioned oxidation step, the protective group may be removed at any point before or after the oxidation.

This makes it possible to produce the iptides represented by the formula (M-α) or (door-b) much more advantageously than in conventional methods.

Component C used as a starting material in this method, that is, the peptide of formula (1) of the present invention or its functional group-protected derivative, is a novel substance that has not been described in the conventional literature. , for example, the liquid phase synthesis method well known in the field of peptide chemistry (see 76-1 of "The Paptt-d#19" above).
(see page 36). for example,
By a well-known liquid phase synthesis method, a peptide represented by the following formula % formula % where Xl and X have the above-mentioned meanings or a functional group protected derivative thereof and a peptide represented by the following formula % formula It can be produced by synthesizing a functional group-protected derivative thereof, condensing the two, and further oxidizing if necessary to form a disulfide bond between the 1st and 7th positions.

Component C that can be produced in this way is generally represented by the following formula, where R, X, and x2 have the above meanings, and Yl, Y
7,Yo,Y4,]'! + and Y6 each represent a hydrogen atom or a protective group.

the lower alkyl group in the above formula (1) (
7? ) has up to 6 carbon atoms, preferably 1 to 4
For example, methyl, ethyl, n-propyl, 9-butyl groups, and the like, among which methyl and ethyl groups are preferred.

In addition, in the above formula Ov), X, , X, and Y1 to Y
As the protecting group represented by 6, any functional group protecting group commonly used in the field of peptide chemistry can be used, but the above-mentioned protecting groups can be advantageously used.

Accordingly, a particularly preferred group of compounds among the peptides of formula (1) or functional group-protected derivatives thereof of the present invention include the following formula % formula % () where R has the above meaning, XII and X and 1 each represent a trityl group or an acetamidomethyl group,
Or Xl, and X, 1 together represent a single bond, Y 1
1 H hydrogen atom, t-butyloxycarbonyl group, p-
represents a methoxybenzyloxycarbinyl group or a trityl group, Y21 represents a hydrogen atom or a t-butyl group, Y31 represents a hydrogen atom or a t-butyl group, Y4I represents a hydrogen atom, a t-butyloxycarbonyl group, a p- This is a peptide represented by: methoxybenzyloxycar represents a nyl group or a trityl group, Y□ represents a hydrogen atom or a t-butyl group, and Y61 represents a hydrogen atom or a t-butyl group.

Representative examples of the compound represented by formula (5)) provided by the present invention are as follows.

(1) R is an ethyl group, X and X2 together represent a single bond, Yl and Y4 are each a t-butyloxycarbonyl group, and Yll is a t-butyl group. , Yl, Y and Y6 each represent a hydrogen atom, (iI) R is a hydrogen atom, xl and X together represent a single bond, and Y1~
A compound of formula (W) where Y6 each represents a hydrogen atom, (lii) R is an ethyl group, xl and X2 each represent an acetamidomethyl group, and Yl and Y4
are t-butyloxycarginyl groups, Y,
is a t-butyl group, and Y2, Y, and Y.

When each represents a hydrogen atom, the compound of formula (), (
iv) R is an ethyl group, Xl and x2 each represent a trityl group, Yl and Y4 are each a t-butyloxycarbonyl group, Y6 is a t-butyl group, Y2, Y3 and Y6 are Compounds of formula (M), (V)J? when each represents a hydrogen atom; is a hydrogen atom,
Xl and x2 together represent a single bond, Yl and Y4 are each a t-butyloxycarbonyl group, and jt
, ya is a t-butyl group, and Y2, Y3 and Y6 each represent a hydrogen atom, a compound of formula (M), (v
i) R is a methyl group, x and X together represent a single bond, Yl and Y4 each represent a t-butyl group, and Y is a t-butyl group; Compounds of the formula (+/) where Ashi, Y2, Y8, Mutsu and Y6 each represent a hydrogen atom, and 6/i) where R is an ethyl group and XI and X2 together form a single bond. A compound of formula (6), where Y and ~Y each represent a hydrogen atom.

The present method has the following advantages over previously known methods for synthesizing the same polypeptide:
It is extremely useful industrially.

b) Due to the nature of the enzymatic reaction, the reaction can be carried out without any side reactions, and furthermore, the product can be synthesized in good yield without producing a racemate, and the product can be easily purified.

b) Unreacted components of both the N-component and the C-component can be completely recovered and reused, which is economically advantageous.

c) Protection of side chain functional groups is unnecessary or may be minimal.

Therefore, the synthesis of raw materials and the final deprotection operation are unnecessary or extremely easy.

2) The reaction takes only a short time, and the reaction equipment can be simple.

The present invention will be further explained below with reference to Examples.

The abbreviations described in the following Reference Examples and Examples are as follows, except for the abbreviations of the amino acids mentioned above.

Boc-: t-butyloxycal? Nyl Z: Penzyloxycal is Nyl Bzl: Benzyl Ac Normal: Acetamide methyl Trt: Trityl OMg: Methyl ester OEt: Ethyl ester OTB: t-Methyl ester 051L: N-Hydroxysuccinimide ester ON
p: Paranitrophenyl ester 0Bzl Nipenzyl ester DMF Nidimethylformamide-F DNSO Nidimethyl sulfoxide DCC Nidicyclohexylcarbodiimide MeOH: Methanol EtOH: Ethanol TaOH: Toluenesulfonic acid AcOH: Acetic acid DCHA Nidicyclohexylamine Reference example (1) 22- 23[,4JBoc-Tyr-Pro-O
MiBoc-Tyr-OH-DCHA46.2Q and l-
Dissolve 16.5 U of Pr o -OMe -HCl in chloroform ZOO- and add 20% DCC while cooling with water and stirring.
, 6 g was dissolved in chloroform 50% and added dropwise. After the dropwise addition, the mixture was stirred under water cooling for about 5 hours, and then the precipitated dicyclotetrahexylurea was separated, and the solution F was concentrated under reduced pressure. The obtained oily residue is dissolved in ethyl acetate, washed with an aqueous citric acid solution and an aqueous sodium bicarbonate solution, and then dried over anhydrous sodium sulfate. The oil obtained by evaporation of the solvent was recrystallized from ether/hexane to give E.

c-Tyr-pro-OMe35+5g (so%) is obtained. Melting point 122-4°C, [α] -29.1° (C-
2, DMF).

Reference example (2) 22-23 CB) Boc-Tyr-Pro-
OHBoc-Tyr-pro-OMe340σ was dissolved in methanol/dioxane (1:1) 160°C, 85 mg of 2N caustic soda was added, and the mixture was left at room temperature for 3 hours. After cooling on ice and neutralizing with 5N hydrochloric acid, the mixture is concentrated under reduced pressure to about 150 ml, and a cold 10% citric acid solution is added to adjust the pH to 2-3.

The precipitated crystals are collected by F, washed with water, and dried to form Boc-Tyr.
31.5 g (96 g) of -pro-OH are obtained. Melting point 114-5℃, Ca〕”,;-2ze° (c;2,
DMF).

Reference example (3) 22-24 (A) Eoc-Tyr-Pr
o-Arg("t)-oxt 3 g of Boe-Tyr-pro-OH3α and 9.8 g of N-hydroxysuccinimide are dissolved in DMF150-, and while cooling with water and stirring, 16.5 g of DCC is dissolved in DMF5G- and added dropwise. After continuing water-cooling and stirring for an hour,
The dicyclotetrahexyl urea is separated from each other, the solution F is concentrated under reduced pressure, the residual oil is dissolved in ethyl acetate, washed with deuterium chloride, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was treated with ethyl acetate/ether/hexane to give powdered Eoe-Tyr-pro-05% 37.0 Il (9
8).

This active ester is converted into H-Ar g (NO,) -0E
t・T80H33,69 and triethylamine IL
Add 2d to 150d of DMFm solution and leave at room temperature overnight. DMF was distilled off under reduced pressure, and the oily residue was dissolved in ethyl acetate, washed with aqueous citric acid and hydrogen chloride, and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure, and the oily residue was dissolved in ethyl acetate. /Eoc-
Tyr-pro-Arg (go2)-oxt4h1y
(ss%) is obtained as an amorphous powder. [α) $-2
41° (c=2, DMF).

Reference example (4) 22-24 (B) H-Tyr-pro-A
rg-(NO,)-0Et-HCI BoC-Tyr-pro-Arg(liO,)-0Et
3a417 about 2-7N hydrogen chloride/ethyl acetate 250
- was added, left at room temperature for 3 hours, and then concentrated under reduced pressure. Ethyl acetate was added to the residue, the vacuum concentration was repeated, and the residue was precipitated with ethanol/ethyl acetate/ether to give H-Ty.
31.3 g (96%) of amorphous powder of r-pro-Arg(gO-0Et-HCl) are obtained.

Reference example (5) 19-21 (A) Boc-Law-Gin-
ThrOMg Z-Gln-Thr-OMgTOgft-butanol/
Dissolve in 6001 ml of water (9:1), add 4 g of 5-thipalladium on carbon, and after catalytic reduction, remove the catalyst and concentrate the ν liquid under reduced pressure. The residue was dissolved in I) MF300- and Boc
Add 3 g of -Law-O5xs and leave at room temperature overnight.

The solvent was distilled off under reduced pressure, ethyl acetate was added to the residue and dissolved by heating, and after cooling, ether was added and the precipitate was collected.
Recrystallize from water containing ethanol to obtain the desired Boc-L.
aw-Gin-Thr-OMgB3.6 (J, C70
Get %>. Melting point 186-7℃, [α, l”, '-2a
o' (C20, DMF).

Reference example (6) 19-21 [B”JBoc-Leu-Gin-
Thr-NHNH.

47.4 g of Boc-Law-Gln-Thr-OMe was dissolved in 300 m of methanol, 85% hydrazine hydra) 40#I74 was added, and the mixture was stirred at room temperature.

After the crystals have precipitated, the mixture is heated overnight, ether is added, and the crystals are separated. The crude crystals are recrystallized from DMF/water to yield 37.6 g (79%) of the title hydrazide. Melting point 203
-4℃, (a] ”D-446° (C=2.80%
A c OH).

Reference example (7) 19-24 [A:] Boc-L
e u -G l n-Thr-Tyr-pro-Ar
g(NO2)0Et B oc -L g w -G l n -Th
r -NHNH21&7g in DMSO50vd! and DM
The solution dissolved in F 150- was cooled to -40°C, and 1
Mix 35d of 72N hydrogen chloride/ethyl acetate with 5.7ml of isoamyl nitrite. After stirring at -20°C for 15 minutes, the reaction solution was cooled to -40°C and triethylamine 13°3
H-T1 obtained in Reference Example (4) was further neutralized by adding m.
1r-pro-Arg (NO,)-0Et-HC1
A solution of 20 g and 5.4 d of triethylamine dissolved in 50 d of DMF was added, and the mixture was stirred overnight under water cooling. Triethylamine hydrochloride is separately added, the p solution is concentrated under reduced pressure, and the resulting oil is applied to a silica dull column and eluted with chloroform/methanol (10:t).

The solvent was distilled off under reduced pressure and the residue was precipitated with methanol/ethyl acetate to obtain the desired Boc-Lau-Gl-Thr-
Tyr-Pr o-Ar g (NO,)-0Et25
．． sg (76%) is obtained as an amorphous powder.

(α) “D-36,4’ (’-1, DMF).

Reference example (8) 19-24 (B) H-Lgw-Gln-
Thr-Tyr-Pro-Arg (No,
) -0E t -HCIBoc-Lelb-Gln-
Thr-Tyr-pro-Argα0□) -0Et
Approximately 2.7N hydrogen chloride/ethyl acetate to 2 s, o g
50- is added and left at room temperature for 3 hours. The solvent was distilled off under reduced pressure, ethyl acetate was added to the residue, and the vacuum concentration was repeated several times. The residue was precipitated with ethanol/ethyl acetate/ether to obtain 214 g of the desired product as an amorphous powder (100%
).

Reference example (9) 17-24 (A '3Z-His-Lye
(Boc)-Leu-Gin-Thr-Tyr-pro
-Arg(No2)-0Et Z-Hi 5-Lys (Bo C)-NHNH210
, 2 g dissolved in DMFBO ml is cooled to -20 DEG C., and 178N hydrogen chloride/ethyl acetate 20-ml and inamyl nitrite 3.15 ml/ml are added. After stirring at -20°C for 15 minutes, the re-core solution was cooled to -40"c', neutralized by adding 7.78" of triethylamine, and H-Leu-Gl
n-Thr-Tyr-pro-,4r g c/vO,
17.7 g of -0Et-HCL and 3.36 d of IJ-r-thylamine are dissolved in 80 dl of DMF and added.

After stirring overnight under water cooling, the triethylamine hydrochloride was removed separately, the liquid was concentrated under reduced pressure, the oily residue was applied to a silica gel column, and chloroform/methanol (S: t) was added.
Elutes with The desired fraction is concentrated under reduced pressure and precipitated with methanol/ethyl acetate to obtain the desired Z-His-Llls.
(Eoc)-Law-Gln-Thr-Tyr-pr
o-ArgOJO,)-OEt19.2g (74%)
obtained as an amorphous powder.

[α] 2 also ’-23,5° (C-2, DMF).

Reference example (10) 17-24 [B 3H-His-Ltta
(Boc) -Leu-Gln-Thr-Tyr-p
ro--4rg-OEt-ACOH z-His-Lys (Boc)-Law-Gln-T
hr-Tyr-pr o-Ar g:(NO,)-
After dissolving 0Et11, Og in 80-thiacetic acid and performing catalytic reduction by adding 5thipalladium carbon Log,
Distribute the catalyst door to door. The P solution is concentrated under reduced pressure, water is added several times and the mixture is concentrated under reduced pressure, and then methanol is added and concentrated under reduced pressure. The resulting oily residue was precipitated with methanol/ether to yield H-Hi a -L ys (Boc)-1,e
v, -Gin-Thr-Tyr-Pro-Arg-OE
An amorphous powder of t-3AcOH tQ,ag (94%) is obtained.

Amino acid analysis Lygl, 10 (1), HisLO2 (1), AT
(lo, '16 (1), TArO, 95 (1), G
l il, 03 (1), Pro 1.15 (1
), Lgul, 00 (1), TyrO, 94 (1)
.

Reference example α1) 4-s (12-1!l) Z-Law-5tt
r-NHNH.

Dissolve 5 g of Z-L e w-2Ser-OMeSL in 40G of methanol and add 85% hydrazine hydra)5
0m was added, the mixture was stirred at room temperature, and after crystallization, the mixture was heated overnight. Add ether to separate the crystals, and recrystallize from methanol to obtain Z-Leg-5s Day NHNH, 45.2 g (87
%). Melting point 176-8℃, (α)F-a6° (
G-1, DMF).

Reference example αgl 12-16(-A]Z-Lttu-5gr-G
In-Glw(OTB)-Law-OleZ-L e
u -S g r -NHNII, 13.9 g to D
The solution dissolved in 00 ml of MFI is cooled to -40'C, and 30 btl of 268 N water/ethyl acetate and 6 g of isoamyl nitrite are added. After stirring at -20'C for 15 minutes, it was cooled to -40°C, and triethylamine 11
, 26 it to neutralize. H-Gt in the reaction solution
n-G1 u (OTE) -Lttlb-OMg 1
Add 50% of DMF solution containing 7.4 g and cool with water.
Stir at night. The reaction mixture was concentrated under reduced pressure, water was added to the residue, the precipitate was collected, and recrystallized from ethanol to obtain the desired Z-Leu-5er-01n-Glu (OTB).
-Lt,rb-OMe2B,Og (93%) is obtained. Melting point 194-196℃, (a,) ”,,” −
21°5° (C-2, D kl F ).

Reference example (131tt-16(, ()Z-4y8 (Eoc)-
Leu-,5er-Gln-G'lu (OTB)-L
m 舊-OMe Z-Lea-5ir-Gln-G11L (OTB)-
Law-OMe23.8gft-butanol/water (9:
1) 250dKll! After catalytic reduction by adding 1 g of 5-thipalladium on carbon, the catalyst is separated and the P solution is concentrated under reduced pressure. Dissolve the residue in DMF 300- and Z-Lys
After adding 15 g of (EaC)-ONp and standing at room temperature overnight, the mixture was concentrated under reduced pressure. The precipitate formed by adding water to the residue is P
and recrystallized from ethanol/water to obtain the desired Z-Ll.
s (Boc)-Lex-5er-Gln-Glu (
OTE)-Leu-OJfe26.31! (85%)
get. Melting point: 215-6℃, : [α]2ken 5-25.0
° (C=2, DMF).

Reference example G41 11-161:B″1Z-Lys (Boc)
-Leu-5er-Gln-GLu (OTB)-L
g w -NHNH.

Z-Lys (Boc) -Law-5ar-Gln-
Glu (OTB)-Law-OMtt1f3.2.1
was dissolved in 00 ml of DMF1, 15 ml of 85% hydrazine hydra was added, and after standing at room temperature for 2 days, the reaction solution was concentrated under reduced pressure. The residue is washed with ethanol to obtain the desired hexapeptide hydrazide 16.7y (92%). Melting point 2
26-7°C (decomposition), [α]V-45,00 (j=2.
80% AcOH).

Reference example αs) xl-z4CA]z-r,ys (Boe)-
Lew-5er-Gln-Glw (OTB)-Let
s-His-Lya (Boc)-Lgu-Gln-T
hr-Tyr-pro-ArQ-OEt Z-Lys (Boe)-Law-5ur-Gln-G
l u (OTB) -L g w -NHNH,5
, 21 is dissolved in DMF50st, the solution is cooled to -40'C, and 27N hydrogen chloride/ethyl acetate 5- and isoamyl nitrite α8- are added. After stirring at -20°C for 15 minutes, it was cooled again to -40°C, and triethylamine 1
Add 9m to neutralize. In this solution, H obtained in Reference Example α
-Hi 5-Lys (Boc)-Law-Gln-
Tr-Tyr-pro-Arg-OEt-3AcOH
Add 3o- of DMF solution containing 5,41, Trier 7mine L4- and stir overnight under water cooling. The reaction solution was concentrated under reduced pressure, and the residue was applied to a silica column and eluted with chloroform/methanol/water (70:30:5). The desired tridecapeptide is methanol/ethyl acetate/
Precipitation with ether gives 5.4 g (60%) of an amorphous powder.

Amino acid analysis: L y a zz 4 (!:), H41LO8 (
1n, A r g 0.99 (1), rho, 5s
(t), S e r O, 89 (1), G tu a
, 17 (3), Prol, 06 (1), Lgu:x
oo(3), 7yrα94(1).

Reference example αQ 4-6 [A] Z-Law-5ar-ThrOMg Z -L eu -Ser - obtained in Reference example α0
Dissolve NHNH225,,6Q in DMF 200-,
After cooling to -40°C, add 60% of 2.35N hydrogen chloride/ethyl acetate and 11.6% of inamyl nitrite.
After stirring for 15 minutes at °C, the mixture was cooled again to -40 °C and neutralized by adding 19.8-triethylamine.

Add H-Thr-OMtt (Z-Thr-On
A solution prepared by catalytically reducing 7 g of GIA) in 50 g of DMF was added, and stirring was continued overnight under water cooling. Concentrate the solvent under reduced pressure, add water to the oily residue and stir. This was recrystallized from methanol/ethyl acetate to obtain the desired Z-Ltts-5er-Thr-0Mg26.6Q.
(81%).

Melting point 185-6°C, (a) "p-'L 8° (
C=1, DMF).

Reference example α? ) 3-6 [A) Z-Asn-Law-Ear-T
nr-OMg Z-Law-5et-Thr-0Mg13. , Sg to t
-Suspended in 40G-butanol/water (9:1), 5%
After catalytic reduction is performed by adding 2 g of palladium on carbon, the catalyst is separated and the F solution is concentrated under reduced pressure. The residue was dissolved in DMF80, 2 g of Z-Asn-ONpIL was added, and the mixture was left at room temperature for two nights. The precipitated crystals were dissolved by heating, concentrated under reduced pressure, and the residue was recrystallized from DMF/methanol to obtain the desired Z-Aan-Lttu-5er-Thr-0Mg13.
Obtain 7g (81ch). Melting point 218°~222°C, (a
) 'p −2fs−0° (c=2, DMF).

Reference example α8) 3-6 [B) H-Aan-Les-5er-T
hr-OMg Z-Aays-Lew-Ear-Thr-0Mg5.8
g was suspended in 150ml of t-butanol/water (9:1) and
After catalytic reduction by adding 0.6 g of Chinoradium carbon, the catalyst was separated and the filtrate was dried under reduced pressure.

The crystalline product is used immediately in the next reaction.

Reference example α9) 1-2 [A) Boc-Cya (Actn)-
5er-OMtt Boc-Cys (Acm)-0H-1) CHA&8
g (0,03-T-ru) and H-5g r-OMt
t-HC14717 (0.03 mol) and glue were dissolved in Roform Zoom, and 18 g of DCCa was added while cooling with water and stirring.
(α03 mol) is added. After reacting for 3 hours under water cooling and 2 hours at room temperature, the precipitated dicyclohexyl urea was washed door to door with Shirishiroform. The F1 washing solution is washed with an aqueous citric acid solution and deuterium chloride water, and then dried over anhydrous sodium sulfate.

The solvent was distilled off to obtain an oily target product9. Get sg (sgchi).

Reference example @ (i l-2 (B) Boe-Cys (Asm)-5
#r-NHNH.

BoC-Cya (Acm)-5ttr-01g9゜6
g was dissolved in DHFsO-, the container was purged with nitrogen gas, 15 m of 85 hydrazine hydrate was added, and the mixture was allowed to react overnight in a refrigerator. The reaction solution was dried under reduced pressure, and the remaining crystals were recrystallized from methanol to obtain 7.3 g (7 seconds) of the desired product. Melting point: 199-zoo℃, (a)'', j-gto
'(C=2.80% AcOH).

Reference example 61!1) 1-6CA, Boc-Cya (Acm)
-5ar-Aan-Leu-5pr-ThrOMg Boc-Cys (Acm) -5e r -NHN
H.

After heating and dissolving 3.94 g in a mixed solvent of I) MF: I) MSO (2: 1) 60-, it was cooled to -20°C, and while stirring, 20 m of λ72JY hydrogen chloride/ethyl acetate and 137 m of isoamyl nitrite were added. Add and stir for 15 minutes.

The reaction solution was cooled to -40°C, and 7.62 d of triethylamine was added.
Add to neutralize. Add H-A obtained in Reference Example α to this solution.
sn-Lex-5ar-Thr-0Mm441gI)
Add vinegar solution dissolved in MF 30 ad, and cool for 20 min under water cooling.
Allow time to react. Triethylamine hydrochloride is separated, the solvent is dried under reduced pressure, and the residue is recrystallized from methanol to obtain 6.1 g (75 g) of the desired title compound. Melting point 208
-211℃, (a 〕”, knee 22.0° (C20
, I) MF).

Reference example @1) 1-6 [E) Boc-Cya (Acm)-5
er-Asn-Leu-5ar-Thr-NHNH.

Boc-Cya (Acm)-5er-Asn-Lgu
-5ar-Thr-OMe5g in DMF50ad! K.W.
Is(,,85*Hit''-Jinhi V2-) 4gmg was added, left overnight in a refrigerator under a nitrogen atmosphere, concentrated under reduced pressure, and the residue was washed with methanol to obtain 47g (94g) of the desired product.
%). Melting point 187-188℃, ("α) height -34
5° (c=2.80q6AeOH).

Reference example H7-to(:, <lBo e -(1'y s (A
cm)-Val-Lew-Gly-OEt Boa-Cys (Acm)-OH-DCHA29.3
4g and H-Val-Law-Gly-OEtHC12L
8Q was dissolved in 360 dlC of chloroform and added to DC while cooling with water and stirring. A solution of 'l'l (l) dissolved in 30 ml of chloroform is added dropwise. After stirring for 3 hours under water cooling and overnight at room temperature, the precipitated dicyclotetrahexyl urea is separated and washed with dichloroform. After washing with an acid aqueous solution and a sodium bicarbonate solution, drying with anhydrous sodium sulfate.

Chloroform is distilled off and the residue is recrystallized from ethyl acetate to obtain the desired protected tetrapeptide 29.39 (80%). Melting point 140-141℃, [α,: 1%'-1a5°
(C20, DMF).

Reference example @4) T-LO[B)H-Cys (Acm)-Va
l-Lea-Gly-OEt -HCooHBoc-C
ys (7cm)-Val-Lttw-Gly-OEt
Dissolve 5.9 g in 100 ad of 98 thiformic acid and leave at room temperature overnight. The residue was thoroughly washed with ether and dried to obtain 5.3 g (100 g) of the desired product as a powder.

Reference example 'J 1-10(,4)Bc+c-Cya (Acm
)-5er-Aan-Lttu-Ear-Thr-Cy
s (Acm)-Val-Leu-Gly-OEt Reference Example 6! Boc-Cys (, Jcm) -5
g r -A s n -L g u -S e r
-T h r -NHNH,4,059 in DMF3θd
Dissolve by heating.

2. Cool to below 0°C and stir. ,'l 2N hydrogen chloride/
Add 10d of ethyl acetate, then cool to -20°C, add inamyl nitrite, trnl, and heat to -15°C to -1.
Stir for 10 minutes at 0°C.

- Cool to 40°C, neutralize by adding triethylamine &81m, and add H-Cya (Acm)-VaL-Le
w-Gly-OEt-HC00HL71 in DMF30
-Dissolve in 2. A solution neutralized with 0.74% of triethylamine is added and reacted at 0°C for 20 hours.

Triethylamine hydrochloride is delivered door to door, the solvent is concentrated under reduced pressure,
The residual liquid was crystallized with DMF/methanol, and then further diluted with DMF/methanol.
Recrystallization from water yields 2-7 g (40 g) of the desired product.

Melting point 242-244℃ (decomposition), [α]V-2&5°
(C=1, I)MF).

Reference example @fj) 1-10 (B: IBOc-Cya (Acm
)-5ar-Aan-Law-5ttr-Thr-Cy
a (Aem)-Val-Lau-G I Y -NH
N.H.

Boc-Cys (Acm)-5ar-Asn-Lex
-5ttr-Thr-Cya (Acm)-Vat-L
Dissolve 14.52 g of aw-GLy-OR in 30 m of DMF, add 1.5 liters of 85es hydrazine hydrogen, and react overnight in the refrigerator. Concentrate hydrazine hydroxide and DMF under reduced pressure, wash the residue with methanol, and get the desired product! Obtain 25 g (90 g).

Melting point 236-237℃ (decomposed), [α]V-42L3°
(C=0.3.80chi AcOH).

Reference example 6175 22-23 Bee-Tyr (Bgl)-
ro-OR Boc-Tyr (BgJ)-0Sx85g in DMF6
00- solution and proline 2B (l to water 100+d
28 yd of triethylamine was added dropwise while stirring under cooling with water. After completion of the dropwise addition, the mixture was stirred at room temperature for 3 hours, and then concentrated under reduced pressure. The resulting syrupy residue was dissolved in 300 ml of water and acidified with citric acid. The precipitated product was extracted with ethyl acetate, and the ethyl acetate layer was washed with water, dried,
Concentrate to approximately % and add hexane to precipitate crystals. 7
2g (85ch). Melting point 141-2℃, [α]V-21
3° (C-1, EtOH).

Reference example (28122-24BoC-Tya (Bzl)-Pr
o -Ar g (NO,) -0Et Reference example @ Bo e-Tyr (Bct) -pro o-0
H33gXH-ArlJ (NOx)-0Et・Ts
OH2Q,2 (lz N-hydroxysuccinimide 9.0g and triethylamine 1O09- in DMF3
50J17! DCC16,2 while cooling with water and stirring.
A solution of g dissolved in 50 ml of DMF is added dropwise. After the completion of the dropwise addition, stirring was continued at room temperature for 12 hours, and the precipitated crystals were separated, and the solution F was concentrated under reduced pressure to obtain a syrupy residue. Dissolve this in ethyl acetate, wash with 4 citric acid, 4 strips of sodium bicarbonate solution, and water in this order, and dry.

The solvent was distilled off, and the resulting residue was subjected to silica dull column chromatography (eluent: chloroform/methanol lO:l) and recrystallized from ethyl acetate/hexane to obtain the target product 4.
317 (79%). Melting point 89-90℃, ('')
”p −17−2 m (c = 1, MeOH).

Reference example @s) 19-211:, 4) Z-Lttu-Gln
-Thr-OMg Z-Gln-Thr-0Mg 39.5g was mixed with t-butanol/water (9:1) 1! 5% palladium on carbon 2
After catalytic reduction, the catalyst is separated and the p liquid is concentrated under reduced pressure. The residue was dissolved in 300 ntl of DMF, and Z-Lgu-
Add ONp8B, 3ct, and leave at room temperature overnight. The solvent was distilled off under reduced pressure, and the remaining crystals were thoroughly crushed with ethyl acetate. Recrystallization from DMF-ethyl acetate gave the desired Z-Lgs-Gin-Thr-0Mg42fi (83
%).

fi point 203-4℃, (a) "D-1 &3'
(C=1, DMF).

Reference example CIIQI 19-21(B) Z-Leu-Gln-
Thr-NHNH.

DM Z-Leu-Gln-Thr-0Mm25.49
Dissolve in F 300- and add 30 g of 85% human zinc hydroxide at 0°C, leave to stand overnight at room temperature, concentrate under reduced pressure, and thoroughly mash the residue with cold water. Recrystallization from DMF/water yields 23 g (9 o) of the title hydrazide. Melting point 219-220°C, [a:]”D-I
Z 1° (c = L, D MF ).

Reference example 0υ 19-24 Z-Law-Gin-Thr-Ty
r(Btl)-pro-Arg(IiO,)-0E
The tripeptide Eoc-Tyr (BgJ
)-pro-Arg (No2)-0Et71 at 98%
Dissolve in 40 g of formic acid, leave at room temperature overnight, and then dry under reduced pressure. The residue was dissolved in cold water, triethylamine was added little by little to adjust the pH to 9, the precipitated product was extracted with ethyl acetate, the organic layer was dried, and the solvent was distilled off to give H-Tyr (B
zL)-pro-A r g (1(0,) -0E
get t. On the other hand, the hydrazide Z-La obtained in Reference Example 00)
x-Gin-T5r-NHNH,5,19 in DMFlo
Suspend in od and dilute with zaN hydrogen chloride/ethyl acetate at 0°C.
0 mg was added and dissolved, and the mixture was further cooled to -15°C and 2 g of Inamyl nitrite L was added. After 15 minutes, it was cooled to -50°C and 9.7d of triethylamine was added, and the above H-T
71r (Ex 1)-Pro-ArQ(NO,)-0
A solution of Et in DMF30- is added, and stirring is continued at 0° C. for 16 hours. The precipitate was separated and the F solution was dried under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform/methanol 10:1), and both desired fractions were crystallized with ether to obtain the title hexapeptide Z-.
Leu-Gin-Thr (Bz l) -pro
-Ar g (NO,)-0Et 7.5 g (70
%). Melting point 120-2℃, [α] sweetened 37.2°
(C=1, EtOH).

Reference example <32 17-24 Z-His-Lys (Boc)
-Law-Gin-Thr-Tyr-prO-Aτ to 1
-OEt Z-Law-C obtained in Reference Example 01); 1n-Thr-T
yr (Bg l) -Pro o-ArEi (A'
□d-0Et20 (Dissolve 1 in 500I of 80% acetic acid and add 2g of 5-thipalladium on carbon for catalytic reduction. After completion, remove the catalyst, dry the ν solution under reduced pressure, and mix the residue with ether.) Crush and measure , dry under vacuum over sodium hydroxide. 9.2517 of this product was dissolved in DMFsO- and neutralized with triethylamine. On the other hand, Z-His
-Lye (Boa)-NHNH, a 36 f DM
F 10 oI11tK solution 5L - 20°C 2.8N hydrogen chloride/acetic acid xfk20d, then isoamyl nitrite L2
Q was added, and after 10 minutes the mixture was cooled to -50°C, triethylamine 6.4- was added thereto, the above hexapeptide solution in DMF was added, and stirring was continued at 0°C for 16 hours. Insoluble matter was removed separately, the filtrate was dried under reduced pressure, and the residue was applied to a silica solution (70:30:1 form/methanol/water).
Elute in 2). Both desired fractions were concentrated and precipitated with ether to obtain the title Z-His-Lye (Boc)-Law-
01%-Thr-T11r-pro-Arg-OEt1
α2Jy (78%) is obtained.

[α: 17-g & 7° (c=1, DMF).

Reference example C1: i1o-1t(A) Z-Gly-Lys-(
Boc)-0Mg H-Lys (BoC)-0Mm2.6y was dissolved in 300ml of ethyl acetate, 33g of Z-Gll-ONp was added, and the solution was left at room temperature for 16 hours.
Wash sequentially with water, dry, concentrate the solution back to 1, and add hexane. b. Crystals precipitate. The crystals were separated and recrystallized from ethyl acetate/hexane to obtain the title dipeptide 4 (
Obtain L6g (90%). Melting point 74-5℃, [α)
%O-&s.

(c=1, MeOH).

Reference example G14 1O-11(B:l Z-Gly-Lye(B
oc) -NHNH.

Reference example 0 Z-Gly-Lye (Boc)-0 obtained in the field
After reacting 36 g of Mg with I) MP 300-medium hydrazine human sheet 30g overnight, it was concentrated under reduced pressure, and the residue was recrystallized from methanol/water to obtain the desired hydrazine YZ-Gl.
y-Lys(Boc)-NHNH.

Obtain 32 g (89%).

Melting point 139-140℃, (a) %'-xa, s° (C
= 1, MgOH).

Reference example (u9 1G-16(,4) Z-Gly-Lys(B
oc)-Lew-5er-Gln-Glu (OTB)
-Law-OMg Z-Gly-Lye (Eoe)-N obtained in Reference Example
Dissolve 27.5 g of HNH in aoo- DMF, -1
While cooling to 5° C. and stirring, 30 ml of Z3N hydrogen chloride/ethyl acetate and then 6.6 g of isoamyl nitrite were gradually added. After 10 minutes, cool to -50'C and add triethylamine9.
Drop 7d. On the other hand, pentaizotide Z-Ltt-5ar-Gln-Glx (OT
B)-Law-0Mg8(J.

6g of t-tutanol/in the presence of 5-Δradium carbon*3y
The product obtained by catalytic reduction in 350°C of water (9:1) was
The solution was dissolved in MFloo- and added to the above azide solution, and stirring was continued at 0°C overnight. The reaction mixture was dried under reduced pressure, the residue was thoroughly crushed with water, the precipitate was washed with water, and after drying, DMF was added.
/ recrystallized from ethanol to obtain the title Z-Gly-Ly
e (Bec)-Lgs-Ear-Gin-Gls (
OTB)-Law-0Mg 45.3 g (84%) are obtained. Melting point: 224-5°C (decomposed), (a]',,'-
2ts.

(C=1, DMF).

Reference Example 96) 1O-16(B) Z-Gly-Lye(Bo
c) -Law-5er-Gin-Glu (OT B
) -L g u -NHNH.

Reference Example 0 43g of hegtapeptide obtained from DMFS O
After reacting with 30 m of hydrazine hydride in 0IILt at room temperature for 24 hours, it was concentrated under reduced pressure, and the residue was recrystallized from DMF/ethanol to obtain the desired Z-Gly-Lys (
Boc)-Law-5ar-Glx-Glw (OTB
)-LelL-NHNH, 38 g (88%) is obtained. Melting point 225-6℃ (decomposition), (α)'', 7-t7.g
o (c=IXDMF).

Reference example 917) 1G-24Z-Gly-Lye (Boc)
-Leu-5er-01n-Glu (OTB)-La
w-His-Lys (Boc)-Leu-Gln-T
20 g of hr-Tyr-pro-Arg-OEt To-Arg-OEt is dissolved in 400 d of 80% acetic acid, and 2 g of 5% radium carbon is added for catalytic reduction. After the reaction is completed, the catalyst is separated and the liquid F is dried under reduced pressure.The residue is thoroughly crushed with ether, removed and dried over sodium hydroxide under reduced pressure. Of this product, 6.8 gf: DJ (F5
Dissolve in 0ml and neutralize with triethylamine.

On the other hand, Bebutapeptide hydrazide Z obtained in Reference Example C3φ
-Gly-Lys (Boc)-Lew-5er-Gl
n-Glx (OTB)-Lea-NHNH, 6.5g
100#I7! 2.3N hydrogen chloride/ethyl acetate (15In11) was added thereto while stirring at -15°C, and then 77g of isoamyl nitrite α was added. -50 after 10 minutes
The mixture was cooled to 0.degree. C., neutralized with 48 g of triethylamine, added with the DMF solution of the above octapeptide, and continued stirring at 0.degree. C. for 16 hours. Separate the insoluble matter, dry the lachrymal fluid under reduced pressure, and apply the residue to a silica gel column (eluent: chlorotetraform/methanol/water 70:30:3). Concentrate the desired components and mash with ether. and the title Z-Gly-Lys
(Boc)-Leu-5er-Gln-Glu (O
TB)-Law-H4s-Lys (Boc)-Le1
4-Gin-Thr-Tyr-pro-Arg-oEt
8. sct (7s%) is obtained as an amorphous powder. [α] Dorsal -148° (c=1XDMF).

Reference example G38)? -9Trt-Cys (Trt)-Val
-La1L-OMe; l-Val-Law-OMg37.8g with methanol-7
Dissolve in A/300〆 and 5N hydrogen chloride/methanol 5㎜, add 4 g of 5% A radium on carbon, and perform catalytic reduction.

After the reaction, the catalyst was separated, the tear fluid was dried under reduced pressure, the residue was dissolved in aOOgo DMF, and Trt-Cys (7' r t
) -08u-CnHa 78 y was added and then heated to 0°C.
Cool and add 14 grams of triethylamine. After standing overnight at room temperature, the mixture is dried under reduced pressure, the residue is dissolved in chloroform, and the organic layer is washed with 4% citric acid, 4% sodium bicarbonate, and water in this order, and dried. The solvent was distilled off, and the residue was recrystallized from methanol to obtain the title Trt-Cys (Trt)-Val-Lt.
, 71 g (85%) of rb-ONg are obtained. Melting point 108
-9℃, (a:] 'D +2 Z 3° (C=2
, chloroform).

Reference example C49) 1-2(A) Eoc-Cys (Trt)
-3ttr-OMg Boa-Cys (Trt)-OH-DCHA65Q and H-5er-OMtt-HC115.6g for 500d
DCC2α6 was dissolved in chloroform and stirred while cooling with water.
A solution of g dissolved in 50 ml of chloroform is added dropwise. After dropping, stir at room temperature for 15 hours, remove insoluble matter,
The filtrate is dried in the following order: 4 citric acid, 4 sodium bicarbonate, and water. The solvent was distilled off and the residue was recrystallized from ethyl acetate/hexane to give the title Boa-Cys (Trt)-5ur-OMt.
t39.5g (70 parts) is obtained. Melting point 181-2℃, [
α]V+23.7° (C-1, MttOH).

Reference example 60) 1-2 [B:] Boc-Cys (Trt)
−5εdayNHNH.

Eoc-Cys (Trt)-5e obtained in Reference Example 09)
t -0Mg 56.4 g methanol 500rJ
Dissolve hydrazine hydrogen chloride at 0°C 2 s *tJ+
After leaving at 0℃ for 16 hours, the precipitated crystals were separated, washed with water, and dried to obtain the desired hydrazide Boc-Cys (Tr).
t)-5er-NHNH251 g (90%) are obtained.

Melting point 104-8℃, [α)7+0.76'(C-1, J
f e OH).

Reference example ρυ 1-6(A) Boc-C1/s (Tri)-
5er-Aan-Leu-5er-Thr-Mg Hydrazide Boc-Cya (Tr
t) -5sr -NHNH,32g'IrDMF25
23N hydrogen chloride/with stirring at -30°C.
Ethyl acetate 5011I/, then isoamyl nitrite 8g
Add gradually. After 10 minutes, the mixture was cooled to -50°C and neutralized by adding triethylamine 1al-.

On the other hand, the tetrapeptide Z-λsn-L obtained in Reference Example α
ttu-5er-Thr-0Mg 33.1g in t-butanol/water (9:1) 800-5% palladium on carbon 1
．． Catalytic reduction is carried out using 5 g. After the reaction is completed, the catalyst is distributed door to door.
The filtrate was dried under reduced pressure, and the residue was dissolved in DM7'100m, added to the above azide solution, and stirred at 0°C for 20 hours. The reaction mixture was dried to dryness under reduced pressure, and 400 g
m, thoroughly mash the precipitate, wash with water, and dry.

Recrystallize from DMF/water to obtain the desired BoC-Cys (
Trt)-5ar-Ass-Law-5ar-Thr-
OMe 4 Z8 g (76,7%) are obtained.

Melting point 212-4℃, [α:]'D + 0-44°
(C=1, DMF).

Reference example (Af!J l-6CB) Eoc-Cya (Trt
)-5ttr-Aan-Law-5ttr-THr-
NHNH.

Reference example Hexapeptide 64 obtained with αυ. ? Q55
0 mef) Hydrazine Hitlerde 60i and 0 in DMF
After reacting overnight at ℃, the mixture was poured into 3 tons of ice water.

The precipitate was separated, washed with methanol and then ether, and the title hydrazide Boc-Cys (Trt)-5er-
Astr, -Leu-5er-Thr-NHNH,s
6 g (86%) are obtained.

Melting point 205-6℃, [α] back +6.26 (e = 1,
DMF).

Reference example @3) 1-9(,4:] Boc-Cys (Trt
)-5ur-Asn-Law 5et-Thr-Cys
(Trt) -Meal-Lax-Me Hydrazide Boe-Cys (Trt) obtained in Reference Example α-Me
-5ar-Asn-Lgu-5er-T h r -N
Dissolve HNH,29,4Q in DMF 35 G-,
2-3N hydrogen chloride/ethyl acetate with stirring at 30°C
-1 Then 4.2 g of isoamyl nitrite is gradually added.

After 10 minutes, cool to -50°C and add 9.7 m of triethylamine.
Add to neutralize.

On the other hand, the tripeptide Trt-Cya obtained from Reference Example 0
(Trt)-Vat-Lgw-OMtt30g to 80%
Dissolve in acetic acid and leave at room temperature for 3 hours, then add 160 ml of water. The resulting precipitate was separated, the P solution was dried under reduced pressure, saturated 11 water (2 GG) was added to the residue, and the precipitated oil was extracted with ethyl acetate. The organic layer was dried and the solvent was distilled off, and the resulting oil was dissolved in DMF 100- and added to the above azide solution.

The reaction solution was stirred at 0°C for 2 days, then dried under reduced pressure, and the resulting precipitate was washed with water, dried, and recrystallized from chloroform/methanol to obtain the desired B.
oC-Cys (Trt), -5er-Asn-Law
-Ear-Thr-Cys (Trt)-Val-La
w-0Mg26.4y (57%) is obtained. Melting point 227
-230℃ (decomposition), [α]V-7.7' (c=1,
DMF).

Reference example @4 1-9(B:l BaC-Cys (Trt)-
Ear-Asn-Law-5ur-Thr-Cys (
Trt)-Vat-Leu-NHNH.

Reference example: 1g of nonapeptide 2a obtained from α-bu was added to DMF340.
The mixture was dissolved in DMSO1'IO and DMSO, and human 2-zine hydra-tro3i was added thereto, and the mixture was left at 0°C for 2 days. The reaction solution was poured into ice-cooled saturated saline 21, and the precipitate was washed with water and dried to obtain the desired hydrazide Boc-Cya (Trt).
-5ur-Aan-Leu-5ur-Thr-Cys
(Trt)-V a l -L e w -NHNH
, 26g (100%) is obtained. Melting point 242-5°C (decomposed), [α]V-a5° (c=1.DMF).

Example I Boc-Cya (Acm)-5ur-Asn-Le
w-5er-Thr-Cys (Acm)-Val-L
eu-Gly-Lys (Boc)-Law-5er-
Gln-Qlu (OTB)-Leu-His-Lys
(Boc)-Lgsc-Gin-Thr-Tyr-P
ro-Arg-OEt Reference Example 2.5 g of the protected tetradecapeptide obtained in α5) is dissolved in 40 g of 80-thiacetic acid, and 5-thipalladium on carbon is added and hydrogenated. After the reaction is completed, the catalyst is separated and dried under reduced pressure, and the residue is thoroughly washed with ether to form a powder. On the other hand, reference example @ 2.25 g of boiled decapeptide hydrazide was added to I) MP-DMSO (1:1) 40
Dissolve in water, cool to 20°C, add 5d of 2.72N hydrogen chloride/ethyl acetate and 0°23g of inamyl nitrite, and add 1.
After stirring for 5 minutes, the mixture was cooled to -40°C and neutralized by adding 9m of triethylamine. A solution prepared by dissolving the above reduction product in DMF 30d and adding 0.37-triethylamine was added thereto, and stirring was continued overnight under water cooling. The precipitated triethylamine hydrochloride was separated and concentrated under reduced pressure, and the residue was dissolved in nxp-α5 mol acetic acid (1:1) and filtered through Sephadex LH-2O Cassim.

Elute with the same solvent, concentrate the desired fraction under reduced pressure, l) MF/
Re-precipitate from ethyl acetate to obtain the title compound! 35
g'&: get.

[α]υ−1&0° (C=2, DMF).

Example 2 -Val-Lex-Gly-Lys (Boc) -L
eu-5er-Gln-Glrb (OTB)-Lew
-His-Lys (BoC)' -Leu-GltL
-Thr-Tyr-pro-Arg-OEt 300 mL of protected tetracosapeptide obtained in Example 1 I) 3 ml of MF
Dissolve in water and dilute with 30% methanol. This solution is added dropwise to a solution of iodine 400 M9 dissolved in methanol 40 at room temperature with vigorous stirring. 1 more after Kamashita ends
After stirring for a certain period of time, the mixture was cooled with ice and IN-sodium thiosulfate was added dropwise until the bath liquid was decolored. Concentrate methanol under reduced pressure and add ice water to remove the resulting precipitate, dry and refrigerate from methanol/ether. This is DMF-'0
．． Sephadex Lll- in a system of 5 molar acetic acid (1:1)
The product was filtered with 20 glue and reprecipitated from methanol/ether to obtain 280 da of the desired product.

(ff)”,’+20.0° (C=2, DMF).

Amino acid analysis: LysL98 (2), Hiso, e i (i)
, Ar go, 9e (z)1,4SpO,59(
1), T h r 1.82 (2), S a r Z
51 (a), Glw2-93 (3), P r OO
, 9g (1), G11LO1 (1), C11B 0.
89 (1), rcli, oO(1), L e u 5
．． 0? (5), T y r 1.01 (1).

Example 3 H-Cya (Aem)-5ir-Aan-Lgs-5
ur-Thr-Cya (Acm)-Val-Lgw-
Gly-Lya-Lgu-5ttr-GLts-Glw
-Lgw-His-Lys-Lgu-Gln-Thr-
Tyr-pro-Arg-OH The protected nato-2cosapeptide 5Qq obtained in Example 1 was dissolved in 5 ml of DMlo and 0.3 m of α5 mol Tris-HCl buffer (pH 6,0), and 21 v of trypsin was added thereto at 30°C for 1 hour. react. The reaction solution was dried under reduced pressure, the residue was dissolved in 3H trifluoroacetic acid containing 0.5-anisole, reacted at room temperature for 40 minutes, and then dried under reduced pressure. After washing the residue with ether, 0°2N
Dissolve in acetic acid and add Sephadex G-25 (2゜5X10
The desired fractions were collected and lyophilized to obtain the title compound 39FF.

[α]v-73.7° (c=0.76, H2O).

Example 4 Boc-Cys (Trt)-3er-Asn-Lea
-5er-Thr-Cys (Trt)-Val-Le
u-Gly-Lys (Boc)-Lerb-5er-
Gln-GilL(OTB)-Lew-His-Lys
(Boc)-Lau-Gln-Thr-Tyr-pr
Pentadecapeptide Z-Gly-Lys (Boc)-Leu-5er obtained in o-Arg-OEt Reference Example 07)
-Gln-Glu (OTB)-Lttrb-His-
Lys(Boc)-Leu-Gln-Thr-T11r
-Pro-Arg-OEt 6.6g in 80% acetic acid 150%
5% palladium on carbon for catalytic reduction. After the reaction is completed, the catalyst is separated, the F solution is dried under reduced pressure, and the residue is thoroughly washed with ether to form a powder.

On the other hand, nonapeptide hydraziYBoe obtained from Reference Example 0
-Cys (Trt) -5er-Aan-Leu-5
er-Thr-Cys (Trt)-Va l -L
e w -NHNH, 46g in DMF100st and 'l
JDME 0100 r! Dissolve LlK and gradually add 10IIlt of Z3N hydrogen chloride/ethyl acetate and 0.36 g of isoamyl nitrite while stirring at -10'C. 15
After a few minutes, it is cooled to -50°C and neutralized with triethylamine 3.2-. A solution prepared by dissolving the above reduction product in 50 m/ml of DMF and adding 0.42 rtrl of triethylamine was added thereto, and stirring was continued at 0°C for 16 hours. Insoluble matter was separated, the tear fluid was dried under reduced pressure, and the residue was coated with 1"k of silica,
17 ram (eluent: ritroform/methanol/
water (70:30:3), the desired components were concentrated and powdered with ether to obtain the title tetracosapeptide a9g (64ti). [α)%G-&00 (c=1, -DMF).

Example 5 -Val-La%-Gly-Lys (Roc)-Le
s-5er-Gin-Glw (OTB)-Leu-H
is-Lye (Boc) -Lew-Gin-Thr
-Tyr-pro-Arg-OEtThe tetracosapeptide 7409 obtained in Example 4 was dissolved in 50-
Add this solution to 5101Ili+ of iodine and 50r of methanol.
The solution was added dropwise to the solution dissolved in 20°C while stirring at 20°C. 5
After a minute, cool on ice, add IN-sodium thiosulfate dropwise to decolorize, and pour into 1 liter of ice water. The resulting precipitate is collected by centrifugation, dissolved in ethanol, and concentrated. Finally, dissolve in ethanol and add ether, and dry the precipitate several times to obtain the title target compound 52019 (sa%). (a
) ”n + t 9.s '(' =, DMF)
.

This compound was found to be identical to the product of Example 2 from analysis by high performance liquid chromatography and thin layer chromatography.

Example 6 -1'al-Law-Gly-Lys (Boc) -
1, stt, -5er-Gln-Gls (OTB)
-Law-His-Lye (Roe) -Lau-G
ln-Thr-Tyr-Pro-Arg-OH Example 5
Tetracosapeptide 100~ obtained in DNllm and α
Dissolve in 5M Tris-HCl buffer (pH No.) α6-, add trypsin 4IR9, and react at room temperature for 1 hour. The reaction solution was directly transferred to Sephadex LH-20 (2,5
Load the column onto a 10.0× column and elute with DMFlo, 5 molar acetic acid (1:1). Both desired fractions were collected, concentrated under reduced pressure, and washed with ether to obtain the desired title compound 9011.
is obtained as a white powder.

(α) , , o 10(, 2° (C-1, DMF).

Example 7 Val-Lerb-Gly-Lys-Leu-5er-
Gln-Glw-Le? b-His-Lys-Ltt
w-Gln-Thr-Tyr-Pro-Arg-OEt The tetracosapeptide IQ obtained in Example 5 was added to anisole 3
Tn1. The solution was dissolved in 30 ml of trifluoroacetic acid containing the following ingredients, left at room temperature for 30 minutes, and then concentrated under reduced pressure.

Pour the residue into 30 tl of 0.2N acetic acid! Dissolved in ether 50
Add - and mix. After separating the aqueous layer and concentrating it under reduced pressure to about half its volume, Sephadex G-25 (45xloOm) was used.
The desired fractions were collected and concentrated under reduced pressure, and powdered with ethanol/ether to obtain the title membrane-free tetracosapenotide ester 730.

[α'l ”n −1a 5° (C=0.25, so
%AcOH).

Example 8 Vat-Law-Crly-Lys-Lttu-5g
r-Gl n-Glw-Lgu-His-Lys-L
eu-Gln-Thr-Tyr-pro-Arg-OH The protected tetracosapeptide 9QIv obtained in Example 6 was dissolved in trifluoroacetic acid containing anisole III+/, reacted for 40 minutes at room temperature, and then concentrated under reduced pressure. Wash the residue with ether. This is dissolved in 0.2N acetic acid, mixed with Sephadex G-25 (2.5 x 100 anam), and the desired two fractions are collected and lyophilized to obtain the title completely unprotected tetracosapeptide 789.

Reference example @51 25-32(, () Z Thr-Aan-T
hr-Gty-5er-Gly-Thr-pro-N
H.

Z −T h r −A s x −T h r −G
ly-NHNH.

Dissolve 5.4 g in I) MF50d, cool to -20°C, add 20d of 27ON hydrogen chloride/ethyl acetate and nitrite inaminium #L55-, keep at -20°C and stir for 10 minutes, then lower to -40°C. Cooled triethylamine 7.56-
Add to neutralize. Then H-5er-Gly-Thr
-Pro-NH13,7TQt-DMFloyd was added to the solution and stirred overnight under water cooling. The precipitated triethylamine hydrochloride is separated and concentrated under reduced pressure, and the residue is repeatedly reprecipitated from methanol to obtain the target product hsg.

(:α)%”-azoo (c; 1, DMF).

Reference example (4425-12 (B, l H-Thr-Asn-Th
r-Gly-5er-Gly-Thr-p r o −
NH, -AcOH Reference example @ 2 g of protected octapeptide amide obtained in 80
Catalytic reduction is carried out in the presence of 4 g of 5% palladium-carbon α in 40% acetic acid. After the reaction is completed, the catalyst is separated and dried under reduced pressure, and the residue is washed with ether to obtain the target product LTg in powder form. (α
,)"p-4ta" (c=2.80% AcOH).

Reference example @'6 1-sz [A″l Boc-Cys-5ur-
Val-Law-Gly-Lyal, B.

c) -Leg-5er-C; in-Glu(OTB)-
Law-His-Lytt (Boc)-Law-Gi
n-Thr-Tyr-Pro'-Arg-Thr-Aa
n-Thr-Gig-5ir-G111-Thr-Pr
o-NH.

The protected tetracosapeptide 160 Da obtained in Example 2 and the reference example @ boiled octapeptide amide 315 Da were mixed in DMF.
15 meters! Dissolve in trypsin 10~ and rpcxo
, xINg in α3 mohetris hydrochloride buffer (p#
aO) Add LO- and stir at room temperature for L5 hours. The reaction was stopped by adding 2 m of glacial acetic acid, and 1) after adding MF 2-, it was loaded onto a Sephadex LH-20 (L5 x 100 m) O column and extracted with DMF/α5 molar acetic acid (1:1). The desired fractions are collected and concentrated under reduced pressure, and the residue is washed with ether to obtain the desired white powder of protected dotriacontapeptide amide '12119. [:cr:1%”-
316° (c=2, DMF/H20 (7:3)).

Val-Law-Gly-Lye-Law-5er-G
ln-GLu-Law-H4s-Lye-Lew-Gl
n-Thr-Tyr-pro-Arg-Thr-Aan
-Thr-Gly-5ttr-Gly-Thr-pr
o -NH2 Reference example (100 μl of protected dotriacontapeptide amide obtained in April was mixed with trifluoroacetic acid and
5- and left at room temperature for 45 minutes, ether was added to obtain a precipitate of about 10019. This is dissolved in IN acetic acid and passed through a small column of Amberlite IRA-410 (acetic acid form) and the effluent is lyophilized. This was dissolved in 0.2N acetic acid, treated with P# (P) using Sephadex G-25 (3,8 x 50 cIn), and the desired two portions were lyophilized to obtain the desired title dotriacontapeptide amide 55.

(α)”, j-4LO° (C=1.50%Ac0H
).

Amino acid analysis: L 78 L 99 (2), Hi a LO1 (1)
, A rQ L 01 (1), A a p Z 0
? (2), T h r 467 (5), S # r
3.61 (4), Glu&03 (+1), Prozt
s(2), Gig2.92(3), Cv a O, 91
(1), Val 1.02 (1), Le u s,
o o (s), T vrl, OO (1).

Patent applicant: Teikoku Zoki Seiyaku Co., Ltd. Procedural amendment (spontaneous) February 7, 1985 Manabu Shiga, Commissioner of the Patent Office Indication of 1981 patent application No. 268537 2, title of invention Novel peptide 3, Relationship with the case of the person making the amendment Patent applicant address 4th representative, 5'41 Akasaka 2-chome, Minato-ku, Tokyo 107 Phone: 585-2258 (and 1 other person) 6. Subject of amendment (1) Specification No. On page 7, line 9, there is “I” (2) (2) On page 10, line 7, there is [-NH-CH, -CO-
J and (3) In the second line from the bottom of page 12, the phrase "N component" is corrected to "N component."

(4) In the 8th line of page 16 of the same document, the phrase ``the present invention'' is corrected to ``method J''.

(5) On page 23, line 5 [Y2. Toaruwo-Thr
-j “Y, correct it as 1.

-Thγ-'' (6) r Ar g: (A'O
t) Correct J to IrArQ (A'02) j.

(7) On page 46, line 8 of the same page, replace r(Aem)J with F.
(AC Bait)” I am corrected.

(8) rTyr on page 53, line 5 of the same page says “Tv8”
Correct it with J.

(9) In the third line from the bottom of page 65, after the words [in the order of water], add ``wash''.

顛 On page 68, line 6 of the same page, replace “-TH” with f −Th.
Correct it as j.

αυ In the second line of page 69 of the same page, the text "Leu 5er-" is corrected to "-Lerb-5ttr-".

α2 On page 78, line 6 of the same page, replace the text "(C2," with 'I(
Correct c=1, j.

Ren 3 On page 81, line 2 of the same page, it says “obtain” and then “[α
)”-13,2° (C=0.25.50chi ACOR)
. ” to join.

α.W.
Correct it to Z-Thr-J.

On page 82, line 3 from the bottom, there is “Ltts-(Bo J
Correct the statement as [”Lys(Bo j).

Written amendment to the above procedure (c) June 27, 1985 Manabu Shiga, Director General of the Patent Office1, Indication of the case 1982 Patent Review No. 2685372, Title of the invention Novel peptide3, Person making the amendment Relationship Patent applicant address: 2-5-1-5 Akasaka, Minato-ku, Tokyo; date of amendment order: B-Ichikuchi-O-Yog-me 2-trillion (issued on the 7th day) as shown in the attached sheet.

(1) On page 12 of the specification, lines 6-8 [("THE...
),” [1 “The Enzymes” Volume ■ (
“T HE E N Z Y M E S” + V
of, n[), 746 pages, 1971 Academic
Press (ACADEMIC PRESS publication reference)],
I am corrected.

(2) On page 1.9, line 7, “He1v, Chim,
ActaJ and Aruo [Helveti Power, Kimi Power, Acta (
Helv.

Chim, Acta)j.

(3) On page 19, line 9, “J-Amer, Chem
.

Sac, , J and ``Journal of the American Chemical Society (J, Amer.

Chem, Soc, ), correct J.

(4) In the 4th to 2nd lines from just below No. 19, “E, Sc1+r
Published by E. Schr6der and Kay Lübke.
K, Lubke) Wr.
Volume 1, pp. 3-75, 196
5 years academic press (A cade+nic P
ress) issued,” is corrected.

(5) On page 20, lines 10-11, “”TheP ep
t : de's” J is corrected to “[The Peptides”].

that's all

Claims (1)

[Claims] Formula % Formula % In the formula, R represents a hydrogen atom or a lower alkyl group, and xl
and X each represent a thiol protecting group, or Xl and X together represent a single bond, or a functional group-protected derivative thereof.