JPH07138298A - Production of sulfate derivative of hirudin analog or its salt - Google Patents

Abstract

PURPOSE:To obtain a new derivative, containing a specific amino acid sequence in a part thereof, capable of remarkably enhancing the antithrombin activities and good in mass productivity by sulfating phenolic hydroxyl group of the tyrosine residue in the molecule of a hirudin analog. CONSTITUTION:This new hirudin analog sulfate is obtained in a large amount with good reproducibility by treating a hirudin analog which is peptide, containing an amino acid sequence expressed by the formula (X1 is Pro or Glu; X2 is Glu, Glu-Asp, Glu-Tyr or Tyr) and having <=3000 molecular weight with reagents for protecting functional groups, protecting side chains and carboxyl group at the C-terminal contained in the hirudin analog with protecting groups, providing a substitution product of the hirudin analog with the protecting groups, then reacting phenolic hydroxyl group of the tyrosine residue contained in the substitution product of the hirudin analog with the protecting groups with a sulfur trioxide (SO3) complex, converting the phenolic hydroxyl group into sulfate and subsequently releasing the protecting groups. The resultant hirudin analog sulfate has remarkably enhanced antithrombin activities.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a hirudin analog showing high similarity to a part of the amino acid sequence of natural hirudin. In particular, it relates to a method for producing a hirudin analog which is similar to the amino acid partial sequence of the C-terminal region of natural hirudin and has a molecular weight of half or less of that of natural hirudin.

[0002]

Natural hirudin is a polypeptide having antithrombin activity, which is isolated from the secretion fluid from the salivary glands of medicinal leech. It is known that natural hirudin has three kinds of mutants whose amino acid sequences are similar to each other. Of the three variants, the first isolated one is called HV-1 with 65 amino acid residues. Also,
HV-1 has the same 65 amino acid residues as HV-1, and its amino acid sequence differs from HV-1 by 9 amino acids.
The variant called -2 also has 66 amino acid residues, which has one more amino acid than HV-2, and its N-terminal Ser
Up to ³² , the same as HV-2, at the 63rd position in the C-terminal region
When Ala is considered to be added, its amino acid sequence differs from HV-2 by 10 amino acids, HV-3.
Have been isolated as mutants, respectively. The native hirudin normally exists only once in its C-terminal region.
It is known that it is secreted as a sulfate ester form of the phenolic hydroxyl group of the Tyr residue. This sulfated form is reported to have about 10 times higher antithrombin activity than the non-sulfated form [PJ.
Braun et al. Biochemistry vol.27 p.6517-6522 (198
8)].

Hirudin is, due to its antithrombin activity,
A pharmacological action that inhibits or suppresses blood coagulation is expected, and its application as an anticoagulant is being promoted. However, since hirudin is a substance that does not exist in the body, an antibody against it is generated, which may induce various allergic symptoms caused by an antibody reaction. Further, in order to prevent the induction of allergic symptoms, it is necessary to limit the dose. The present inventors have found that hirudin analogs, that is, peptides consisting of an amino acid sequence similar to a part of the amino acid sequence possessed by hirudin and having a smaller molecular weight than hirudin, or a compound modified with a protective substituent thereof are It was found that a compound having a thrombin activity and a sulfated tyrosine residue of the above-mentioned hirudin analog had a high antithrombin activity, and was proposed as a compound having a novel antithrombin activity (Japanese Patent Application No. 3-63909). And International Patent Publication WO92 / 15610). Furthermore, in the invention of the above-mentioned prior application, as a method for producing a hirudin analog in which the tyrosine residue is sulfated (sulfate ester), a hirudin analog not previously sulfated is prepared, and the hirudin analog is sulfated. A method of esterification was proposed.

The inventors of the present invention continued their studies on this production method. According to the above method, in addition to the sulfated esterified product of the target hirudin analog, the amino acid located at the C-terminal of the peptide was It was found that the product converted into the optical isomer was also obtained as a by-product. This by-product itself is a compound having a high antithrombin activity, and is as useful as the target sulfated derivative of the hirudin analog, but when both are mixed, it is applied as a medicine. Therefore, it has been difficult to determine the dose required to obtain the desired pharmacological effect. That is,
Since there is a slight difference in the antithrombin activity of the both,
There is a problem that the dose cannot be determined unless the mixing ratio is accurately measured. Furthermore, although the mixture ratio of the produced mixture is within a certain range, there are some fluctuations,
This has been a major obstacle in producing products of uniform quality with good reproducibility. Therefore, there has been a demand for a production method capable of suppressing the production of the above-mentioned byproducts and producing only the sulfated esterified product of the target hirudin analog with high purity.

[0005]

The present invention has solved the above problems. That is, an object of the present invention is to produce only a sulfated esterification product of a specific hirudin analog selectively, and not contain a by-product in which an amino acid is converted into another optical isomer, and to obtain a high-purity hirudin analog. Another object of the present invention is to provide a method for producing a sulfuric acid esterified body or a salt thereof in a high yield. Further, another object of the present invention is to achieve mass production by using a chemical synthesis method by a liquid phase method without applying a solid phase method in a process for producing the sulfated ester of hirudin analog or a salt thereof. Another object of the present invention is to provide a method for producing a sulfated esterified product of a hirudin analogue and a salt thereof which are excellent in

[0006]

The present invention provides a hirudin analog which is a peptide containing a part of the amino acid sequence represented by the following general formula (I) and having a molecular weight of 3000 or less. In producing a hirudin analog sulfate ester or a salt thereof by sulfate-forming a phenolic hydroxyl group of a tyrosine residue contained in the body, first, the side chain and the C-terminal carboxyl group contained in the hirudin analog are Characterized in that it is protected with a protecting group to prepare a protected group substituted with a hirudin analog, and then the phenolic hydroxyl group of the tyrosine residue contained in the substituted substituted group is sulfated, and then the protective group is eliminated. The present invention relates to a method for producing a sulfated hirudin analogue or a salt thereof. Phe-Glu-X1-Ile-Pro-X2-Tyr-Tyr- (I) (wherein X1 is Pro or Glu, X2 is Glu, Glu-Asp,
Means Glu-Tyr or Tyr, respectively. Also, the formula (I)
The amino group of the peptide may be protected in advance).

The protective group-substituted product can be, for example, an ester such as benzyl carboxylate in which a benzyl group or the like is introduced as a protective group into the carboxyl group. In addition, the amino group existing in the amino acid contained in the hirudin analog, for example, the α-position amino group of the amino acid located at the N-terminal is previously converted to the tertiary-butyloxycarbonyl group (B
oc group) and succinyl group (Suc group). Also when the protective group-substituted product is prepared in advance by synthesis, the carboxyl group and the amino group are appropriately substituted with a protective group in order to form a desired peptide bond. INDUSTRIAL APPLICABILITY The present invention is a method that can be suitably applied to hirudin analogs that have a high homology with natural hirudin in their amino acid sequences. In particular, it is suitable for hirudin analogues containing the above-mentioned amino acid partial sequence of general formula (I) in a region of 15 amino acid residues or less from the C-terminal, and further, hirudin having 20 or less total amino acid residues. It is more suitable for analogs. Further, as a hirudin analog, it is suitable for a peptide containing two tyrosine in the amino acid sequence of the peptide and having an amino acid at the C-terminal as an optical isomer. The optical isomers of amino acids (L) and (D) are distinguished from each other according to the common notation (D)
Specify only body.

In the present invention, the sulfate esterification reaction is
In the presence of a Lewis base type solvent such as pyridine or dimethylformamide, the protective group-substituted product is added in an excess amount, preferably 1
It can be carried out by allowing 0 to 500 equivalents of sulfatrioxide (SO ₃ ) complex to act at around room temperature. As the sulfa-trioxide (SO ₃ ) complex, those soluble in the Lewis base type solvent are preferable, for example, pyridine, dioxane, trimethylamine, triethylamine, dimethylaniline, thioxane, bis (2-chloroethyl) ether, Examples thereof include a complex of 2-methylpyridine, quinoline, dimethylformamide and the like and sulfatrioxide (SO ₃ ). Further, it is preferable to carry out the catalytic reduction method using palladium carbon (Pd / C) or the like as a catalyst in order to remove the protective group after the protective group-substituted product is converted to a sulfuric acid ester.

Further, the sulfate esterified product obtained by the above-mentioned production method can be purified and isolated as a salt thereof and a base or an acid. That is, salts of various cations with ionized carboxyl groups and sulfate groups present in the amino acid side chains and C-terminals contained in the peptide of the compound, for example, sodium salt, calcium salt, magnesium salt, barium salt, ammonium salt, piperidine. Salts, morpholine salts, dimethylamine salts, diethylamine salts and the like can be formed. In addition, salts of ionized amino groups present in the amino acid side chains contained in the peptide of the compound and various anions, such as trifluoroacetate, acetate, tartrate, oxalate, lactate, succinate, and malon. Acid salts, formates, p-toluene sulfonates, phosphates, hydrochlorides, sulphates can be formed.

[0010]

In the production method of the present invention, the side chain and C contained in the hirudin analogue are subjected to the sulfuric acid esterification reaction.
Since the terminal carboxyl group has a substituted protecting group, it is possible to suppress the isomerization reaction between the (L) form and the (D) form of the C-terminal amino acid. That is, since the sulfuric acid esterification reaction can be carried out by suppressing the isomerization reaction between the (L) form and the (D) form, the reaction product obtained by removing the protecting group after that is only the target optical isomer. Becomes

Further, in the production method of the present invention, the protective group substitution product of the hirudin analogue can be obtained by a liquid phase synthesis method, for example, without depending on a solid phase synthesis method using a resin, and Since the reaction of sulfuric acid esterification is also a liquid phase reaction, it becomes possible to carry out a uniform reaction, which is excellent in mass productivity.

The manufacturing method of the present invention will be described below with reference to Examples.
Further description will be made. In these examples, DMF is dimethylformamide, HOBt is 1-hydroxybenzotriazole, EDC is N-ethyl-N '(3-dimethylaminopropyl) carbodiimide, and HONB is N-hydroxy-5-norbornene-.
2,3-dicarboximide, AcOEt is ethyl acetate, THF
Is tetrahydrofuran, Pac is phenacyl, DCC
Indicates dicyclohexylcarbodiimide, and DCHA indicates dicyclohexylamine.

[0013]

Example 1 The following is a hirudin analog (A) Suc-Phe-G
Lu-Pro-Ile-Pro-Glu-Tyr-Tyr-Leu-Gln-OH amino acid residue side chain and C-terminal carboxyl group protected by a benzyl group Suc-Phe-Glu (OBzl) -Pro-Ile
-Pro-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl, and the sulfated esterification product of hirudin analog (A) Suc-Phe-Glu-Pro-Ile
The _{-Pro-Glu-Tyr (SO 3} H) -Tyr (SO 3 H) -Leu-Gln-OH process of manufacturing will be described.

(1) Production of protective group substitution product of hirudin analogue (A) Step (1-1): Production of Boc-Gln-OBzl Raw material Boc-Gln-OH (5.0 g, 20.01 mmol) is used as a solvent dimethylformamide. A solution dissolved in 30 ml of (DMF) was prepared, the solution was cooled to freezing point, K ₂ CO ₃ (1.66 g, 12.01 mmol) and benzyl bromide (2.38 ml, 22.01 mmol) were added and mixed. The mixture was cooled to freezing and stirred overnight (about 14 hours). Then, the mixture was filtered, the solvent DMF was removed and the mixture was concentrated. A 5% NaHCO ₃ aqueous solution was added to the concentrated solution to give a solid reaction product. The filtered solid of the reaction product was treated with 5% NaHCO ₃
Batch washing was sequentially performed using an aqueous solution, a 5% aqueous citric acid solution, and water. The obtained reaction product was dried, then dissolved again in a solvent of 50 ml of toluene, and purified by recrystallization. The weight of the obtained reaction product Boc-Gln-OBzl after purification was 4.74 g, and the yield was 71%. The reaction product Boc-Gln-OBzl showed Rf = 0.39 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent) in thin layer chromatography.

Step (1-2): Preparation of Boc-Leu-Gln-OBzl Boc-Gln-OBzl (4.74g, 1) obtained in the above step (1-1)
4.11 mmol) in 4N hydrogen chloride / dioxane solution (35 ml, 141.10
mmol) and the solution was stirred at room temperature for 2 hours. After that, the residue obtained by concentrating the solution under reduced pressure was used as a solvent DMF 20.
It was dissolved in ml and triethylamine (1.96 ml, 14.11 mmol) was added to neutralize. Cool the resulting solution to freezing and
-Leu-OH ・ H ₂ O (3.52g, 14.11mmol), HOBt (1.90g, 14.11m
mol) and EDC.HCl (2.96 g, 15.52 mmol) were added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then the mixture was filtered and concentrated by removing the solvent DMF. A 5% NaHCO ₃ aqueous solution was added to the concentrated solution to give a solid reaction product. The filtered solid of the reaction product was sequentially washed in batch with 5% aqueous NaHCO ₃ solution, 5% aqueous citric acid solution, and water. The obtained reaction product was dried, then dissolved again in a toluene-ethanol solvent, and purified by recrystallization. The obtained reaction product Boc-Leu-Gln-OBzl weighed 3.72 g after purification, and the yield was 59%. Reaction product Boc-Leu-Gln-O
Bzl is Rf = 0.40 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent)
showed that.

Step (1-3): Preparation of Boc-Tyr-Leu-Gln-OBzl Boc-Leu-Gln-OBzl (3.72) obtained in the above step (1-2)
g, 8.29 mmol) was added CH ₂ Cl ₂ (20 ml) and 4N hydrogen chloride / dioxane solution (21 ml, 82.90 mmol), and the solution was stirred at room temperature for 2 hours. Then, the residue obtained by concentrating the solution under reduced pressure was washed with ether. The residue obtained is treated with the solvent DMF 20.
It was dissolved in ml and triethylamine (1.15 ml, 8.29 mmol) was added to neutralize. The solution was cooled to freezing point and triethylamine (1.15 ml, 8.29 mmol) and Boc-Tyr-ONB [previously Bo
It was prepared using c-Tyr-OH (2.79 g, 9.94 mmol), HONB (1.78 g, 9.94 mmol), and EDC.HCl (2.09 g, 10.94 mmol). ]
Was added and mixed. The mixture was stirred overnight (about 14 hours). Then, the mixture was filtered and concentrated by removing the solvent. The solution obtained by adding the solvent AcOEt to the concentrated solution was sequentially subjected to batch cleaning using 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water. The AcOEt solution was concentrated and dried by removing the solvent, and isopropyl ether was added to the resulting residue to give the reaction product Boc-T.
A solid of yr-Leu-Gln-OBzl was obtained. The solid is converted to THF-AcOE
It was purified as a reprecipitate using t and isopropyl ether. The weight after purification was 5.03 g, and the yield was 100%. The reaction product Boc-Tyr-Leu-Gln-OBzl has Rf = 0.
34 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent).

Step (1-4): Boc-Tyr-Tyr-Leu-Gln-OBz
Preparation of Boc-Tyr-Leu-Gln-OBzl obtained in the above step (1-3)
(5.03 g, 8.29 mmol) was added with 30 ml of trifluoroacetic acid, and the solution was stirred at room temperature for 2 hours. Then, the residue obtained by concentrating the solution under reduced pressure was washed with ether. The obtained residue was dissolved in 20 ml of solvent DMF, and triethylamine (1.15 m
l, 8.29 mmol) was added to neutralize. The solution was cooled to freezing point and triethylamine (1.15 ml, 8.29 mmol) and Boc-T
yr-ONB [previously Boc-Tyr-OH (2.79 g, 9.94 mmol), HONB
(1.78g, 9.94 mmol) and EDC HCl (2.09g, 10.94mmo
l) was prepared. ] Was added and mixed. The mixture was stirred overnight (about 14 hours). Then, the mixture was filtered and concentrated by removing the solvent. 5% NaHC in the concentrate
The reaction product Boc-Tyr-Tyr-Leu-Gln-O was added by adding an aqueous solution of O _3.
A solid of Bzl was obtained. Further, the filtered solid of the reaction product was subjected to sequential batch washing with 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water. After drying, the solid
Purified as a reprecipitate using THF and isopropyl ether. The weight after purification was 4.25 g, and the yield was 66%. The reaction product Boc-Tyr-Tyr-Leu-Gln-OBzl is Rf
= 0.23 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent).

Step (1-5): Boc-Glu (OBzl) -Tyr-Tyr-L
Preparation of eu-Gln-OBzl Boc-Tyr-Tyr-Leu-Gln-OBz obtained in the above step (1-4)
30 ml of trifluoroacetic acid was added to l (4.25g, 5.48 mmol),
The solution was stirred at room temperature for 2 hours. Then, the residue obtained by concentrating the solution under reduced pressure was washed with ether. The obtained residue was dissolved in 20 ml of a solvent DMF, and triethylamine (0.7
6 ml, 5.48 mmol) was added to neutralize. The solution was cooled to the freezing point, Boc-Glu (OBzl) -OSu (2.86 g, 6.58 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours).
Then, the mixture was filtered and concentrated by removing the solvent. A 5% NaHCO ₃ aqueous solution was added to the concentrated solution to obtain a solid reaction product Boc-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl. Further, the filtered solid of the reaction product was subjected to sequential batch washing with 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water. After drying, the solid was washed with MeOH and AcOEt.
Was purified as a reprecipitate. Weight after purification is 3.72
and the yield was 68%. Reaction product Boc-Glu (O
Bzl) -Tyr-Tyr-Leu-Gln-OBzl has Rf = 0.31 (volume ratio CHCl
₃ : MeOH = 9: 1 mixed solvent). In addition, amino acid analysis (6N HCl hydrolysis method) of the reaction product was performed, and the amino acid content was measured to be Glx: Leu: Tyr = 1.90: 1.00: 2.00, which is predicted (Glu + Gln): Leu: It was in good agreement with Tyr = (1 + 1): 1: 2.

Step (1-6): Preparation of Boc-Ile-Pro-OPac The raw material Boc-Ile-OSu (6.44 g, 19.63 mmol) was used as the solvent DMF 40.
The solution dissolved in ml was cooled to the freezing point, and H-Pro-OH (4.52 g,
39.30 mmol) and triethylamine (5.45 ml, 39.30 mmol)
An aqueous solution in which the salt of was dissolved in 10 ml of H ₂ O was mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture was filtered and concentrated by removing the solvent.
5% of the solution obtained by adding the solvent AcOEt to the concentrate
Washing was sequentially performed using an aqueous citric acid solution and water.
The obtained AcOEt solution was dried and concentrated to give the intermediate product Boc-Ile-
I got Pro-OH. A solution of this intermediate product Boc-Ile-Pro-OH in 20 ml of DMF was cooled to the freezing point, and triethylamine (2.71 ml, 19.63 mmol) and bromoacetophenone were added.
(4.05g, 20.61mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture was filtered and concentrated by removing the solvent. The solution obtained by adding the solvent AcOEt to the concentrated solution was washed successively with 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water. The AcOEt solution was dried and concentrated to give the reaction product Boc.
-Ile-Pro-OPac was obtained as a residue. Further, it was purified by silica gel column chromatography (volume ratio CH ₂ Cl ₂ : MeOH = 80: 1 mixed solvent). The weight after purification was 6.85 g, and the yield was 83.
%Met. The reaction product Boc-Ile-Pro-OPac has Rf = 0.
75 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent).

Step (1-7): Preparation of Boc-Pro-Ile-Pro-OPac Boc-Ile-Pro-OPac (2.23) obtained in the above step (1-6)
g, 5.00 mmol) in 4N hydrogen chloride / dioxane solution (12.5 m
l, 50.0 mmol) and the solution was stirred at room temperature for 2 hours. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in 20 ml of a solvent DMF, and triethylamine (0.69 ml, 5.00 m
(mol) was added to neutralize. The obtained solution was cooled to the freezing point, and Boc-Pro-OBt (5.00 mmol) [Boc-Pro-OH (1.07 mmol
g, 5.00 mmol), HOBt (0.68 g, 5.00 mmol) and EDC ・ H
Prepared using Cl (1.05 g, 5.50 mmol). ] Was mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then the mixture was filtered and concentrated by removing the solvent DMF. The solution obtained by adding the solvent AcOEt to the concentrated solution was washed successively with 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water. The AcOEt solution was dried and concentrated to give the reaction product Boc-Pro-Ile-Pro-OPac.
Was obtained as a residue. In addition, silica gel column chromatography
(Volume ratio CH ₂ Cl ₂ : MeOH = 100: 1 mixed solvent). The weight after purification was 1.18 g, and the yield was 44%. The reaction product Boc-Pro-Ile-Pro-OPac has Rf = 0.66.
(Volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent).

Step (1-8): Boc-Glu (OBzl) -Pro-Ile-P
Preparation of ro-OPac Boc-Pro-Ile-Pro-OPac obtained in the above step (1-7)
(3.40 g, 6.25 mmol) in 4N hydrogen chloride / dioxane solution
(15.6 ml, 62.5 mmol) and the solution was stirred at room temperature for 2 hours. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF 40 ml, and triethylamine (0.87 ml,
6.25 mmol) was added to neutralize. The resulting solution was cooled to freezing point, Boc-Glu (OBzl) -OH (2.11g, 6.25 mmol), HOBt.
(0.84g, 6.25mmol) and EDC ・ HCl (1.44g, 7.50mmo
l) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture is filtered,
It was concentrated by removing the solvent DMF. The solvent AcOE
The solution obtained by adding t was washed successively with 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water.
The AcOEt solution was dried and concentrated to give the reaction product Boc-Glu (OBz
l) -Pro-Ile-Pro-OPac was obtained as a residue. The weight after dryness was 4.57 g, and the yield was 96%. Reaction product Boc-
Glu (OBzl) -Pro-Ile-Pro-OPac has Rf = 0.58 (capacity ratio CH
Cl ₃ : MeOH = 9: 1 mixed solvent).

Step (1-9): Boc-Phe-Glu (OBzl) -Pro-I
Preparation of le-Pro-OPac Boc-Glu (OBzl) -Pro-Ile-P obtained in the above step (1-8)
ro-OPac (4.50g, 5.90mmol) was added to 4N hydrogen chloride / dioxane solution (14.8ml, 59.0mmol) and the solution was stirred at room temperature for 2
Stir for hours. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF (40 ml), and triethylamine (0.8
2 ml, 5.90 mmol) was added to neutralize. The resulting solution was cooled to freezing point, Boc-Phe-OH (1.57g, 5.90 mmol), HOBt.
(0.80g, 5.90 mmol) and EDC ・ HCl (1.36g, 7.08 mmo
l) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture is filtered,
Concentrated by removal of the solvent DMF. The solvent AcO
The solution obtained by adding Et was washed successively with 5% NaHCO ₃ aqueous solution, 5% citric acid aqueous solution, and water.
The AcOEt solution was dried and concentrated to give the reaction product Boc-Phe-Glu.
(OBzl) -Pro-Ile-Pro-OPac was obtained as a residue. Furthermore, silica gel column chromatography (volume ratio CH ₂ Cl ₂ : MeOH = 100: 1
(Mixed solvent). The weight after purification was 3.04 g, and the yield was 57%. Reaction product Boc-Phe-Glu (OBz
l) -Pro-Ile-Pro-OPac has Rf = 0.59 (volume ratio CHCl ₃ : MeOH
= 9: 1 mixed solvent).

Step (1-10): Boc-Phe-Glu (OBzl) -Pro-I
Preparation of le-Pro-OH Boc-Phe-Glu (OBzl) -Pro-I obtained in the above step (1-9)
le-Pro-OPac (3.00 g, 3.30 mmol) was dissolved in 50 ml of acetic acid, and the solution was treated with 1N hydrochloric acid to activate activated zinc powder (10.79 g,
(165 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, the solution was filtered to remove zinc dust, and concentrated by removing the solvent acetic acid. The solution obtained by adding the solvent AcOEt to the concentrated solution was washed successively with a 1N aqueous hydrochloric acid solution and water. The AcOEt solution was dried and concentrated to give the reaction product Boc-Phe.
-Glu (OBzl) -Pro-Ile-Pro-OH was obtained in the residue. Ether was added to the residue for recovery and purification. Weight after purification is 2.
It was 00 g and the yield was 77%. Reaction product Boc-Phe
-Glu (OBzl) -Pro-Ile-Pro-OH has Rf = 0.33 (volume ratio CHCl
₃ : MeOH = 9: 1 mixed solvent). In addition, amino acid analysis (6N HCl hydrolysis method) of the reaction product was performed to find that the amino acid content was Glx: Pro: Ile: Phe = 1.09: 1.97: 1.00: 1.0.
It was determined to be 3 (Glx indicates Glu or Gln) and was in good agreement with the predicted Glu: Pro: Ile: Phe = 1: 2: 1: 1.

Step (1-11): Boc-Phe-Glu (OBzl) -Pro-I
Preparation of le-Pro-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl Boc-Glu (OBzl) -Tyr-Tyr-L obtained in the above step (1-5)
eu-Gln-OBzl (1.20g, 1.21 mmol) in trifluoroacetic acid 1
0 ml was added and the solution was stirred at room temperature for 1 hour. Then, the residue obtained by concentrating the solution under reduced pressure was washed with ether. The residue was dissolved in the solvent DMF 10 ml, and triethylamine (0.17 ml, 1.21 mmol) was added to neutralize. The resulting solution was cooled to freezing point and obtained in step (1-10) above.
Boc-Phe-Glu (OBzl) -Pro-Ile-Pro-OH (1.25 g, 1.57 mm
ol), norborn-5-ene-2,3-dicarboximidodiphenyl phos
phate (0.65 g, 1.57 mmol) and triethylamine (0.3
9 ml, 2.78 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixed solution was filtered to remove the solvent DMF and concentrated. Water was added to the obtained concentrated liquid to give the reaction product Boc-Phe-Glu (OBzl) -Pro-I.
A solid of le-Pro-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl was produced. After washing the solid with MeOH and drying, the weight obtained was 1.17 g and the yield was 58%. Reaction product B
oc-Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr-Tyr-Leu
-Gln-OBzl is, Rf = 0.31 (volume ratio CHCl _3: MeOH = 9: 1 mixed solvent) showed. In addition, amino acid analysis of the reaction product
(6N HCl hydrolysis method) was performed and the amino acid content was Glx:
Pro: Ile: Leu: Tyr: Phe = 3.08: 1.98: 1.00: 1.00: 2.03: 1.0
Measured and predicted to be 1 (Glu + Gln): Pro: Ile: Leu: Ty
It was in good agreement with r: Phe ＝ 3: 2: 1: 1: 2: 1.

Step (1-12): Suc-Phe-Glu (OBzl) -Pro-I
Preparation of le-Pro-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl Boc-Phe-Glu (OBzl) -Pro-I obtained in the step (1-10)
le-Pro-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl (0.25 g, 0.1
3 ml of trifluoroacetic acid was added to (5 mmol) and the solution was stirred at room temperature for 1 hour. Then, the residue obtained by concentrating the solution under reduced pressure was washed with ether. The residue is solvent DM
Dissolve in 3 ml of F, triethylamine (21 μl, 0.15 mmol)
Was added to neutralize. The resulting solution was cooled to freezing and succinic anhydride (18 mg, 0.18 mmol) and triethylamine
(21 μl, 0.15 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. During this time,
The Boc protecting group that protects the N-terminal Phe amino group is Suc
Substituted with a protecting group. Then, the solvent DMF was removed from the mixed solution and concentrated, water was added to the obtained concentrated solution, and the reaction product Suc-Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr- Tyr
-A solid of Leu-Gln-OBzl was produced. The solid was purified as a reprecipitate using MeOH and ether. After drying
The weight of the purified product was 242 mg, and the yield was 97%. Reaction product Suc-Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -T
yr-Tyr-Leu-Gln-OBzl has Rf = 0.12 (volume ratio CHCl ₃ : MeO
H = 9: 1 mixed solvent).

(2) Production of Sulfate Ester of Hirudin Analog (A) Protecting group-substituted Suc-Phe-Glu (OBzl)-of the hirudin analog (A) produced by the process described in (1) above. Pro-Ile-Pro-
Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl (100 mg, 0.06 mmo
l) was dissolved in 4 ml of 20% pyridine / DMF solution. Add pyridine-sulfur-trioxide [Pryzine
sulfurtrioxide complex] (1 g, 6.3 mmol) was added and the resulting solution was stirred at room temperature for 4 hours. During this period, the phenolic hydroxyl groups present in the two Tyr residues of the protective group-substituted product are each sulfated to form a disulfate esterified product. After that, the solution was dissolved in a 50% MeOH / H ₂ O mixed solvent, and the desired reaction product, a disulfate ester Suc.
-Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr (SO ₃ H) -Tyr
(SO ₃ H) -Leu-Gln-OBzl was recovered and purified by preparative HPLC.
The preparative liquid containing the above-mentioned disulfate ester was concentrated to about 10 ml, 10 mg of 10% -Pd / C powder was added, and hydrogenolysis was carried out at room temperature and atmospheric pressure for 7 hours. During this process, the benzyl group of the protecting group is released, and the desired final product Suc-Phe-Glu is removed.
-Pro-Ile-Pro-Glu- Tyr (SO 3 H) -Tyr (SO 3 H) -Leu-Gln-OH is obtained. After filtering the reaction solution containing the final product,
It was desalted and purified by preparative HPLC and gel filtration to obtain an eluate containing only the final product. After 10% ammonia water was added to the eluate to adjust the pH to 7 to 8, the final product was recovered as an ammonium salt by freeze-drying. As a result of repeating the above purification operation twice, the purified product (ammonium salt)
Had a weight of 14.8 mg and a yield of 16%.

[0027]

Example 2 The following is the hirudin analog (B) Suc-Phe-G
lu-Pro-Ile-Pro-Glu-Tyr-Tyr- (D) Leu-OH amino acid residue side chain and C-terminal carboxyl group protected by benzyl group Suc (OBzl) -Phe- Glu (OBzl) -Pro
-Ile-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl, and the sulfate esterification product of hirudin analog (A) Suc-Phe-Glu-Pro-I
le-Pro-Glu-Tyr ( SO 3 H) -Tyr (SO 3 H) - (D) illustrating the process of manufacturing the Leu-OH.

(1) Production of Protected Group Substitute of Hirudin Analog (B) Step (2-1): Preparation of Boc- (D) Leu-OBzl Raw Material Boc- (D) Leu-OH (4.0g, 16.04) mmol) as solvent DMF 20
Make a solution dissolved in ml, cool the solution to freezing point, and add K ₂
CO ₃ (1.33 g, 9.62 mmol) and benzyl bromide (2.10 ml, 1
7.64 mmol) was added and mixed. The mixture was cooled to freezing and stirred overnight (about 14 hours). Then the mixture was filtered and concentrated by removing the solvent DMF to give an oily residue. A solution of the oily residue in ethyl acetate was added to 5%
Washing was sequentially performed using a citric acid aqueous solution, a 5% NaHCO ₃ aqueous solution, and a 5% saline solution. Anhydrous magnesium sulfate was added to the solution and dried. After that, the solvent ethyl acetate was removed from the filtered solution by distillation under reduced pressure, and further dried under vacuum to recover a reaction product Boc- (D) Leu-OBzl. The recovered weight was 5.13 g and the yield was 99%. The reaction product Boc- (D) Leu-OBzl has Rf = 0.51 (volume ratio CHC
l _3: MeOH = 9: 1 mixed solvent) showed.

Step (2-2): Preparation of Boc-Tyr- (D) Leu-OBzl Boc- (D) Leu-OBzl (5.13g, obtained in the above step (2-1)
15.96 mmol) in 4N hydrogen chloride / dioxane solution (39.9 ml,
159.6 mmol) and the solution was stirred for 1 hour while cooling to freezing. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in 40 ml of a solvent DMF, and triethylamine (2.22
ml, 15.96 mmol) was added to adjust the pH to 8. The resulting solution was cooled to freezing point and Boc-Tyr-OH (14.49 g, 15.96 mm
ol), HOBt (2.16 g, 15.96 mmol) and DCC (3.95 g, 1
9.15 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then the mixture was filtered and concentrated by removing the solvent DMF. A precipitate formed by adding a 5% citric acid aqueous solution to the concentrated solution was washed successively with a 5% citric acid aqueous solution, a 5% NaHCO ₃ aqueous solution, and 5% water. The obtained reaction product was purified as a reprecipitate using THF / ether as a solvent. The obtained reaction product Boc-Tyr- (D) Leu-OBzl had a weight after purification.
It was 4.85 g and the yield was 62.7%. Reaction product Boc-
Tyr- (D) Leu-OBzl has Rf = 0.58 (volume ratio CHCl ₃ : MeOH =
9: 1 mixed solvent).

Step (2-3): Boc-Tyr-Tyr- (D) Leu-OBzl
Preparation of Boc-Tyr- (D) Leu-OBzl (4.
75 g, 9.80 mmol) in 4N hydrogen chloride / dioxane solution (24.5
ml, 98.0 mmol) was added, and the solution was stirred for 1 hour while cooling to a freezing point. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in 40 ml of a solvent DMF, and triethylamine (1.36
ml, 9.80 mmol) was added to adjust the pH to 8. The solution was cooled to freezing point, Boc-Tyr-OH (2.76g, 9.80 mmol), HO
Bt (1.32g, 9.86 mmol) and DCC (2.43 g, 11.76 mmo
l) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture was filtered and concentrated by removing the solvent. The solution obtained by adding the solvent AcOEt to the concentrated solution was added with 5% citric acid aqueous solution, 5% NaHCO ₃
Washing was sequentially performed using an aqueous solution and 5% saline. Anhydrous magnesium sulfate was added to the solution and dried. Further filtration was performed and the solvent AcOEt was removed from the AcOEt solution by vacuum distillation. From the residue obtained after concentration, using ethyl acetate and ether, the reaction product Boc-Tyr-Tyr- (D)
Leu-OBzl was purified as a reprecipitate. Weight after purification is 2.
It was 21 g, and the yield was 34.8%. Reaction product Boc-T
yr-Tyr- (D) Leu-OBzl has Rf = 0.43 (volume ratio CHCl ₃ : MeOH
= 9: 1 mixed solvent). In addition, amino acid analysis (6N HCl hydrolysis method) of the reaction product was performed, and the amino acid content was predicted to be (D) Leu: Tyr = 1.00: 1.96.
It was in good agreement with (D) Leu: Tyr = 1: 2.

Step (2-4): Boc-Glu (OBzl) -Tyr-Tyr-
Preparation of (D) Leu-OBzl Boc-Tyr-Tyr- (D) Leu-OBzl obtained in the above step (2-3)
(1.81 g, 2.79 mmol) in 4N hydrogen chloride / dioxane solution (2
(4.5 ml, 98.0 mmol) was added, and the solution was stirred for 1 hour while cooling to the freezing point. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in 40 ml of a solvent DMF, and triethylamine was added.
(0.39 ml, 2.79 mmol) was added to adjust the pH to 8. The solution was cooled to freezing point and Boc-Glu (OBzl) -OH (0.94g, 2.79
mmol), HOBt (0.38 g, 2.79 mmol) and EDC HCl (0.59
g, 3.07 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture was filtered and concentrated by removing the solvent. The solvent ethyl acetate was added to the concentrated solution to dissolve it again. The solution was washed successively with a 5% aqueous citric acid solution, a 5% aqueous NaHCO ₃ solution, and a 5% saline solution, and then dried over anhydrous magnesium sulfate. After further filtration, the solvent A was removed from the AcOEt solution.
cOEt was removed by vacuum distillation. Obtained reaction product B
The solid of oc-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl was purified as a reprecipitate using DMF and ether. The weight after purification was 1.59 g, and the yield was 66.0%. Reaction product
Boc-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl showed Rf = 0.50 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent). In addition, amino acid analysis (6N HCl hydrolysis method) of the reaction product was performed, and the amino acid content was Glx: (D) Leu: Tyr = 1.03: 1.00: 1.
The measured value was 98, which was in good agreement with the predicted Glu: (D) Leu: Tyr = 1: 1: 2.

Step (2-5): Boc-Pro-Glu (OBzl) -Tyr-T
Preparation of yr- (D) Leu-OBzl Boc-Glu (OBzl) -Tyr-Tyr-obtained in the above step (2-4)
4D hydrogen chloride / dioxane solution (4.03 ml, 16.01 mmol) was added to (D) Leu-OBzl (1.40 g, 1.61 mmol), and the solution was stirred for 1 hour while cooling to a freezing point. After that, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF 40 ml, and triethylamine (0.22 ml, 1.61 mmol) was added to adjust the pH to 8.
Was prepared. The solution was cooled to freezing and Boc-Pro-OH (0.
32 g, 1.61 mmol), HOBt (0.22 g, 1.61 mmol) and DCC
(0.33 g, 1.93 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. afterwards,
The mixture was filtered and concentrated by removing the solvent. The solvent ethyl acetate was added to the concentrated solution to dissolve it again. The solution was 5% aqueous citric acid, 5% aqueous NaHCO ₃ , and 5%
After sequentially washing with a saline solution, anhydrous magnesium sulfate was added and dried. Further filtration was performed and the solvent ethyl acetate was removed from the ethyl acetate solution by distillation under reduced pressure. The obtained reaction product Boc-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-
The OBzl solid was purified as a reprecipitate using ethyl acetate and ether. The weight after purification was 0.78 g, and the yield was 50.3%. Reaction product Boc-Pro-Glu (OBzl) -Tyr
-Tyr- (D) Leu-OBzl has Rf = 0.58 (volume ratio CHCl ₃ : MeOH =
9: 1 mixed solvent).

Step (2-6): Boc-Ile-Pro-Glu (OBzl) -T
Preparation of yr-Tyr- (D) Leu-OBzl Boc-Pro-Glu (OBzl) -Tyr-T obtained in the above step (2-5)
yr- (D) Leu-OBzl (0.78g, 0.81 mmol) in 4N hydrogen chloride /
A dioxane solution (4.03 ml, 16.01 mmol) was added, and the solution was stirred for 1 hour while cooling to a freezing point. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF (40 ml), and triethylamine (0.11 ml, 0.81 mmol) was added to adjust the pH to 8. The solution was cooled to freezing point and Boc-Ile-
OH (0.17 g, 0.81 mmol), HOBt (0.11 g, 0.81 mmol) and DCC (0.20 g, 0.97 mmol) were added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture was filtered and concentrated by removing the solvent.
The obtained oily residue was again dissolved in the solvent DMF, and washed successively with 5% citric acid aqueous solution, 5% NaHCO ₃ aqueous solution, and water. The solvent was removed from the resulting solution by vacuum distillation. Furthermore, the reaction product Boc-I obtained as a residue
le-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl was purified by column chromatography (volume ratio CHCl ₃ : MeOH = 40: 1 mixed solvent). The weight after purification was 0.20 g, and the yield was 23.3%. Reaction product Boc-Ile-Pro-Glu (OBzl) -Tyr-Tyr- (D)
Leu-OBzl showed Rf = 0.33 (volume ratio CHCl ₃ : MeOH = 9: 1 mixed solvent). In addition, amino acid analysis of the reaction product (6
N HCl hydrolysis method) and amino acid content is Glx:
The measured (D) Leu: Pro: Tyr = 1.02: 1.00: 0.94: 1.96 was in good agreement with the predicted Glu: (D) Leu: Pro: Tyr = 1: 1: 1: 2.

Step (2-7): Boc-Glu (OBzl) -Pro-OH
Preparation of DCHA Raw material Boc-Glu (OBzl) -OSu (5.00 g, 11.51 mmol) as solvent
Dissolve in 40 ml of DMF, cool the obtained solution to freezing point, and
c-Pro-OH ・ Et ₃ N salt (23.01 mmol) [Boc-Pro-OH
(2.65 g, 23.01 mmol) and Et ₃ N (3.20 g, 23.01 mmol)
Was prepared as an aqueous solution. ] And triethylamine (3.20 g, 23.01 mmol) were added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the mixture was filtered and concentrated by removing the solvent. The obtained oily residue was dissolved again in the solvent ethyl acetate, and the obtained solution was washed successively with 5% citric acid aqueous solution, 5% NaHCO ₃ aqueous solution, and 5% saline solution. Anhydrous magnesium sulfate was added to the solution and dried. After filtration, the solvent ethyl acetate was removed from the solution by distillation under reduced pressure to give the intermediate product Boc-Glu (OBzl) -Pro-OH as a residue. Intermediate products Boc-Glu (OBzl) -Pro-OH and DCHA (2.29
ml, 12.66 mmol) formed with Boc-Glu (OBzl) -P
The ro-OH-DCHA was purified and crystallized using a solvent combination of n-hexane and ethyl acetate. Weight after purification is 6.60
The yield was 93.1%.

Step (2-8): Boc-Glu (OBzl) -Pro-OPac
Preparation of Boc-Glu (OBzl) -Pro-OH obtained in the above step (2-7)
Dissolve DCHA (5.30 g, 8.60 mmol) in the solvent DMF 40 ml,
Further acetophenone bromide (1.88 g, 9.46 mmol) was added and the solution was stirred at room temperature for 1 hour. Then, the mixture was filtered and concentrated by removing the solvent. The obtained oily residue was again dissolved in the solvent ethyl acetate, and washed successively with 5% citric acid aqueous solution, 5% NaHCO ₃ aqueous solution, and 5% saline solution. Anhydrous magnesium sulfate was added to the solution and dried. After filtration, the solvent ethyl acetate was removed from the solution by distillation under reduced pressure, and the reaction product Boc-Glu (OBzl) -Pro-OPac obtained as a residue was further purified as a reprecipitate using ether. The weight after purification is 3.76 g,
The yield was 79.2%.

Step (2-9): Boc-Phe-Glu (OBzl) -Pro-O
Preparation of Pac Boc-Glu (OBzl) -Pro-OPac obtained in the above step (2-8)
(3.50 g, 6.30 mmol) in 4N hydrogen chloride / dioxane solution (15.
75 ml, 63.0 mmol) and 15 ml of methylene chloride,
The solution was stirred for 1 hour keeping it at freezing. After that, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF 40 ml, and triethylamine (0.88 ml, 6.30 mmol) was added thereto.
The pH was adjusted to 8. The resulting solution was cooled to freezing point and Boc-
Phe-OH (1.56 g, 6.30 mmol), HOBt (0.85 g, 6.30 mmol)
And EDC * HCl (1.32 g, 6.90 mmol) was added and mixed.
The mixture was stirred overnight (about 14 hours) while keeping the freezing point. The mixture was then concentrated by removing the solvent DMF. The solution obtained by adding the solvent ethyl acetate to the obtained oily residue was washed successively with 5% citric acid aqueous solution, 5% NaHCO ₃ aqueous solution, and 5% saline. Anhydrous magnesium sulfate was added to the solution and dried. After filtration, the solvent ethyl acetate was removed from the solution by vacuum distillation, and the reaction product Boc-Phe-Glu (OBz
A solid (precipitate) of l) -Pro-OPac was obtained. The weight after vacuum drying was 2.07 g, and the yield was 46.9%.

Step (2-10): Suc (OBzl) -Phe-Glu (OBzl)
Preparation of -Pro-OPac Boc-Phe-Glu (OBzl) -Pro-O obtained in the above step (2-9)
4N hydrogen chloride / dioxane solution in Pac (2.07g, 2.96mmol)
(7.40 ml, 29.6 mmol) was added and the solution was stirred for 1 hour keeping the freezing point. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF 40 ml, and triethylamine (0.41 ml, 2.96 mmol) was added to adjust the pH to 8.
The resulting solution was cooled to freezing point and Suc (OBzl) -OH (1.85 g,
4.44 mmol), HOBt (0.48 g, 3.55 mmol) and DCC (0.8
5 g, 4.14 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. During this period, the Boc protecting group that protects the amino group of N-terminal Phe is changed to Suc (OBz
l) group. The mixture was then concentrated by removing the solvent DMF. A solution obtained by adding the solvent ethyl acetate to the obtained oily residue was added with a 5% aqueous citric acid solution and a 5% Na solution.
Washing was sequentially performed using an HCO ₃ aqueous solution and 5% saline. Anhydrous magnesium sulfate was added to the solution and dried. After filtration, the solvent ethyl acetate was removed from the solution by vacuum distillation. Furthermore, the reaction product obtained as a residue
Suc (OBzl) -Phe-Glu (OBzl) -Pro-OPac was subjected to silica gel column chromatography (volume ratio CH ₂ Cl ₂ : AcOEt = 2: 1 mixed solvent).
Purified by. The weight after purification was 1.66 g, and the yield was 6
It was 4.1%.

Step (2-11): Suc (OBzl) -Phe-Glu (OBzl)
Preparation of -Pro-OH Suc (OBzl) -Phe-Glu (OBzl) obtained in the above step (2-10)
-Pro-OPac (1.66 g, 2.03 mmol) was dissolved in 50 ml of acetic acid,
Zinc dust (6.64 g, 101.5 mmol) was added to the solution with a 1N aqueous hydrochloric acid solution.
After adding 50 ml of the solution, keep it at the freezing point 1
Stir for hours. Then, the solution is filtered to remove zinc dust,
The residue obtained by concentration under reduced pressure was dissolved in the solvent ethyl acetate. The ethyl acetate solution was washed successively with a 1N hydrochloric acid aqueous solution and 5% saline. Anhydrous magnesium sulfate was added to the solution and dried. After filtration, the solvent ethyl acetate was removed from the solution by vacuum distillation. Furthermore, the reaction product Suc (OBzl) -Phe-Glu (OBzl) obtained as a residue
-Pro-OH with silica gel column chromatography (volume ratio CHC
I ₃ : AcOEt = 2: 1 mixed solvent). The weight after purification was 0.15 g, and the yield was 10.3%.

Step (2-12): Suc (OBzl) -Phe-Glu (OBzl)
Preparation of -Pro-Ile-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl Boc-Ile-Pro-Glu (OBzl) -Ty obtained in the above step (2-6)
4N hydrogen chloride / dioxane solution (5.00 ml, 20.0 mmol) was added to r-Tyr- (D) Leu-OBzl (0.20 g, 0.19 mmol), and the solution was stirred for 1 hour while keeping the freezing point. Then, the residue obtained by concentrating the solution under reduced pressure was dissolved in a solvent DMF 10 ml, and triethylamine (0.03 ml, 0.19 mmol) was added to adjust the pH to 8. The obtained solution was cooled to the freezing point and the Suc (OBzl) -Phe-Glu (OBzl) -P obtained in the step (2-11) was obtained.
ro-OH (0.15 g, 0.21 mmol), HOBt (0.03 g, 0.21 mmol)
Also, EDC / HCl (0.05 g, 0.27 mmol) was added and mixed. The mixture was stirred overnight (about 14 hours) while keeping the freezing point. Then, the residue obtained by concentrating the mixed solution under reduced pressure was dissolved in the solvent ethyl acetate. The ethyl acetate solution,
Washing was sequentially performed using a 5% citric acid aqueous solution, a 5% NaHCO ₃ aqueous solution, and a 5% saline solution. Anhydrous magnesium sulfate was added to the solution and dried. After filtration, the solvent ethyl acetate was removed from the solution by vacuum distillation. Reaction product Suc (OBzl) -Phe-Glu (OBzl) -Pro-I obtained as a residue
The solid of le-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl (precipitate) was further purified by use of ether as a reprecipitate. The weight after purification was 0.25 g, and the yield was 71.8%. The reaction product Suc (OBzl) -Phe-Glu (OBzl) -Pro-I
Amino acid analysis of le-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl
(6N HCl hydrolysis method) was performed and the amino acid content was
x: Pro: Ile: (D) Leu: Tyr: Phe ＝ 2.20: 2.00: 0.90: 1.00: 1.9
Glu: Pro: Ile: (D) Leu: Ty measured and predicted to be 0: 1.10.
It was in good agreement with r: Phe ＝ 2: 2: 1: 1: 2: 1.

(2) Production of Sulfate Ester of Hirudin Analog (B) Protecting group-substituted Suc (OBzl) -Phe-Glu (of Hirudin analog (B) prepared by the process described in (1) above. OBzl) -Pro-Il
e-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OBzl (100mg, 0.06 m
mol) was dissolved in 4 ml of 20% pyridine / DMF solution. Add pyridine-sulfur-trioxide [Pryzine
sulfurtrioxide complex] (1 g, 6.3 mmol) was added,
The resulting solution was stirred at room temperature for 4 hours. During this period, the phenolic hydroxyl groups present in the two Tyr residues of the protective group-substituted product are each sulfated to form a disulfate esterified product. Then, the solution was dissolved in a mixed solvent of 50% MeOH / H ₂ O, and the target reaction product, a disulfate ester, was formed.
Suc (OBzl) -Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr
(SO ₃ H) -Tyr (SO ₃ H)-(D) Leu-OBzl was collected and purified by preparative HPLC. About 1 aliquot containing the above-mentioned disulfate ester
After concentrating to 0 ml, 10% -Pd / C powder 10 mg was added, and hydrogenolysis was carried out at room temperature and normal pressure for 7 hours. During this process, the protecting group, benzyl group, is released and the desired final product Suc
-Phe-Glu-Pro-Ile-Pro-Glu-Tyr (SO ₃ H) -Tyr (SO ₃ H)-(D) Le
u-OH was obtained. The reaction liquid containing the final product was filtered, and then desalted and purified by preparative HPLC and gel filtration to obtain an eluate containing only the final product. After 10% ammonia water was added to the eluate to adjust the pH to 7 to 8, the final product was recovered as an ammonium salt by freeze-drying. The weight of the purified product (ammonium salt) was 13.8 mg, and the yield was
It was 15.7%.

[0041]

Example 3 Protecting group-substituted Suc-Phe-Glu (OBzl) -Pro-Ile-Pro of the hirudin analog (A) described in Example 1 above.
-Glu (OBzl) -Tyr-Tyr-Leu-Gln-OBzl, and the protective group-substituted Suc (OBzl) -Phe of the hirudin analog (B) described in Example 2.
-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr-Tyr- (D) Leu-OB
After the reaction of sulphating zl, the products in each of the resulting mixtures were analyzed by reverse phase HPLC. In addition, reverse phase HP
The LC measurement was performed under the following conditions. Column: Waters μ-Bondapak C ₁₈ (3.9 × 300 m
m) Solvent: Solvent A 0.1% trifluoroacetic acid aqueous solution Solvent B 0.1% trifluoroacetic acid aqueous solution and acetonitrile mixed gradient: Acetonitrile 10-60% / 50 min (concentration change rate of solvent B) Flow rate: 1.5 ml / min Detection: 230 nm (absorbance)

FIG. 1 shows the analysis result of the mixed solution obtained after the reaction of converting the protected group of the hirudin analog (A) of Example 1 into a sulfate group. A new peak corresponding to the reaction product was found in the mixture at an elution time of 35.7 minutes. The eluate of this peak was collected and the reaction solution containing the final product obtained by hydrogenolysis was analyzed and the results are shown in FIG.
Shown in. A new peak corresponding to the reaction product was found in the reaction solution at an elution time of 16.9 minutes.

On the other hand, International Patent Publication WO 92/15610
Hirudin analogue (A) Suc-Phe-Glu-Pro-Ile-Pro-Glu-Tyr-Tyr-Leu prepared in advance according to the solid-phase synthesis method described in 1.
-Gln-OH and its C-terminal Gln-substituted compound (D) Gln Suc-Phe-Glu-Pro-Ile-Pro-Glu-Tyr-Tyr-Leu- (D) Gln-
Using OH, the reaction liquid containing the disulfated ester compound treated under the reaction conditions of the above-mentioned sulfuric acid esterification of Example 1 was subjected to reverse phase HPLC analysis under the above measurement conditions. As shown in FIG. 3, the reaction solution obtained by treating the hirudin analog (A) under the reaction condition of sulfate esterification corresponds to the reaction product, the main peak corresponding to the elution time of 16.6 minutes, and the associated elution time of 17.3 minutes. Two minute peaks were found. In addition, the reaction solution obtained by treating the compound in which Gln at the C terminus of the hirudin analog (A) was replaced with (D) Gln under the reaction conditions of sulfate esterification, the elution time was
A major peak of 17.3 minutes and two peaks with an associated elution time of 16.6 minutes were found. The peak with an elution time of 16.6 minutes is
The peak which is a disulfate ester of hirudin analog (A) and has an elution time of 17.3 minutes is C of hirudin analog (A).
It can be seen that this is a disulfate esterified compound of the compound in which the terminal Gln is replaced with (D) Gln.

The compound found as a peak was fractionated at an elution time of 16.9 minutes shown in FIG. 2, and 30 μg of the compound and 30 μg of the above-mentioned hirudin analog (A) disulfated ester compound were collected.
G and g were mixed in an equal amount and subjected to reverse phase HPLC analysis. As shown in Fig. 4, both of them have a single peak and elution time of 16.7.
Eluted in minutes. Further, 30 μg of the compound found as a peak at the elution time of 16.9 minutes shown in FIG. 2 and the compound obtained by substituting the C-terminal Gln of the above-mentioned hirudin analogue (A) with (D) Gln Equal amounts of 30 μg of the product were mixed and subjected to reverse phase HPLC analysis. As shown in FIG. 5, both were eluted as a single peak at the elution times of 16.8 minutes and 17.4 minutes, respectively. From the above, the elution time shown in Fig. 2
The compound found as a peak at 16.9 minutes was Suc-Phe-Glu-Pro-I, a disulfate ester of the hirudin analog (A).
it is found that le-Pro-Glu-Tyr ( SO 3 H) -Tyr (SO 3 H) -Leu-Gln-OH.

FIG. 6 shows the analysis result of the mixed solution obtained after the reaction of converting the hirudin analogue (B) -substituted protective group of Example 2 into a sulfuric acid ester. A new peak corresponding to the reaction product was found in the mixture at an elution time of 47.3 minutes. The eluate of this peak was collected, and the reaction solution containing the final product obtained by hydrogenolysis was analyzed. As a result, a new peak corresponding to the reaction product was found in the reaction solution. But,
The elution time was found at 19.8 minutes. That is, the C-terminal is (D) Leu
It can be seen that this is due to the disulfate esterified product.

On the other hand, according to the solid-phase synthesis method described in International Patent Publication WO 92/15610, the hirudin analogue (B) prepared in advance was used and treated under the reaction conditions for the sulfate esterification of Example 1 above. The reaction liquid containing the esterified disulfate was subjected to reverse phase HPLC analysis under the above measurement conditions. As shown in FIG. 7, the reaction solution obtained by treating the hirudin analog (B) under the reaction condition of sulfate esterification corresponds to the reaction product, the main peak corresponding to the elution time of 19.9 minutes, and the associated elution time of 20.7 minutes.
Two minute peaks were found. Each of the two peaks is a disulfate esterified product, and is a hirudin analog (B).
Was a disulfate esterification product corresponding to the two optical isomers whose C-terminals were (L) Leu and (D) Leu.

From the above comparison, it can be seen that only one desired optical isomer can be selectively produced by the method of the present invention. Furthermore, there are two types of C-terminal amino acids, which are found when a hirudin analog that has not been sulfated by a solid-phase synthesis method is prepared in advance, and then a disulfate ester is prepared by the reaction of sulfate esterification. It can be seen that the side reaction of becoming the optical isomer of is suppressed.

[0048]

INDUSTRIAL APPLICABILITY In the production method of the present invention, the side chain and the C-terminal carboxyl group contained in the hirudin analogue have a substituted protecting group during the reaction of sulfuric acid esterification, so that the C-terminal amino acid There is an advantage that the isomerization reaction between the (L) form and the (D) form can be suppressed. Furthermore, in the amino acid residue having a carboxyl group in the side chain (L) body- (D)
It also has the advantage of suppressing interbody isomerization reactions. That is,
By suppressing the isomerization reaction between the (L)-and (D) -forms, the sulfate esterification reaction can be performed with good reproducibility. Only the body. Furthermore, in the production method of the present invention, the protective group substitution product of the hirudin analog can be prepared by a liquid phase synthesis method without relying on, for example, a solid phase synthesis method utilizing a resin, and a reaction of its sulfate esterification. Since it also uses a liquid phase reaction, it is possible to carry out a uniform reaction, and there is an advantage that it has excellent mass productivity.

[Brief description of drawings]

FIG. 1 shows the reverse phase H of the resulting mixed solution after the reaction of converting the hirudin analog (A) -substituted substituent of Example 1 into a sulfuric acid ester.
PLC analysis results, reaction product disulfate ester Suc (O
Bzl) -Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr (SO ₃ H)
-Tyr (SO ₃ H) elution peak of -Leu-Gln-OBzl - shows a click.

2 is a disulfate ester Suc (OBzl) -Phe of Example 1. FIG.
-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr (SO ₃ H) -Tyr (SO ₃
The figure which shows the reverse phase HPLC analysis result of the deprotected reaction product after the hydrogenolysis of (H) -Leu-Gln-OBzl.

FIG. 3 shows the results of reverse-phase HPLC analysis of the resulting mixed solution after the reaction of sulfidation of the hirudin analog (A) of Example 1, showing the disulfated ester of Suc-Phe-Glu-Pro-Ile-Pro. -Glu
_{-Tyr (SO 3 H) -Tyr (} SO 3 H) -Leu-Gln-OH elution peak - click and its attendant eluted peak - shows a click.

4 is a disulfate ester Suc (OBzl) -Phe of Example 1. FIG.
-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr (SO ₃ H) -Tyr (SO ₃
(H) -Leu-Gln-OBzl deprotected reaction product after hydrogenolysis and disulfate Suc-Phe-Glu-Pro-Ile-Pro-Glu
_{-Tyr (SO 3 H) -Tyr (} SO 3 H) -Leu- (L) shows the results of those equal amounts mixed and Gln-OH was reversed phase HPLC analysis.

FIG. 5: Disulfate esterified product Suc (OBzl) -Phe of Example 1
-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr (SO ₃ H) -Tyr (SO ₃
(H) -Leu-Gln-OBzl deprotected reaction product after hydrogenolysis and disulfate Suc-Phe-Glu-Pro-Ile-Pro-Glu
_{-Tyr (SO 3 H) -Tyr (} SO 3 H) -Leu- (D) shows the results of those equal amounts mixed and Gln-OH was reversed phase HPLC analysis.

FIG. 6 shows the reverse phase H of the resulting mixed solution after the reaction of converting the hirudin analogue (B) -substituted substituent of Example 2 into a sulfuric acid ester.
PLC analysis results, reaction product disulfate ester Suc (O
Bzl) -Phe-Glu (OBzl) -Pro-Ile-Pro-Glu (OBzl) -Tyr (SO ₃ H)
_{-Tyr (SO 3 H) - (} D) elution peak of Leu-OBzl - shows a click.

FIG. 7 shows the results of reverse-phase HPLC analysis of the resulting mixed solution after the reaction of sulfidation of the hirudin analog (B) of Example 2, the disulfated ester Suc-Phe-Glu-Pro-Ile-Pro. -Glu
_{-Tyr (SO 3 H) -Tyr (} SO 3 H) - (D) elution peak of Leu-OH - click and its attendant eluted peak - shows a click.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Tadanori Morikawa 530 Chokeiji Temple, Takaoka City, Toyama Prefecture Fuji Pharmaceutical Co., Ltd.

Claims (3)

[Claims] 1. A tyrosine residue containing a hirudin analogue, which is a peptide having a molecular weight of 3000 or less, which contains an amino acid sequence represented by the following general formula (I) as a part thereof. In order to produce a hirudin analogue sulfate ester or its salt by sulfuric acid esterification of the phenolic hydroxyl group of, the first step is to protect the side chain and C-terminal carboxyl group contained in the hirudin analogue with a protecting group. Body-protecting group-substituted product, then the phenolic hydroxyl group of the tyrosine residue contained in the protecting-group-substituted product is sulfated, and then the protecting group is eliminated, whereby a sulfated ester of a hirudin analogue is characterized. Or the manufacturing method of the salt. Phe-Glu-X1-Ile-Pro-X2-Tyr-Tyr- (I) (wherein X1 is Pro or Glu, X2 is Glu, Glu-Asp,
Means Glu-Tyr or Tyr, respectively. Also, the formula (I)
The amino group of the peptide may be protected in advance). 2. The method according to claim 1, wherein the hirudin analog contains two tyrosines in the amino acid sequence of the peptide, and the C-terminal amino acid is an optical isomer. 3. A method comprising reacting a substitution product of a protective group for a hirudin analog with a sulfa-trioxide (SO ₃ ) complex in the presence of a Lewis base type solvent to esterify a phenolic hydroxyl group of a tyrosine residue. The manufacturing method according to claim 1 or 2.