JPS6023680B2 - Decapeptide derivatives and their production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to decaptide derivatives and methods for their production.

More specifically, the present invention is based on the general formula <・) [wherein, Often, Y3 is NQ or a carboxyl group protecting group, Z is H or a side chain hydroxyl group protecting group, or -S03× (× is K, N
represents an alkali metal such as a, or a base such as triethylamine or cyclohexylamine), Z2 represents H or a protecting group for the side chain hydroxyl group, and P,
Gin, Asp, T's, Thr, GIy, Trp, Me
t and Phe represent pyroglutamic acid, glutamine, aspartic acid, tyrosine, threonine, glycine, tryptophan, methionine, and phenylalanine residues, respectively], salts thereof, and methods for producing them.

The decaptide derivative represented by the general formula (1) is a new substance, and is used, for example, as a raw material for producing caerulein, which is known as a physiologically active peptide.

Cerulein is known as a physiologically active peptide, and its common name is ceruletide, diethylamine [chemical name: L-pyroglutaminyl-L-glutaminyl-L-as/glutyl-○-sulfo-L-tyrosyl-L-threonyl-glysilyl-L] It is a common name for tryptophanyl-L-methionyl-L-as/glutyl-L-phenylalaninamide tridiethylamine salt trihydrate, and is a useful substance as a biliary tract and biliary function test drug.

Conventionally, the formula Pyr-Gin has been used to produce cellulein.
Tetrabeptide azide represented by -Asp-Tyr-N3 is condensed with hexabebutide represented by the formula (wherein Ac represents an acyl residue of a fatty acid having 1 to 4 carbon atoms); Decabeptide shown by is obtained and treated with anhydrous pyridine-hydro-sulfuric acid complex at low temperature to give the formula ? y
r-Gin-Asp-Tyr(S03H)-Thr(A
c) -GIy-Trp-Met-Asp-Phe-NH
A method is known in which cerulein is obtained by obtaining 2 and subjecting it to alkaline hydrolysis under mild conditions (Japanese Patent Publication No. 44-2507y).

This conventional method has drawbacks such as racemization, occurrence of side reactions, and difficulty in purifying the target product, and there is a desire to develop an even better method for producing cerulein.

As a result of various studies on decaptide conductors, the present inventors found that, for example, in the general formula (1),
Penzyloxycarbonyl group (Z), Y, = Y2 = penzyl group (Bzl), Y3 = NH2, Z = S03 days, Z2
Decabeptide derivatives that are two Bzl {[Z-Pyr-G
in-Asp(0&1)-Tyr(SQH)-Thr(
Bzl)-GIy-Trp-Met-Asp(OBzl
)-Phe-NH2]・(C2day5)2NH}[Decabebutide derivative A] When deprotection is performed, problems such as racemization and occurrence of side reactions in conventional methods are improved, and They found that caerulein could be purified easily. The decapeptide derivative A is a peptide derivative that simultaneously contains a methionine residue and a chemically unstable sulfate ester group in the hydroxyl group of a tyrosine residue. In the case of peptide derivatives, it is difficult to remove the protecting group by catalytic reduction, and there is no example of removing the protecting group by catalytic reduction from a peptide derivative containing both a methionine residue and a sulfate group. be. Surprisingly, the present inventors were able to selectively remove the protective group by catalytic reduction from decaptide derivative A containing sulfate esters at the same time in both methionine and tyrosine residues without removing the unstable sulfate esters. It was discovered that the desired caerulein could be obtained smoothly and in high yield by deprotecting it.

Originally, sulfate esters are extremely unstable to acids, and the commonly used removal of protecting groups with acids cannot be applied to the removal of protecting groups from peptide derivatives containing sulfate esters, so the conventional method described above is not applicable. In this method, a method for eliminating the ○-acyl group using an alkali is employed. However, under alkaline conditions, racemization of some amino acid residues and side reactions originating from aspartic acid residues cannot be avoided, making it difficult to obtain highly pure caerulein. In this respect, the synthesis of caerulein by catalytic reduction of a decaptide derivative represented by the general formula (1), for example, decaptide derivative A, has fewer side reactions, is easy to purify, and can yield highly pure and highly active cerulein. It is far above the law. Of course, among the decaptide derivatives represented by the general formula (1), for example, decaptide derivatives other than decaptide derivative A can also be deprotected by conventional methods in peptide chemistry, but in this case, a method that allows the S03 group to be eliminated is adopted. When this is done, for example, caerulein can be obtained by introducing an SQH group after deprotection. Below, the general formula (
The decaptide derivatives and salts shown in 1), their production methods, and the production method of caerulein from the decaptide derivatives in the present invention will be explained respectively.

The decaptide derivative in the present invention is a novel substance represented by general formula (1).

In the general formula (1), Protecting groups for amino groups such as butyloxycarbonyl group (c) and tosyl group (Tos), Y, Y2 may be the same or different, and include methyl and ethyl esters, benzyl esters, t-butyl esters, P-nitro Carboxyl group protecting group such as benzyl ester, Y3 is N ratio or Y,
, Y2 is a carboxyl group protecting group, and Z is a side chain hydroxy group protecting group such as acetyl, trifluoroacetyl, benzyl, t-butyl, trityl, etc., or -S03
× (× means an alkali metal such as K or Na or a base such as triethylamine or cyclohexylamine), Z2 is H or a side chain hydroxyl group such as acetyl, trifluoroacetyl, benzyl, t-butyl, trityl, etc. The protecting groups are shown respectively. The decaptide derivative represented by the general formula (1) has protective groups (X,, Y,, Y2, Y3
, B, Z2), depending on the type, a representative example is the general formula (1) where X,=Z(
benzyloxycarbonyl group), Y,=Y2=Bzl(
benzyl group), Y3=NH2, Z,=day, Z=Bzl(
benzyl group).

The physical and chemical substances of the above substance (hereinafter referred to as decaptide derivative B) are as follows.

{1) Melting point 202-20400 {2) [Q] Crab=1260 (N・N-dimethylformamide, C=0.098)'31 Elemental analysis value (%) C day N Analysis value 61.52 5.83 10.60 calculated value
61.64 5.90 10.74 [4' amino acid analysis value (carp in HCI, 2 unique hydrolysis at 11000) A
sp (2.0) (the number indicates the number of molecules), Thr (1
．． 1), Gin (2.0), GIy (1.2), Met
(0.8), Tyr (0.9), Phe (1.2).

Furthermore, other decaptide derivatives include the above-mentioned decaptide derivatives.

The decaptide derivative represented by general formula (1) can be produced by the following method.

That is, general formula (0) (wherein, X, Y, Z have the same meanings as above, and Y4 is O
H or a carboxyl group protecting group such as methyl ester or ethyl ester); and a hexabeptide derivative represented by the general formula (m) (wherein Y2, Y3, and Z are the same as defined above). Alternatively, by fragment condensation with an acid salt thereof, a decaptide derivative represented by the general formula (1) can be obtained.

The tetrapeptide derivative represented by the general formula (0), which is used as a raw material, is a new substance, and representative examples thereof include, in the general formula (b), =penzyl group (Bzl), Y4=O
CH3(OMe), Z, = day, i.e., the tetrapeptide derivative represented by the formula (tetrapeptide derivative 1
) can be given.

This tetrapeptide derivative 1 can be synthesized by a conventional peptide synthesis method, and specifically can be produced by the method shown in Reference Example 1 below. Tetrapeptide derivatives other than tetrapeptide derivative 1 can also be produced according to Reference Example 1. Also,
The hexabeptide derivative represented by the general formula (m) used as the other raw material is also a new substance, and representative examples thereof include, in the general formula (m), Y2=penzyl group (Bzl), Y3: N ratio, Z2=penzyl group (Bzl
), that is, hexabeptide derivatives represented by the formula (hexabeptide derivative (1)).

This hexabeptide derivative 1 can be synthesized by a conventional peptide synthesis method, and specifically can be produced by the method shown in Reference Example 2 below.
Hexabeptide derivatives other than Hexabeptide Derivative 1 can also be produced according to Reference Example 2. In the fragment condensation for producing the decaptide derivative in the present invention, the hexabeptide derivative represented by the general formula (m) is used in equimolar to 2 times the molar amount of the tetrabeptide derivative represented by the general formula (D). By this, the desired decaptide derivative can be effectively obtained.

The reaction is carried out by adding 0.1 to 0.5 mmol of a tetrapeptide derivative or a hexabeptide derivative or a salt thereof in an aqueous medium with a pH of 2.0 to 11.0 in the presence of a proteinase at a temperature that does not deactivate oxygen, usually at room temperature. Fragment condensation is carried out for about several tens of seconds to 2 seconds, or according to a conventional peptide synthesis method.

In the method using oxygen, the proteinase is
For example, acidic protease NO. 306 [Agr. B
iol. Chem. 39, 423 (1975)] (Ac
rocylindrimm species, manufactured by Kyowa Hakko Kogyo KK),
Pepsin, thermolysin, prolysin, trypsin,
Any acidic, neutral, or alkaline protease such as Q-chymotrypsin or papain can be used. In particular, in the present invention, as shown in Examples below, proteases of microbial origin, whose use in fragment condensation has not been known in the past, can be effectively used. The amount of proteinase used can be arbitrarily determined depending on the reaction raw materials and the like. The reaction proceeds smoothly in an aqueous solvent containing water or a water-compatible organic solvent such as methanol, ethanol, dioxane, NON-dimethylformamide, etc., and the product is usually obtained as a precipitate. To recover the product (decabeptide derivative) from the reactant, the precipitate is collected by a conventional method and washed with a weakly acidic aqueous solution, a weakly alkaline aqueous solution, and water as appropriate to obtain a high-purity target product. be able to.

Furthermore, the decabeptide derivative represented by the general formula (1) is represented by the general formula (W) (wherein Y5 is OH or a protecting group for a carboxy group such as methyl ester or ethyl ester, and other symbols are the same as above). It can also be produced by fragment condensation of a putabeptide derivative and a tribetide derivative represented by general formula (V) or an acid salt thereof under the same conditions as the method described above.

The heptabeptide derivative represented by the general formula (W) is a new substance, and a representative example thereof is the heptabeptide derivative represented by the formula (heptabeptide derivative 1)
can be given.

This heptabebutide derivative 1 can be synthesized by a conventional peptide synthesis method, and specifically can be produced by the method shown in Reference Example 3 below. Heptabeptide derivatives other than heptabeptide derivative 1 can also be produced according to Reference Example 3. As the tribeptide derivative represented by the general formula (V),
For example, (tribeptide derivative 1) is mentioned.

This tribeptide derivative 1 is a known substance
ofChemicaISocietyP. 555 (19
66)], and can be produced, for example, by the method shown in Reference Example 2 below. Products other than the above-mentioned tribeptide derivative 1 can also be produced according to the method of Reference Example 2. Fragment condensation of the heptabeptide derivative represented by the general formula (W) and the tribetide derivative represented by the general formula (V) or its acid salt is
It can be carried out according to the conditions for condensation of the tetrapeptide derivative and hexabeptide derivative described above.

In the production of decaptide derivatives in the present invention, when fragment condensation is performed without using an enzyme, a conventional condensation method used for peptide synthesis can be used.

The decaptide derivative thus obtained can be easily converted to caerulein, for example, by the method exemplified in Reference Example 4 below.

Examples and reference examples are shown below.

Example 1 Z-Pyr-Gin-Asp(OBzl)-Tyr (Reference Example 1) 189 criminal 9 (0-25 skin mole) and Th
r(Bzl)-GIy-Trp-Met-Asp(OB
zl) -Phe-NH2.trifluoroacetate (Reference Example 2) 9 of 260 (0.25 mole) was dissolved in N.N-dimethylformamide (DMF) and the solution was adjusted to pH 4.
0.0 of 0.2M acetate buffer 5' and INNaOHO. 25
After adding acid protease No. 306
(AcrocylindrMm species, Kyowa Hakko Kogyo K.K.
9 of 20 was added and shaken at 30 qo for one period.

The white precipitate thus precipitated is taken out by the furnace, and this is
．． It was washed with 8K citric acid, water, 4% NaHCo3, and water in this order, and dried under vacuum. Next, this was dissolved in N·N-dimethylformamide 5', and ethyl acetate was added thereto. The thus produced next starch is collected in a furnace and vacuum dried to obtain the target product Z-PM-Gin-Asp(OBzl)-T.
yr-Thr(Bzl)-GIy-Trp-Met-A
An sp(OBzl)-Phe-N ratio of 9 of 15.7 was obtained. The physical and chemical properties of this product were as described above. Example 2 Z-Pyr-Gin-Asp(OBzl)-TM-OM
e (Reference Example 1) 9 of 193 (0.25 mole) and Thr(Bzl)-GIy-Trp-Met-Asp(
OBzl)-Phe/N trifluoro.

Acetate (Reference Example 2) 9 of 260 (0.25 mole
) was dissolved in Z of N・N-dimethylformamide 5, and I
N-NaOHO. 25', 0.2MCac120.5
After adding 5 parts of a 0.2 M Tris-HCl buffer solution, pH 9.0, 9 parts of Q-chymotrypsin (crystals) [manufactured by Worthington Co., Ltd., 10,000 U/9 (activity)] of 10 parts,
Shake at room temperature for about 5 minutes. India CIO. After adding 05' and stopping the reaction, 2
Adding twice the amount of water produces a white precipitate. This precipitate was taken out in a furnace, thoroughly washed with 0.8M citric acid, water, 4% NaHCo3, and water in that order, and then dissolved in N.N-dimethylformamide and added with ethyl acetate to form a pillow dens. The product was collected in an oven and dried under vacuum to obtain the same target product 131M as in Example 1 (yield 31%). Example 3 Z-Pyr-Gin-Asp (OBzl)-Tyr (reference example) 304-9-Thr (BZ1)-G1y-Tr
p-Met-ASp(OBzl)-Phe-NH2.Trifluoroacetate (Reference Example 2) 9 of 388 was dissolved in 4 of N-N-dimethylformamide, and 0.4 of methylmorpholine [(0 Add .405 evening), 11o
Cool to o0 and add 1-hydroxybenzotriazole 9
2 of 9, N.

Add N'-dicyclohexylcarbodiimide (92) and incubate for 2 hours. Then, I left it in the refrigerator for 4 hours. The resulting insoluble material (N·N'-dicyclohexylurea) was removed from the furnace, 20 parts of ethyl acetate was added dropwise to the furnace solution, and the mixture was left in a cold room for 1 hour. The thus-formed starch was taken out of the furnace and washed with about 100% of a 0.9M citric acid aqueous solution, then sequentially washed with water, 4% NaHC03, and 50% of water, then washed with 10% of ether and dried. do. A decaptide derivative B similar to that in Example 1 is thus obtained. Yield: 9 of 360 (yield chord %). Example 4Z-Py
r-Gin-Melon p (OBZI)-TM-Thr (Bzl
)-GIy-Trp-OMe (Reference Example 3 below) 589 females (0.05 mole) and Met-Asp (OBzl
)-Phe-NQ・HC1 (Reference Example 2 described later) 26.8 ridges 9 (0.05 mmole) were dissolved in 1 part of N・N-dimethylformamide, and 0.2M CaC120 was added to this solution.
．． 0.05' and 0.2M Tris-HCl buffer, pH 9.0 1
After addition of chlorine, Q-chymotrypsin 25c was further added, the reaction was carried out at room temperature with vigorous shaking for 10 minutes, and decabeptide derivative B was prepared from the reaction solution in the same manner as in Example 1.
got a 9 of 4.7.

Yield 10%. Example 5 The same Z-PM-Gi as in Example 4 was used as a raw material.
n-Asp(OBzl)-Tyr 1cho hr(Bzl)-GIy-Trp-〇Me and Met-Asp(〇Bzl)
Using -Phe-NH2.HCI, reaction and purification were carried out according to the synthesis method in Example 3 to obtain the same decaptide derivative B as in Example 1.

Reference example 1 Z-PM-Ginichiro p (OBZI)-TM-OMe and Z-Pyr-Gin-Asp (OBzl)-Tyr
Synthesis: Kawa Z-PM-Gin-OH (manufacturing method is described later) 3
．． 91 Yu (10 moles) and Niichi Asp (OBzl)
)-TM-OMe trifluoroacetate [R. Day. Ma
zur, J. M. Schlatter and AH.
Gbldkamp, J. A. C. S. 91, 2684gu 69)] 5.15 evening (low mole) N●N-
Dissolve in 15 parts of dimethylformamide.

Add 1.01 g of methylmorpholine to this solution,
After cooling to 0.00C, 2.30 hours of 1-hydroxybenzotriazole and 2.27 hours of N.N'-dicyclohexylcarbodiimide (DCC) were added, and the mixture was left overnight. The resulting insoluble matter (N・N'-dissic.hexylurea) was removed, and the supernatant liquid obtained in this way was diluted with 0.3M citric acid 10
Drip into 0'. The white starch produced in this way was collected in a furnace, thoroughly washed with 0.8M citric acid, water, 4% NaHC03, and water in that order, and then dissolved in N·N-dimethylformamide and water was added to crystallize it. Z-Pyte-Gin-Asp
(OBzl)-TM-OMe was obtained. Yield 4.54 evenings (
yield 59%). The melting point of this tetrapeptide derivative is 19
9-20r○, [Q] Color 2 = 142.90 (N-N-dimethylformamide, C = 2.04) Elemental analysis value (%)
are analytical values C: 60.50, H: 5.71, N: 9.11
, calculated value C: 60.54, H: 5.6, N: 9.0
5, and the infrared absorption spectrum (based on the KB Kamehishi method) (hereinafter referred to as IR spectrum) is shown in FIG. 1, and the nuclear magnetic resonance spectrum (NMR spectrum) is shown in FIG.

‘.

)Z-Pyr-Gin-Asp(OBzl)-Tyr-
OMe to Z-Pyr-Gin-Asp(OBzl)-
Conversion to Tyr was performed as follows. Z-Pyr
-Gin-pine p (OBZI)-Tyr-OMe2.50
The solution (3.2 moles) was dissolved in 50 moles of N.N-dimethylformamide and mixed with 50 moles of 0.2 M Tris-HCl buffer, pH 8.7.

Subtilisin BPN' (trade name: Nagase, manufactured by Nagase Sangyo Co., Ltd.) 20 parts 9 was added to this, and after standing at room temperature for several minutes, P
When H is adjusted to 2 with a state CI of 2, white makuraden is produced. This was taken out of the furnace, thoroughly washed with water, and then recrystallized from hot methanol to obtain Z-PM-Gin-Asp (OB
zl)-Tyr was obtained. Yield: 2.35 yen (yield: 97%)
. The physical and chemical substances of this substance are as follows. (11 Melting point 203-204℃ [21 [Q] Customer = -25.30 (
N.N-dimethylformamide, C=1.02)'31
Elemental analysis value (%) C day N analysis value 59.83 5.45 9.06 calculated value
60.07 5.44 9.22 [4] The IR spectrum is shown in Figure 3.

The '51 NMR spectrum is shown in FIG.

Shiichi Synthesis of Z-PM-Gin-OH: Z-PM-OH
25 evening, N-oxysuccinimide 10.91 evening, N.
To 19.57 ml of N'-dicyclohexylcarbodiimide, 175 ml of dioxane and 25 ml of chloroform were added, and the mixture was stirred overnight while cooling to 110 qo.

The produced insoluble matter (N-N'-dicyclohexylurea) is removed by furnace, the furnace liquid is evaporated to dryness, the residue is dissolved in ethyl acetate, the insoluble matter is removed, and the furnace liquid is concentrated to obtain crystals. Ta. Yield: 27.1 evenings (yield: 79.2%). The entire amount of the crystals obtained above was dissolved in 400% acetonitrile, and 13% of Gin (glutamine) was added to this solution. Madarayu and NaHCo
Add dropwise an aqueous solution containing 37.98 ml and let stand overnight at room temperature.

Then, on the same day, the pH of this reaction solution was adjusted to 1 using CI and concentrated to produce a white precipitate. The precipitate was kept on ice overnight, then filtered and the crystals were purified with ether. The crystals were dissolved in hot methanol, kept under ice cooling overnight, heated in an oven, and concentrated under reduced pressure to obtain 15.7
62 (yield 54.0%) of Z-PM-Gin-OH was obtained. The physical and chemical properties of this product were as follows. '1
' Melting point 181-18 is 0 [2} [Q] Cost = -17.70 (N-N-dimethylformamide, C = 1.01) {3' Elemental analysis value (%) C day N Analysis value 54.96 5.49 10.52 Calculated value
55.24 5.41 10.74 Reference example 2 (E Thr (Bzi) - GIy - Trp - Met - Asp
(OBzl)-Synthesis of Phe/NH2: Boc-Thr
(Bzl)-GIy-Trp (produced by the method described below)
524 o (1 mole) and Met-pine p (OBz
l) -Phe-NH2.HC1 (manufactured by the method described below) 537 o (lm mole) was dissolved in N-N-dimethylformamide 2', INNaOHI', 0
．． After adding 0.2 M Ca (OCOC melon) 21 [, pH 7.0 Mcnvaine buffer solution], thermolysin (neutral protease) (manufactured by Daiwa Kasei Co., Ltd.) (9390p) was added.
u/9) 200 9 was added and shaken at 30 pm for 20 hours.

The white precipitate thus precipitated was collected in a furnace and the precipitate was reduced to 0.
It was purified in the order of 9M citric acid, water, 4% NaHC03, and water, then dissolved in N.N-dimethylformamide, and crystallized by adding ethyl acetate.
)-Gin-Trp-Met-Asp(OBzl)-P
he-NH2 was obtained. Yield: 461 (yield: 45%).
The physical and chemical properties of this substance are as follows. (1' Melting point 204-205 o C ■ [Q] Deprivation = -26.9o (N.N-dimethylformamide, C = 1) '3' Elemental analysis value (%) C day N Analysis value 62.53 6. 42 10.61 calculated value
62.41 6.51 10.62■ Amino acid analysis value (in carp HCI, 2 unique hydrolysis at 110qo) As
p(1.03) (molecule), Thr(1.0), GIy(
1.0), Met (0.86), Phe (1. female).

[B'8X-Thr(Bzl)-GIy-Trp-Me
Thr-(B
zl)-GIy-Trp-Met-Asp(OBzl)
-Synthesis of Phe/NH2: BM-Thr(Bzl)-G
Iy-Trp-Met-Asp(0&1)-Phe-N
Dissolve 0.51 ml of formic acid in 50% of formic acid and soak at room temperature for 2 hours.

The mixture was then evaporated to dryness under reduced pressure at room temperature, and ether (anhydrous) was added to the remaining groove to form a powder, which was then dried under vacuum. Thus Thr(Bzl)-GIy-Trp-Met-A
sp(OBzl)-Phe-N ratio/HCOOH was obtained.
Yield: 0.36 hours (yield: 74%). The IR spectrum of this product is shown in FIG.

Synthesis of Shiichi BX-Thr(Bzl)-GIy-hp:
2.43% of European c-Thr(Bzl)-GIy-Trp-OMe (see below) was dissolved in 25% of N.N-dimethylformamide, and 0.2M Tris-HCl buffer (pH 8) was added to this.
．． 8) Add 50M, and further add 10 females of Nagase (manufactured by Nagase Sangyo Co., Ltd.) (102 x 1 pi PU/9), and stir at room temperature for about 2 hours. Then, the pH of the solution was adjusted to C on the same day under ice cooling.
The solution was adjusted to 2 with I and extracted with 500 ml of ethyl acetate. The extracted ethyl acetate layer is dried with anhydrous sodium sulfate. The ethyl acetate layer is then concentrated and triturated with anhydrous ether.

The resulting white powder is filtered and dried under vacuum, resulting in BX
-Thr(Bzl)-GIy-Trp is obtained. Yield: 1.77 hours (yield: 55.7%). The IR spectrum and NMR spectrum of this product are shown in FIGS. 6 and 7, respectively.

B B ratio-Thr(Bzl)-GIy-Trp-OMe
Synthesis: B ratio-GIy-OH0,350 evening, day one Trp
1OMe. Hcl o. 473 was dissolved in N-N-dimethylformamide of 5, and to this was added 0.110 of N-methylmorpholine, 0.230 of hydroxybenzotriazole, and DCCO. Add 277 evenings and worship at -15℃ for 2 hours.

Then, the mixture was left to stand overnight at room temperature, and the insoluble matter (N·N'-dicyclohexylurea) formed was filtered off. The furnace solution was concentrated to dryness and the remaining residue was dissolved in ethyl acetate.Then, the ethyl acetate solution was diluted with 0.9 M citric acid solution, water, 4% NaHCo3,
Wash the rice bran with water.

The ethyl acetate solution is dried over anhydrous sodium sulfate, then concentrated and triturated with anhydrous ether. The resulting precipitate was placed in a furnace and dried under vacuum to form &c-G
Iy-Trp-OMe was obtained. Yield: 0.464 min (yield: 65.1%). The physical and chemical properties of this substance are as follows. '11 Melting point 13-1320 [2] [Q] Deprivation =
12-80 (methanol, C: 1-0) (3' elemental analysis value (%) C day N analysis value 60.76 6.78 11.18 calculated value
60.78 6.7111.19 &c-01y-Trp-OMe (approximately 0.464 m) obtained above is dissolved in 98% formic acid and stirred for 2 hours at room temperature.

It is then evaporated to dryness under reduced pressure at room temperature, and then triturated with anhydrous ether. The resulting white powder is filtered, collected and dried under vacuum. Thus H-0
1y-Trp-OMe・HCOOHO. 321 yen (yield 84.6%) was obtained. Then, B obtained as above
oc-Thr (BZI)-OH3.09 evening, day GIy
Dissolve 2.91 parts of 1000 ml of OMe HCOOH in 20 parts of N-N-dimethylformamide, add 1.10 parts of N-methylmorpholine, 230 parts of hydroxybenzotriazole, and 2.27 parts of DCC to make 110 parts. I simulated Azusa for 5 hours.

Then, the mixture was incubated overnight at room temperature, and the resulting residue (N·N'-dicyclohexyl urea) was removed in an oven. The furnace solution was concentrated to dryness, the residue was dissolved in 200% ethyl acetate, and then 0.0% ethyl acetate was dissolved.
Refine in the following order: 9M citric acid, water, 4% NaHCo3, water. The ethyl acetate solution is dried over anhydrous sodium sulfate, then concentrated and triturated with anhydrous ether. The resulting white powder was separated by furnace and collected and dried under vacuum to give BM-Thr(Bzl)-GIy-hp-
Obtained OMe. Yield: 5.32 hours (yield: 98.8%). The physical and chemical properties of this substance are as follows.

{1} Melting point 224-225qo ■ [Q] Color 2 = 19.10 (methanol, C = 1-〇) {3' Elemental analysis value (%) C day N analysis value 69.32 10.9612.33 calculation value
69.31 10.6612.51 [4] The NMR spectrum is shown in FIG.

Thr(Bzl)-GI from Boc-Thr(string 1)-01y-Trp-OMe obtained in the above method
The synthesis of y-Trp-OMe follows the above rule c-GIy-Tr
This was carried out according to the method for synthesizing H-GIy-Trp-OMe from p-OMe.

■ Met-Asp(OBzl)-Phe-N ratio/Hc
Synthesis of l: Pmz-Met-Asp(OBzl)-Ph
e-NH2 (synthesized by the method described below) 0.405 m to 1
0w'3. Dissolve in HCl-ethyl acetate and stir at room temperature for about 1 hour.

The solution is then concentrated and the concentrate is triturated with anhydrous ether. When the obtained powder is filtered and dried in vacuum, Met-Asp(OBzl)-Phe-NH2.H
clo. 286 units (yield 87.4%) are obtained. The physical and chemical properties of this substance are as follows.

'11 Melting point 201-2020'21 [Q] Color 2=
-8. ? (N.

N-Dimethylformamide, C 1.0)'31 Elemental analysis value (%) C day N Analysis value 55.64 6.16 10.23 Calculated value
55.91 6.19 10.43'41 The NMR spectrum is shown in FIG.

N PmseMet-Asp(OBzl)-Phe-N
Synthesis of H2: Pmz-Asp(OBzl)-OH3.7
0 evening, H-Phe-NH21.64 evening, 10'N.
Dissolve N-dimethylformamide, add 2.30 hours of hydroxybenzotriazole and 2.27 hours of N.N'-dicyclohexylcarbodiimide, and make 100ml of N-dimethylformamide.
I prayed for about 4 hours.

Next, I worshiped the hawk overnight at room temperature. The generated insoluble matter is removed from the furnace, and the furnace liquid is concentrated at room temperature. Approximately 300 people remain [
of ethyl acetate, and the ethyl acetate solution was dissolved in 300' of 0.9 M citric acid aqueous solution, 400' of water, 300' of water.
Wash with 4% NaHco3 solution and 400 ml of water. Next, this ethyl acetate solution is dried with anhydrous sodium sulfate, concentrated to dryness, and the remaining bamboo is dissolved in about 40 ml of hot methanol. If this solution is stored in the refrigerator (5°C) overnight, crystals will crystallize. This is passed through an oven and dried under vacuum.
Yield 3. Banyu (yield 77.2%). Crystal obtained above 3. The scales were dissolved in a mixed solution of 40 parts trifluoroacetic acid and 8 parts anisole, and stirred at room temperature for about 1 hour. The solution is then concentrated at room temperature. The concentrated residue was triturated with 200 ml of anhydrous ether, and the resulting crystals were filtered and dried under vacuum.
)-Phe-N ratio/trifluoroacetate is obtained. Yield 93.4%. The NMR spectrum is shown in FIG.

H-rudder p(OBzl)-Phe N France obtained above
Trifluoroacetate 0.467m, P blood-Met-OH
O. 296, N-methylmorpholine 0.110',
Hydroxybenztriazole 0.230m, DCO
O. On the evening of 227, add DMF5.0 and heat for 1 hour at -10℃.

The generated insoluble matter is removed from the furnace, and the furnace liquid is concentrated at room temperature. The residue was dissolved in 100% ethyl acetate and 200% ethyl acetate.
9M citric acid aqueous solution, 200' water, 4% of 200' water
The mixture is washed with aqueous sodium bicarbonate and 200 ml of water, and then the ethyl acetate layer is dried over anhydrous sodium sulfate and concentrated. 10% concentrated residual bamboo
When dissolved in hot methanol and stored overnight in the refrigerator (15°C), it crystallizes. The crystals are filtered and dried under vacuum. When recrystallization is repeated in the same manner with 1 methanol hot methanol, 0.405 pm of Pm Lee Met-Asp (OBzl) is obtained.
-Phe-N ratio is obtained.

(Yield: 60.9%) The physicochemical properties of this product are as follows.

[11 Melting point 181-1820'21 [Q] Color 2:
114.5o (methanol, C=1-〇)'3l Elemental analysis value (%) C day N Analysis value 61.45 6.02 8.43 Calculated value
61.36 6.01 8.52 Reference example 3Z-Pyr
-Gin-Asp(OBzl)-Tyr-Thr-(&
1) Synthesis of -GIy-Trp-OMe: Z-Pyr-G
in-Asp(OBzl)-Tyr (Reference Example 1) 76 females (0.1 mole) and Thr(Bzl)-GIy-
Trp-OMe・P-toluenesulfonate (Reference Example 2
) Mouse (0.1 mole) was dissolved in 0.6' of N.N-dimethylformamide, and 1.2' of Mcllvaine buffer, pH 4.0, was added to this solution, and then acidic protease No. Add 30610 females, 300
The reaction was carried out under shaking at 0 for 1 hour.

The white starch precipitated in this way was taken out by the furnace, and it was mixed with 0.8M
Wash your knees in the following order: citric acid, 4% NaHCo3 water, and water.
Column chromatography was performed using Sephadex LH-20 equilibrated with N-dimethylformamide, the active fraction was concentrated, and the residue was lyophilized to yield 9 of 40 (yield: 3
2.8%) Z-PM-Gin-$p(OBZI)-T
×-Thr (Side 1)-GIy-Trp-OMe・Day 20
I got it. The physical and chemical properties of this product were as follows.
{1) [Q] Customer = -5.60 (C: 1.0, N・N-
Dimethylformamide) {2} Elemental analysis value (%) C day N Analysis value 61.64 5.81 10.14 Calculated value
61.70 5.84 10.28 [3' amino acid analysis value (same day CI, 2 hours hydrolysis at 11000) G
・A2.09 (Molecular) Aspl. 06, Tyro. 6
5, Thrl. 00, GIyl. 00.

In the above method, mr(Bzl)-GIy-Trp
-Thr instead of -OMe.P-toluenesulfonate
The reaction was carried out in the same manner except that -GIy-Trp-OMe-P-toluenesulfonate was used, and Z-PM-Qn-A
sp(OBzl)-Tyr-Thr-GIy-Trp-
OMe.H20 was obtained.

The physical and chemical properties of this product were as follows. '1' Melting point 196-20100 [2] [Q] Poly=-14.70 (C: 1.0, N・N
(dimethylformamide)'3} Elemental analysis value (%) C day N Analysis value 59.02 5.59 10.82 Calculated value 59.20 5.77 11.09 Reference example 4 Production of caerulein from decabetide derivative B: decaceptide Derivative B (Example 1) 142 cape was dissolved in N.N-dimethylformamide (DMF) (anhydrous), 2 pyridine was added thereto, and then anhydrous pyridine-sulfuric anhydride complex (crystal) 800/9 A solution of DMen 4' and pyridine 4' was mixed and incubated at room temperature for 1 hour.

Thus, the hydroxyl group of the tyrosine residue of decaptide transferor B becomes a sulfate ester (SQH). Then, this reaction solution was mixed with 0.0% diethylamine. Neutralize by dropping Madarayu into a solution of 5 DMF.

The resulting gel-like precipitate is removed by centrifugation and treated. This supernatant solution is passed through a Sephadex LH-20 column equilibrated with N.N-dimethylformamide.

After collecting and concentrating the active fraction flowing out first, anhydrous ether (50%) was added to precipitate a white powder, which was collected and vacuum-dried to obtain [decabeptide transfer A] 13 product 9. Obtained.

Yield 90%. IR spectrum of decapeptide transfer A (
The characteristic absorption in Fig. 13) is 1250 and 1050.
Characteristically observed in Aku-1, the hydroxyl group of the tyrosine residue is S
It was confirmed that the condition was QH.

Decabeptide derivative A50 female, Pd-C (palladium carbon) 9 of 300, N.N-dimethylformamide 5
In addition to secretion, reduction is carried out for one period under hydrogen atmosphere, normal pressure, and room temperature. The solution portion is then taken out of the oven and passed through a column of Sephadex LH-20 (manufactured by Pharmacia) equilibrated with DMF. Add 10 parts of methanol to the first 30 active fractions (3 bottles of fractions No. 6-8 and 10). The resulting insoluble matter is then removed in the oven, and 10 parts of ether is added to the oven solution. The resulting makuden is collected by centrifugation. The precipitate is dissolved in water and freeze-dried. The cellulein thus obtained was dissolved in water, and an ion exchange resin [Amberlite CG-
50 (days)] for deionization.

36. The cerulein-containing aqueous solution thus obtained is concentrated and the residue is vacuum-dried. 9c (yield 90.7
%) product was obtained. The physicochemical properties of the obtained compound are as follows. [1' Elemental analysis value (%) CHN Analysis value 51.4 5.313.6 Calculated value 51.5 5.413.5 '21 The m spectrum is shown in FIG.

[31 The nuclear magnetic resonance spectrum is shown in Figure 12.

As the Rf value plate for '4' double-layer chromatography, use silica gel (manufactured by E. Merck), as the solvent ■ Chloroform: methanol: acetic acid: water = 1:1:0.1:
R of the standard sample in thin layer chromatography using a lower layer of 0.5 (volume ratio) and the product obtained by this method.
The f value is 0 (origin does not move), and ■Chloroform: methanol: 28% ammonia water: water = 1:1:0
．． Both Rf values using 1:0.1 are 0. (UV absorption and HBr/ninhydrin color development).

'51 Toyo Roshi No. 1 is used as furnace paper for electrophoresis. 51A, pyridine:acetic acid:water=3:6:141 (volume ratio) as a solvent, a voltage of 2.4 KV was used for about 2 hours, and the specimen showed three bands, and in this method, One band was observed in the product.

{6} Physiological activity test of caerulein (Bioassay
) ■ As the test drug, the product obtained in this example,
As a control, the test drug was dissolved in physiological saline to prepare a solution with a concentration of 100 years/skin (estimated value), and this was divided into melt-sealed ampoules as a stock solution and opened at the time of use. and used it.

@ The animal used is Har0ey female guinea pig 3
00 to 400 evening was used.

■ Test method: Accurately dilute the sample stock solution (1000 Muta titer/skin [estimated value] with physiological saline to give 0.2 Muta titer/I, 0.1 Buddha Misaki titer/Side (estimated value)). , respectively high volume sample solution (TH
) and low dose sample solution (TL).

The standard cerulein was prepared in the same way, and the cholecystectomy specimen and Magnus procedure were all performed in the “Bioassay of Cerulein” (Applied Pharmacology, Vol. 8).
65-870, 1974).

@ Test results Muta titer/guy Test drug (this product) 1149 Specimen (control) 1000

[Brief explanation of the drawing]

Figures 1 and 2 show Z-Pyr-Gin-Asp (OBz
l) IR spectrum and NMR of -TM-OMe, respectively
The spectrum is shown. Figures 3 and 4 show Z-PM-Gin-Asp (OBzl
) shows the IR spectrum and NMR spectrum of one Tyr. Figure 5 shows Thr(B2l)-Cly-Tr
IR spectrum of p-Met-trillion p(OBZI)-Phe-NH2.formate is shown. Figures 6 and 7 are 8X-
The IR spectrum and NM old spectrum of Thr(Bzl)-GIy-Trp are shown, respectively. Figure 8 shows BM-
The NMR spectrum of mr(Bzl)-GIy-Trp-OMe is shown. Figure 9 shows Met-Asp(OBzl)
-Phe-N ratio/Hcl NMR spectrum is shown. Figure 10 shows Asp(OBzl)-Phe・NH2・H
The NMR spectrum of cl is shown. FIG. 11 and FIG. 12 show the m-spectrum and NMR spectrum, respectively, of caerulein. Figure 13 shows the IR of decaptide derivative A.
The spectrum is shown. A picture of a box lord, a carp picture M, a picture of a shrimp, a figure of a doffuku V, a ball box heart moth. Illustration of a boat with a moat illustration, a warizatsu drawing, a snake drawing, a boat drawing, and a bear drawing boat.

Claims (1)

[Claims] 1 Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, Z represents a benzyloxycarbonyl group, and Bz
l represents a benzyl group, Z_1 represents H or SO_3H) or a salt thereof.