JPH08183797A - Production of platelet aggregation inhibiting peptide and peptide to be used therefor - Google Patents

Abstract

PURPOSE: To provide a peptide having excellent platelet aggregation inhibiting capability and safety and producible in a mass by liquid-phase synthesis. CONSTITUTION: This process for producing a platelet aggregation inhibiting peptide expressed by formula Orotyl-Ser-Arg-Gly-Asp-Trp-OH is characterized by (a) the removal of Boc group from Boc-Trp(Z)-OBzl and the coupling with Boc-Asp(OBzl)-OH, (b) the removal of Boc group from the resulting Boc-Asp(OBzl)-Trp(Z)-OBzl and the coupling with Boc-Gly-OH, (c) the removal of Boc group from the resulting Boc-Gly-Asp(OBzl)-Trp(Z)-OBzl and the coupling with Boc-Arg(Z)2 -OH, (d) the removal of Boc group from the resulting Boc-Arg(Z)2 -Gly-Asp(OBzl)-Trp(Z)-OBzl and the coupling with Boc-Ser-(Bzl)-OH, (e) the removal of Boc group from the resulting Boc-Ser(Bzl)-Arg(Z)2 -Gly-Asp(OBzl)-Trp(Z)-OBzl and the coupling with orotic acid and (f) the removal of Bzl group and Z group from the coupling reaction product.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a peptide having an inhibitory action on platelet aggregation and an intermediate used in the method, and more particularly to a compound of the formula: Orotyl-Ser-Arg-Gly-Asp-T.
The present invention relates to a method for producing a peptide having an inhibitory effect on platelet aggregation represented by rp-OH and an intermediate used in the method.

[0002]

2. Description of the Related Art In blood, platelets play a major role in preventing bleeding by adhering to the surface of damaged blood vessels. However, it is known that in a pathological environment, platelet aggregation is the main cause of thrombus formation, and the blood vessel is occluded by the thrombus. This obstruction prevents sufficient supply of oxygen and nutrients to tissues and organs, which is a major cause of ischemic diseases of the circulatory organs such as myocardial infarction and stroke. And
Today, such an ischemic disease has the second highest mortality rate after cancer and is a major social problem.

When performing medical treatment involving circulation of blood to the outside of the body, such as use of a heart-lung machine at the time of surgery or renal dialysis of patients with renal failure, platelet activation is also caused when blood circulates outside the body. Blood coagulation due to platelet aggregation can occur, which is a major obstacle to performing these procedures. Therefore, prevention of these thrombus or blood coagulation is a very important matter for the occurrence of the above-mentioned ischemic disease or the implementation of safe extracorporeal circulation.

Therefore, a platelet aggregation inhibitor has been used conventionally, but especially during extracorporeal circulation and surgery.
Needless to say, when a platelet aggregation inhibitor is used for the purpose of temporarily suppressing the platelet aggregation ability, it is desirable that the agent has excellent platelet aggregation inhibition ability. It is necessary to have a medicinal effect time and to have a property of being excellent in safety in that it is rapidly metabolized into a compound having no side effect after inactivation of the drug.

As described above, the compound has an excellent ability to suppress platelet aggregation, has a structure as close as possible to a natural peptide, and when administered in vivo, it is rapidly metabolized into a compound with no side effect after an appropriate drug efficacy time. Japanese Patent Application Laid-Open No. Heisei 5-23962 discloses a platelet aggregation inhibitor using a peptide having excellent safety characteristics that it loses its activity. Among them, it is disclosed that the peptide represented by the formula: Orotyl-Ser-Arg-Gly-Asp-Trp-OH is highly active.

[0006] However, what is specifically described in Japanese Patent Laid-Open No. Hei5-203962 as a method for producing the above-mentioned peptide is a solid-phase synthesis method, which is not suitable for large-scale synthesis.

[0007]

DISCLOSURE OF THE INVENTION An object of the present invention is to formulate Orotyl-Ser-Arg, which has excellent platelet aggregation inhibitory ability and safety.
It is to provide a method capable of producing a large amount of a peptide represented by -Gly-Asp-Trp-OH by a liquid phase synthesis method. Another object of the present invention is to provide an intermediate useful for producing the above peptide.

[0008]

[Means for Solving the Problems] As a result of earnest research in view of the above problems, the present inventors have found that Boc-Trp (Z) -OBzl is used as a starting material and sequentially coupled by a liquid phase method using a predetermined peptide. It was found that the peptide represented by the formula: Orotyl-Ser-Arg-Gly-Asp-Trp-OH, which is excellent in the ability to suppress platelet aggregation and safety, can be produced in a large amount by carrying out the above, and completed the present invention. .

That is, the present invention provides a method for producing a platelet aggregation-inhibiting peptide represented by the following formula (I): Orotyl-Ser-Arg-Gly-Asp-Trp-OH (I), which comprises: a) Boc -Trp (Z) -OBzl (wherein Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl,
OBzl represents a benzyl ester. ), The Boc group of
Coupling with oc-Asp (OBzl) -OH, b) removing the Boc group of the resulting Boc-Asp (OBzl) -Trp (Z) -OBzl and coupling with Boc-Gly-OH, c) The Boc group of the obtained Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl was removed and coupled with Boc-Arg (Z) ₂ -OH, d) the obtained Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl
Was removed and coupled with Boc-Ser (Bzl) -OH (in the formula, Bzl represents benzyl), and e) the obtained Boc-Ser (Bzl) -Arg (Z) ₂ -Gly. -Asp (OBzl) -Trp
A method for producing a peptide for inhibiting platelet aggregation, which comprises removing the Boc group of (Z) -OBzl and coupling with orotic acid, and then f) removing the Bzl group and the Z group.

The present invention also relates to Boc-Trp (Z) -OBzl Boc.
The peptide represented by the formula: Boc-Asp (OBzl) -Trp (Z) -OBzl obtained by removing the group and coupling with Boc-Asp (OBzl) -OH, the Boc group of the peptide of the peptide Remove and Boc
Formula obtained by coupling with -Gly-OH: Boc-Gly-A
The peptide represented by sp (OBzl) -Trp (Z) -OBzl, obtained by removing the Boc group of the peptide and coupling with Boc-Arg (Z) ₂ -OH, is represented by the formula: Boc-Arg (Z ) ₂ -Gly-Asp (OBzl) -Trp
The peptide represented by (Z) -OBzl and the Boc group of the peptide are removed and obtained by coupling with Boc-Ser (Bzl) -OH, the formula: Boc-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -Trp
It is a peptide represented by (Z) -OBzl (I).

The present invention will be described in detail below. In the present specification, when an abbreviation is used for an amino acid, a peptide, a protecting group, an active group, etc., it follows the rules of the International Union of Pure and Applied Chemistry (IUPAC), International Union of Biochemistry (IBU), or the conventional symbols in the relevant field. And An example is shown below.

Arg: arginine Asp: aspartic acid Gly: glycine Ser: serine Trp: tryptophan Z: benzyloxycarbonyl Boc: t-butoxycarbonyl Bzl: benzyl OBzl: benzyl ester The present invention provides the following formula (I): Orotyl-Ser. A method for producing a platelet aggregation-inhibiting peptide represented by -Arg-Gly-Asp-Trp-OH (I) by liquid phase synthesis. In the present invention, the peptide (I) is synthesized through steps a) to f) below.

A) Removal of the Boc group of Boc-Trp (Z) -OBzl to give B
Coupling with oc-Asp (OBzl) -OH, b) removing the Boc group of the resulting Boc-Asp (OBzl) -Trp (Z) -OBzl and coupling with Boc-Gly-OH, c) The Boc group of the obtained Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl was removed and coupled with Boc-Arg (Z) ₂ -OH, d) the obtained Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl
Boc group was removed and coupled with Boc-Ser (Bzl) -OH, and e) the resulting Boc-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -Trp was obtained.
The Boc group of (Z) -OBzl is removed and coupled with orotic acid, then f) the Bzl and Z groups are removed. In each step, the Boc (t-butoxycarbonyl) group may be removed using hydrochloric acid / dioxane, hydrochloric acid / ethyl acetate, trifluoroacetic acid, etc., preferably 4N hydrochloric acid /
Performed with dioxane solution. The reaction is preferably carried out at a temperature within the range of ice cooling to room temperature, specifically, 0 to 25
It is preferable to carry out at ° C. For example, a 4N hydrochloric acid / dioxane solution is added so that the peptide concentration becomes 5 to 10% by weight, and the mixture is stirred under ice cooling for 1 to 3 hours. Further, if necessary, the mixture is stirred at room temperature for 1 to 3 hours. Then, hydrochloric acid / dioxane is distilled off under reduced pressure.

Coupling in each step includes methylene chloride, chloroform, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone,
Water, methanol or the like can be used as a solvent,
It is preferably carried out in a solvent having a relatively low polarity such as methylene chloride. By using such a solvent, the operability in the post-treatment of the coupling reaction such as evaporation of the solvent is improved.

As the coupling reagent used in each step, dicyclohexylcarbodiimide, water-soluble carbodiimide, 1-propanesulfonic acid cyclic anhydride, diphenylphosphoryl azide, etc., which are known in the art, may be used, among which water-soluble carbodiimide is used. Is preferred. The coupling reagent is 1 for the peptide used.
It is preferable to add 5 to 5 equivalents, and particularly preferably 1 to 3 equivalents.

When carbodiimide is used as the coupling reagent, the coupling reaction can be promoted by adding a reagent such as 1-hydroxybenzotriazole. 1-Hydroxybenzotriazole is preferably added in an amount of 1 to 5 equivalents, more preferably 1 to 3 equivalents, relative to the peptide used. The coupling reaction is
It is preferable to carry out at a temperature within the range of ice cooling to room temperature, specifically at 0 to 25 ° C. To perform the coupling, for example, dissolve the peptide in methylene chloride,
While cooling in an ice bath, adjust the pH to 6 with triethylamine.
7 to 1-hydroxybenzotriazole,
A peptide to be a coupling partner and a water-soluble carbodiimide are sequentially added, and the mixture is stirred under ice cooling for 1 to 5 hours. Further, if necessary, the mixture is stirred at room temperature for 1 to 15 hours.

In each step, in order to prevent the growth of impurities, the obtained intermediate is preferably purified by recrystallization or column chromatography. Whatever method is used, the purification may be carried out by a conventional method. In the case of performing recrystallization, for example, the reaction solution after the reaction is washed twice with 5% citric acid, once with saturated saline solution, twice with 5% sodium hydrogen carbonate solution, and once with saturated saline solution to obtain a product. The organic layer was dried over anhydrous sodium sulfate, etc., and then evaporated under reduced pressure.
The solidified crude product may be recrystallized with methanol or the like.

When performing column chromatography,
For example, Kleselgel 60 manufactured by Merck & Co., Inc. may be used and eluted with chloroform containing 1 to 5% methanol as an elution solvent. By thus purifying the intermediate obtained in each step, the purification of the final product becomes easy. To remove the Bzl and Z groups in step f) it is preferred to carry out catalytic hydrogenolysis. At this time, it is preferable to use 5 to 10% palladium carbon as a catalyst. The catalyst is preferably added in an amount of 5 to 50% by weight, more preferably 20 to 40% by weight, based on the peptide. Catalytic hydrogenolysis may be performed by a conventional method, for example, the peptide is dissolved in a mixed solution of acetic acid / methanol / water, 5 to 10% of palladium carbon is added thereto, and 2 to 10 at 40 ° C. under hydrogen flow. Stir for 48 hours. This reaction may be carried out under atmospheric pressure, or may be carried out by raising the hydrogen pressure using an autoclave.

The peptide obtained by catalytic hydrogenolysis is purified by, for example, reverse phase liquid chromatography, and the desired platelet aggregation-inhibiting peptide: Orotyl-Ser-Arg-Gly-A.
Obtain sp-Trp-OH. Reversed-phase liquid chromatography may be carried out, for example, using a Bondapak C ₁₈ manufactured by Waters as a column and eluting with a gradient of 10 to 25% acetonitrile in 0.1% TFA at a flow rate of 10 to 20 ml / min.

Here, the starting materials used in the present invention
(1) and the intermediate compound (2) obtained in the present invention
(6) is shown below. (1) Boc-Trp (Z) -OBzl (2) Boc-Asp (OBzl) -Trp (Z) -OBzl (3) Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl (4) Boc- Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl (5) Boc-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl (6 ) Orotyl-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -O
Compounds Bzl (1) to (6) are dissolved in a relatively low polar solvent such as methylene chloride to form an amide bond because the nitrogen protons on the indole nucleus of tryptophan and the active protons of the guadinide group are protected. Methylene chloride can be used in the reaction, and the operability of post-treatment after reaction such as solvent removal is good as compared with the case of using dimethylformamide, which has a high boiling point and a high polarity, which is generally used in the field. Is. Further, since the nitrogen proton on the indole nucleus of tryptophan is protected, it is not necessary to use a carbonium ion scavenger such as ethanedithiol when removing the protecting group Boc, and the intermediate purification in each step can be easily performed. be able to. Furthermore, by unifying all the protecting groups of the compounds of (1) to (6) to benzyl (Bzl) and benzyloxycarbonyl (Z), the final deprotection for converting the protected peptide into the final product peptide. In the reaction, the catalytic hydrogenolysis reaction can be carried out under relatively mild conditions.

In the present invention, Boc-Trp (Z) -O is used as a starting material.
Bzl is used, but this Boc-Trp (Z) -OBzl is based on the paper by Kiso et al. (Kiso et al., Chemistry letters, 1983, p739).
And Tilak, Tetrahedron letters,
1968, p6323). In step a), Bo is used as a coupling partner.
c-Asp (OBzl) -OH, step b) with Boc-Gly-OH as the coupling partner, step c) with Boc-Arg (Z) ₂ -OH as the coupling partner, Boc-Ser (Bzl) -OH is used as a coupling partner in d), but these peptides can be obtained from reagent manufacturers such as Watanabe Chemical Industry Co., Ltd.

The platelet aggregation-inhibiting peptide obtained in the present invention: Orotyl-Ser-Arg-Gly-Asp-Trp-OH has an excellent ability to inhibit platelet aggregation and has a structure as close as possible to a natural peptide. When it is administered in vivo, it has an excellent safety characteristic that it is rapidly metabolized into a compound having no side effect and loses its activity after an appropriate drug efficacy time.

[0023]

EXAMPLES The present invention will be described in more detail with reference to production examples and examples, but the present invention is not limited to these production methods and examples. (Production Example 1) (1) Production of Boc-Trp-OBzl Boc-Trp-OH 25.00 g (0.0821 mol) was dissolved in DMF (dimethylformamide) 180 ml, and dicyclohexylamine 14.89 g (0.0821 mol) and benzyl bromide were added to the solution. 14.05g
(0.0821 mol) was added, and the mixture was stirred at room temperature for 40 hours. After the reaction, the generated precipitate was filtered off, and the filtrate was evaporated under reduced pressure. The residue was dissolved in 300 ml of ethyl acetate, the insoluble material at that time was removed by filtration, and then washed with 5% citric acid, water, 5% sodium hydrogen carbonate and saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give Boc-Trp-OBzl. The yield was 30.07 g, and the yield was 92.85%.
The results of mass spectrometry and NMR are as follows. Mass spectrum (C ₂₃ H ₂₆ N ₂ O ₄ : 394.9) FAB-MS: 417 (M + Na) NMR (CDCl ₃ , δ ppm): 1.43 (s, 9H), 3.28 (d, J = 5.
2,2H), 4.64 (dd, J = 5.2,7.8,1H), 5.05 (s, 2H), 6.94
(s, 1H), 7.08-7.24 (m, 2H), 7.31 (d, J = 7.8,1H), 7.54
(d, J = 7.6,1H), 8,45 (s, 1H) (2) Production of Boc-Trp (Z) -OBzl 25.00 g (0.0630 mol) of Boc-Trp-OBzl obtained in (1) Dissolved in 140 ml of methylene chloride, finely divided sodium hydroxide 6.30 g (0.1575 mol), tetrabutylammonium hydrogen sulfite 120 mg (0.63 mmol) and benzyl chloroformate 16.20 g (0.0945 mol) were added sequentially, under an argon stream at room temperature. It was stirred for 1 hour. After the reaction, the insoluble matter was filtered through Celite, and washed twice with ice-cooled 6N hydrochloric acid and saturated saline in this order. The organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the solidified crude product was recrystallized from methanol,
c-Trp (Z) -OBzl was obtained. Yield is 27.75g, yield 7
It was 5.00%. The results of mass spectrometry and NMR are as follows. Mass spectrometry (C ₃₁ H ₃₂ N ₂ O ₆ : 528.2) FAB-MS: 551 (M + Na) NMR (CDCl ₃ , δ ppm): 1.40 (s, 9H), 3.20 (m, 2)
H), 4.69 (dd, J = 5.2,7.3,1H), 5.05 (s, 2H), 5.41 (s, 2
H), 7.17-7.50 (m, 14H), 8.14 (br, 1H) (Example 1) a) Production of Boc-Asp (OBzl) -Trp (Z) -OBzl Boc obtained by repeating Production Example 1 -Trp (Z) -OBzl 70.00g
1000 ml of 4N hydrochloric acid / dioxane solution was added to (0.1352 mol),
After stirring for 1 hour and 10 minutes in an ice bath and further for 3 hours and 30 minutes at room temperature, hydrochloric acid / dioxane was distilled off under reduced pressure. The residue was dissolved in methylene chloride, and triethylamine was added while cooling with an ice bath to adjust the pH to about 7. 1-hydroxybenzotriazole 20.29g (0.1352mol), Boc-Asp (OBzl)
-OH 47.10 g (0.1458 mol) and water-soluble carbodiimide 30.4
8 g (0.1590 mol) was sequentially added, and the mixture was stirred in an ice bath for 1 hour and 30 minutes, and further stirred at room temperature for 15 hours.

After the reaction, the reaction solution was added with 5% citric acid (twice),
The extract was washed with saturated saline, 5% sodium hydrogen carbonate (twice) and saturated saline in this order. The organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the solidified crude product was recrystallized from methanol to obtain Boc-Asp (OBzl) -Trp (Z) -OBzl. The yield was 71.85 g, and the yield was 72.46%. The results of mass spectrometry and NMR are as follows. Mass spectrum (C ₄₂ H ₄₃ N ₃ O ₉ : 733.3) FAB-MS: 756 (M + Na) NMR (CDCl ₃ , δ ppm): 1.35 (s, 9H), 2.65 (dd, J =
5.6,11.6,2H), 2.96-3.08 (m, 1H), 3.16-3.22 (m, 2H)
, 4.95 (dd, J = 4.0,9.2,1H), 5.01 (s, 2H), 5.02 (s, 2
H), 5.41 (s, 2H), 7.11-7.52 (m, 19H), 8.15 (brs, 1H) b) Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl Boc-Asp (OBzl) -Trp (Z) -OBzl 15.00g (0.
200 ml of a 4N hydrochloric acid / dioxane solution was added to (0204 mol), and the mixture was stirred in an ice bath for 40 minutes and further stirred at room temperature for 2 hours, and then hydrochloric acid / dioxane was distilled off under reduced pressure. Dissolve the residue in methylene chloride, add triethylamine while cooling in an ice bath,
The pH was about 7. 1-hydroxybenzotriazole 3.13g (0.0204mol), Boc-Gly-OH 3.93g (0.0224mol)
And 4.70 g (0.0244 mol) of water-soluble carbodiimide were sequentially added, and the mixture was stirred in an ice bath for 45 minutes, and further stirred at room temperature for 15 hours.

After the reaction, the reaction solution was added with 5% citric acid (twice),
The extract was washed with saturated saline, 5% sodium hydrogen carbonate (twice) and saturated saline in this order. The organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the solidified crude product was recrystallized from methanol to obtain Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl. The yield was 13.15 g, and the yield was 79.94%. The results of mass spectrometry and NMR are as follows. Mass spectrometry (C ₄₄ H ₄₆ N ₄ O ₁₀ : 790.3) FAB-MS: 813 (M + Na) NMR (CDCl ₃ , δ ppm): 1.42 (s, 9H), 2.57 (dd, J =
2.4,4.4,1H), 2.65 (dd, J = 2.4,6.3,1H), 2.94-3.00 (m,
1H), 3.13-3.25 (m, 2H), 3.46-3.65 (m, 2H), 4.78-4.89
(m, 2H), 5.02 (s, 2H), 5.06 (s, 2H), 5.41 (s, 2H), 7.1
5-7.51 (m, 19H), 8.13 (brs, 1H) c) Production of Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl b) Boc-Gly- Asp (OBzl) -Trp (Z) -OBzl 5.00g
To (6.30 mmol) was added 4N hydrochloric acid / dioxane solution (100 ml), the mixture was stirred in an ice bath for 3 hours, and then hydrochloric acid / dioxane was distilled off under reduced pressure. The residue was dissolved in methylene chloride, and triethylamine was added while cooling with an ice bath to adjust the pH to about 7. to this
1-Hydroxybenzotriazole 0.97 g (6.30 mmol), Bo
6.69 g (6.96 mmol) of c-Arg (Z) ₂ —OH and 1.45 g (7.59 mmol) of water-soluble carbodiimide were sequentially added, and the mixture was stirred in an ice bath for 3 hours.

After the reaction, the reaction solution was added with 5% citric acid (twice),
The extract was washed with saturated saline, 5% sodium hydrogen carbonate (twice) and saturated saline in this order. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Tr.
A crude product of p (Z) -OBzl was obtained. The yield was 10.48g.
The results of mass spectrometry and NMR are as follows. Mass spectrometry (C ₆₆ H ₇₀ N ₈ O ₁₅ : 1214.5) FAB-MS: 1237 (M + Na) NMR (CDCl ₃ , δ ppm): 1.41 (s, 9H), 1.67-1.72
(m, 4H), 2.62-2.75 (m, 2H), 2.82-2.95 (m, 1H), 3.19
(d, J = 6.5,2H), 3.04 (m, 2H), 3.84 (m, 1H), 4.02 (m, 1H
H), 4.16 (m, 1H), 4.74 (dd, J = 5.9,7.1,1H), 4.85 (dd, J)
= 5.9,7.3,1H), 4.99 (s, 2H), 5.08 (s, 2H), 5.09 (s, 2)
H), 5.20 (s, 2H), 5.38 (s, 2H), 7.11-7.51 (m, 29H),
8.10 (brs, 1H), 9.29 (brs, 1H), 9.37 (brs, 1H) d) Boc-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl
Boc-Arg obtained in the preparation _{c) (Z) 2 -Gly-} Asp (OBzl) -Trp (Z) -OBz
l 10.23 g (8.43 mmol) was added with 4 ml of hydrochloric acid / dioxane solution (150 ml) and the mixture was stirred in an ice bath for 2 hours, and then hydrochloric acid / dioxane was distilled off under reduced pressure. The residue was dissolved in methylene chloride, and triethylamine was added while cooling with an ice bath to adjust the pH to about 7. 1-hydroxybenzotriazole 1.29 g (8.4
3 mmol), Boc-Ser (Bzl) -OH 2.86 g (9.27 mmol) and water-soluble carbodiimide 1.95 g (10.12 mmol) were sequentially added, and the mixture was stirred in an ice bath for 3 hours and further at room temperature for 15 hours.

After the reaction, the reaction solution was added with 5% citric acid (twice),
The extract was washed with saturated saline, 5% sodium hydrogen carbonate (twice) and saturated saline in this order. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give Boc-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp.
A crude product of (OBzl) -Trp (Z) -OBzl was obtained. The yield was 10.06 g, and the crude yield was 85.77%. The results of mass spectrometry and NMR are as follows. Mass spectrometry (C ₇₆ H ₈₁ N ₉ O ₁₇ : 1391.5) FAB-MS: 1414 (M + Na) NMR (CDCl ₃ , δ ppm): 1.43 (s, 9H), 1.58 (m, 4)
H), 2.80-2.95 (m, 3H), 3.20 (d, J = 6.5,2H), 3.46-3.55
(m, 2H), 3.70-3.77 (m, 2H), 3.95 (m, 1H), 4.12 (m, 1)
H), 4.23 (m, 1H), 4.42 (s, 2H), 4.76-4.91 (m, 3H), 5.0
1 (s, 2H), 5.02 (s, 2H), 5.10 (s, 2H), 5.12 (s, 2H), 5.
37 (s, 2H), 7.14-7.51 (m, 34H), 8.08 (brs, 1H), 9.30
(brs, 1H), 9.43 (brs, 1H) e) Orotyl-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -O
Preparation of Bzl Boc-Ser (Bzl) -Arg (Z) ₂ -Gly-Asp (OBzl) -T obtained in d)
rp (Z) -OBzl 9.84 g (7.07 mmol) in 4N hydrochloric acid / dioxane solution
After adding 150 ml and stirring in an ice bath for 2 hours and 30 minutes, hydrochloric acid /
Dioxane was distilled off under reduced pressure. Dissolve the residue in methylene chloride, add triethylamine while cooling in an ice bath, and adjust the pH.
Was set to about 7. 1-hydroxybenzotriazole
3.20 g (21.21 mmol), orotic acid 3.64 g (23.34 mmol) and water-soluble carbodiimide 4.91 g (25.61 mmol) were sequentially added, and the mixture was stirred in an ice bath for 3 hours and 30 minutes, and further stirred at room temperature for 15 hours.

After the reaction, the reaction solution was added with 5% citric acid (twice),
The extract was washed with saturated saline, 5% sodium hydrogen carbonate (twice) and saturated saline in this order. The organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and the solidified crude product was reprecipitated using DMF / ethyl acetate / diethyl ether (5: 2: 1) to obtain Orotyl-Ser (Bzl) -Arg. (Z) ₂ -Gly-Asp (OBzl) -Trp
(Z) -OBzl was obtained. Yield is 5.50g, yield 54.41%
Met. The results of mass spectrometry and NMR are as follows. Mass spectrometry (C ₇₆ H ₇₅ N ₁₂ O ₁₈ : 1429.5) FAB-MS: 1452 (M + N
a) NMR (CDCl ₃ , δ ppm): 1.60-1.81 (m, 4H), 2.79-2.
83 (m, 3H), 3.19 (m, 2H), 3.36-3.48 (m, 2H), 3.54-3.6
2 (m, 2H), 3.70-3.77 (m, 1H), 3.94 (m, 1H), 4.32 (m, 1H)
H), 4.42 (s, 2H), 4.80-5.00 (m, 3H), 5.01 (s, 2H X2),
5.08 (s, 2H), 5.12 (s, 2H), 5.35 (s, 2H), 6.38 (s, 1
H), 7.13-7.52 (m, 34H), 7.95 (brs, 1H), 9.28 (brs, 1)
H), 9.40 (brs, 1H) f) Production of Orotyl-Ser-Arg-Gly-Asp-Trp-OH e) Orotyl-Ser (Bzl)-
Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl 25.0mg (0.0175mm
ol) was dissolved in 7 ml of a mixed solution of acetic acid / methanol / water (4: 2: 1), to which 10 mg of 10% palladium carbon was added,
The mixture was stirred under a hydrogen stream at room temperature for 26 hours. After the reaction, 10% palladium carbon was removed with a centrifugal filtration filter (manufactured by Millipore), and the solvent was distilled off under reduced pressure. The residue was dissolved in 1M acetic acid solution and then purified by high performance liquid chromatography (HPLC). By HPLC, Bondapak C18 (φ19 x 150 mm)
Using a column (Waters) at a flow rate of 17 ml / min, 10% to 25% (20 minutes) acetonitrile in 0.1% TFA was used.
A peak with a retention time of 11.0 was collected by gradient elution.
As a result of purification by HPLC, Orotyl-Ser-Arg-Gly-Asp-T
rp-OH was obtained as the trifluoroacetate salt. Yield 9.7mg
And the yield was 73.43%. In addition, mass spectrometry, N
The results of MR, optical rotation measurement, amino acid analysis and HPLC analysis are as follows. Mass spectrum (C ₃₁ H ₃₉ N ₁₁ O ₁₂ : 757.7) FAB-MS: 758 (M + H) NMR (DMSO-d ₆ , δ ppm): 1.38-1.65 (m, 3H), 1.65
1.84 (m, 1H), 2.45 (dd, J = 7.6,16.0,1H), 2.86 (dd, J =
5.4,16.0,1H), 3.07-3.22 (m, 4H), 3.67-3.74 (m, 4H),
4.28 (dd, J = 7.5,13.0,1H), 4.44 (dd, J = 7.5,14.0,1)
H), 4.47 (dd, J = 7.5,14.0,1H), 4.65 (dd, J = 6.7,14.0,1
H), 6.18 (s, 1H), 6.98 (t, J = 7.3,1H), 7.07 (t, J = 6.8,1
H), 7.15 (d, J = 1.9,1H), 7.34 (d, J = 7.8,1H), 7.49-7.5
8 (m, 2H), 7.99 (d, J = 7.8,1H), 8.10 (d, J = 5.4,1H), 8.1
9 (d, J = 8.1,1H), 8.26 (d, J = 7.3,1H), 8.81 (d, J = 7.3,1)
H), 10.78 (s, 1H), 10.82 (s, 1H), 11.31 (s, 1H) [α] _D ²⁵ : 10.0 ° (C = 0.1, 1N-AcOH) Amino acid analysis (6N HCl + phenol, 24hr, 110 ° C) ): This method cannot be detected because tryptophan is decomposed during acid hydrolysis. Moreover, since the amino acid used as an external standard for quantification was a standard amino acid, orotic acid not contained in the standard amino acid could not be detected.

Trp − (1) Asp 0.90 (1) Gly 1.11 (1) Arg 1.00 (1) Ser 1.00 (1) Orotyl − (1) HPLC analysis: Cosmosil 5C18-AR (φ4.6 × 200 mm) column (Nacalai) 10-40% acetonitrile (60%) in 0.1% TFA at a flow rate of 1.0 ml / min.
Min) gradient elution showed a single peak with a retention time of 20.0 min.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, the formula excellent in platelet aggregation inhibitory ability and safety: Orotyl-Ser-Arg-Gly-Asp-Trp-OH
The peptide represented by can be produced in a large amount by liquid phase synthesis.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Miyazaki 1618 Ida, Nakahara-ku, Kawasaki-shi, Kanagawa Nippon Steel Corporation Advanced Technology Research Center Life Science Research Center

Claims (10)

[Claims] 1. A method for producing a platelet aggregation-inhibiting peptide represented by the following formula (I): Orotyl-Ser-Arg-Gly-Asp-Trp-OH (I), wherein a) Boc-Trp (Z)- OBzl (wherein Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl,
OBzl represents a benzyl ester. ), The Boc group of
Coupling with oc-Asp (OBzl) -OH, b) removing the Boc group of the resulting Boc-Asp (OBzl) -Trp (Z) -OBzl and coupling with Boc-Gly-OH, c) The Boc group of the obtained Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl was removed and coupled with Boc-Arg (Z) ₂ -OH, d) the obtained Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl
Was removed and coupled with Boc-Ser (Bzl) -OH (in the formula, Bzl represents benzyl), and e) the obtained Boc-Ser (Bzl) -Arg (Z) ₂ -Gly. -Asp (OBzl) -Trp
A method for producing a platelet aggregation-inhibiting peptide, which comprises removing the Boc group of (Z) -OBzl and coupling with orotic acid, and then f) removing the Bzl group and the Z group. 2. A water-soluble carbodiimide or a water-soluble carbodiimide and 1-hydroxybenzotriazole are used to carry out the coupling reaction.
A method for producing the described platelet aggregation inhibitory peptide. 3. The method for producing a platelet aggregation-inhibiting peptide according to claim 1, wherein methylene chloride is used as a solvent in the coupling reaction. 4. The method for producing a platelet aggregation-inhibiting peptide according to claim 1, wherein the Boc group is removed using hydrochloric acid / dioxane. 5. The method for producing a platelet aggregation-inhibiting peptide according to claim 1, wherein the Bzl group and Z group are removed by catalytic hydrogenolysis. 6. The method for producing a platelet aggregation-inhibiting peptide according to claim 5, wherein the catalytic hydrogenolysis is carried out in the presence of a 5 to 10% palladium carbon catalyst. 7. The Boc group of Boc-Trp (Z) -OBzl is removed to obtain Boc
Formula obtained by coupling with -Asp (OBzl) -OH: Boc
A peptide represented by -Asp (OBzl) -Trp (Z) -OBzl. 8. The formula: Bo obtained by removing the Boc group of the peptide according to claim 7 and coupling it with Boc-Gly-OH.
A peptide represented by c-Gly-Asp (OBzl) -Trp (Z) -OBzl. 9. A peptide obtained by removing the Boc group of the peptide according to claim 8 and coupling it with Boc-Arg (Z) ₂ —OH.
A peptide represented by the formula: Boc-Arg (Z) ₂ -Gly-Asp (OBzl) -Trp (Z) -OBzl. 10. The compound of the formula: Boc-Ser (Bzl) -Arg (Z) ₂ -Gly obtained by removing the Boc group of the peptide of claim 9 and coupling with Boc-Ser (Bzl) -OH. -Asp (OBzl) -Trp (Z)-
A peptide represented by OBzl.