JPH08217793A - Production of peptide and intermediate used therefor - Google Patents

Abstract

PURPOSE: To produce a large amount of peptide excellent in suppressing action on platelets aggregation and safety by liquid phase synthesis. CONSTITUTION: This method for producing a peptide represented by the formula: Pro-Ser-X-Gly-Asp-Trp-OH comprises removing a Boc group from Boc-Trp(Z)- OBzl and coupling with Boc-Asp(OBzl)-OH, removing a Boc group from the prepared Boc-Asp(OBzl)-Trp(Z)-OBzl and coupling with Boc-Gly-OH, removing a Boc group from the prepared Boc-Gly-Asp(OBzl)-Trp(Z)-OBzl and coupling with Boc-X-OH, removing a Boc group from the prepared Boc-X-Gly-Asp(OBzl)- Trp(Z)-OBzl and coupling with Boc-Ser(Bzl)-OH, removing a Boc group from the prepared Boc-Ser(Bzl)-X-Gly-Asp(OBzl)-Trp(Z)-OBzl and coupling with Z-Pro- OH, and removing a Bzl group and a Z group from the prepared Z-Pro-Ser-(Bzl)- X-Gly-Asp(OBzl)-Trp(Z)-OBzl.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a peptide having biological activity and an intermediate used in the method for producing the peptide, which is represented by the formula: Pro-Ser-X-Gly-Asp-Trp-OH. The present invention relates to a method for producing a biologically active peptide (particularly a peptide having an inhibitory action on platelet aggregation) 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, we have a compound having an excellent ability to suppress platelet aggregation and a structure which is as close as possible to a natural peptide, and when administered in vivo, a compound having no side effect promptly after a proper drug efficacy time. Japanese Patent Application No. 6-235745 proposes a platelet aggregation inhibitor that uses a peptide having excellent safety characteristics that it is metabolized to and loses its activity. Among them, the peptide represented by the formula: Pro-Ser-X-Gly-Asp-Trp-OH is highly active. However, the method specifically described in Japanese Patent Application No. 6-235745 as a method for producing the above-mentioned peptide is a solid-phase synthesis method and is not suitable for large-scale synthesis.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a formula excellent in platelet aggregation inhibitory ability and safety: Pro-Ser-X-Gly-
It is intended to provide a method capable of producing a large amount of a peptide represented by 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.

[0007]

[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. By doing so, it was found that a peptide represented by the formula: Pro-Ser-X-Gly-Asp-Trp-OH, which has excellent platelet aggregation inhibitory ability and safety, can be produced in large amounts,
The present invention has been completed.

That is, the present invention provides the following formula (I): Pro-Ser-X-Gly-Asp-Trp-OH (I) (wherein X represents proline, hydroxyproline, cyclohexylglycine or tertiary leucine). A) Boc-Trp (Z) -OBzl (wherein Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl, and
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) Obtained Boc-Gly-Asp (OBzl) -Trp (Z) -O
Boc group of Bzl was removed and coupled with Boc-X-OH, d) Boc of the obtained Boc-X-Gly-Asp (OBzl) -Trp (Z) -OBzl
The group was removed and coupled with Boc-Ser (Bzl) -OH (wherein Bzl represents benzyl), and e) the resulting Boc-Ser (Bzl) -X-Gly-Asp (OBzl)- Trp (Z) -OB
The Boc group of zl was removed and coupled with Z-Pro-OH, f) the resulting Z-Pro-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp (Z)-
A method for producing a peptide, which comprises removing a Bzl group and a Z group from OBzl.

The present invention also provides the formula: Boc-Asp (OBzl) -Trp.
Peptide represented by (Z) -OBzl, formula: Boc-Gly-Asp (OBzl)
-Trp (Z) -OBzl peptide, formula: Boc-X-Gly-Asp
Peptide represented by (OBzl) -Trp (Z) -OBzl, formula: Boc-Ser
(Bzl) -X-Gly-Asp (OBzl) -Trp (Z) -OBzl peptide and formula: Z-Pro-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp (Z)- OB
It is a peptide represented by zl.

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.

Asp: aspartic acid Gly: glycine Pro: proline Hyp: hydroxyproline Chg: cyclohexylglycine Tle: tertiary leucine Ser: serine Trp: tryptophan Z: benzyloxycarbonyl Boc: t-butoxycarbonyl Bzl: benzyl OBzl: benzyl ester

The present invention is a method for producing a peptide represented by the following formula (I): Pro-Ser-X-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) The Boc group of Boc-Trp (Z) -OBzl was removed to give Boc-Asp (OBzl)
-OH, b) removing the Boc group of the resulting Boc-Asp (OBzl) -Trp (Z) -OBzl and coupling with Boc-Gly-OH,

C) Obtained Boc-Gly-Asp (OBzl) -Trp (Z) -O
Boc group of Bzl was removed and coupled with Boc-X-OH, d) Boc of the obtained Boc-X-Gly-Asp (OBzl) -Trp (Z) -OBzl
The group was removed and coupled with Boc-Ser (Bzl) -OH, e) the resulting Boc-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp (Z) -OB
The Boc group of zl was removed and coupled with Z-Pro-OH, f) the resulting Z-Pro-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp (Z)-
The Bzl and Z groups are removed from OBzl.

When synthesizing a peptide in which X is Hyp (hydroxyproline), the hydroxyl group of Boc-X-OH used in step c) is preferably protected by a protecting group. Such protecting groups include Bzl (benzyl), Bzl (NO ₂ ) (2-
Nitrobenzyl), Bzl (Cl) (chlorobenzyl), Ztf
(1-benzyloxycarbonylamino-2,2,2-trifluoroethyl) and the like can be used, and among them, Bzl (benzyl) is preferably used.

In each step, the Boc (t-butoxycarbonyl) group can be removed by using hydrochloric acid / dioxane, hydrochloric acid / ethyl acetate, trifluoroacetic acid or the like, and preferably
It can be performed using a 4N hydrochloric acid / dioxane solution. The reaction is preferably carried out at a temperature within the range of ice cooling to room temperature, specifically at 0 to 25 ° 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. 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, Kieselgel 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), catalytic hydrogenolysis is preferably carried out. At this time,
Preference is given to using 5-10% palladium on carbon as 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 carried out 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 10 to 40% of hydrogen is added in a hydrogen stream.
Stir at ℃ for 2-48 hours. This reaction may be carried out under atmospheric pressure, or may be carried out by raising the hydrogen pressure using an autoclave.

Peptides obtained by catalytic hydrogenolysis are examples
For example, purify by reverse phase liquid chromatography,
To obtain the peptide: Pro-Ser-X-Gly-Asp-Trp-OH. Reverse
To perform phase liquid chromatography, for example, a column
As Waters Bondapak C ₁₈Flow rate of 10 to 20 ml /
10% to 25% acetonitrile in 0.1% TFA at min
It may be eluted with the gradient of.

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- X-Gly-Asp (OBzl) -Trp (Z) -OBzl (5) Boc-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp (Z) -OBzl (6) Z-Pro-Ser (Bzl ) -X-Gly-Asp (OBzl) -Trp (Z) -OBzl

In the compounds (1) to (6), 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. Further, this protection improves the solubility in a solvent having a relatively low polarity such as methylene chloride, and methylene chloride can be used in the amide bond formation reaction, and has a high boiling point and a high polarity generally used in the art. The operability of post-treatment after the reaction such as evaporation of the solvent is better than that in the case of using dimethylformamide. 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). Also, in step a) as a partner of coupling, Bo
c-Asp (OBzl) -OH, Boc-Gly-OH in step b), Boc-X-OH in step c), Boc-Se in step d).
r (Bzl) -OH is used and Z-Pro-OH is used in step e),
These peptides can be obtained by purchasing from reagent manufacturers such as Watanabe Chemical Industry Co., Ltd.

The platelet aggregation-inhibiting peptide obtained in the present invention: Pro-Ser-X-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.

[0028]

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 spectrometry (C ₂₃ H ₂₆ N ₂ O ₄ : 394.9) FAB-MS: 4
17 (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 Boc-Trp-OBzl 25.00 g (0.0630 mol) obtained in (1) was dissolved in 140 ml of methylene chloride, and 6.30 g of micronized sodium hydroxide was prepared. (0.1575 mol), tetrabutylammonium hydrogen sulfite 120 mg (0.63 mmol) and benzyl chloroformate 16.20 g (0.0945 mol) were sequentially added, and the mixture was stirred at room temperature for 1 hour under an argon stream. 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: 5
51 (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, 2H), 7.17-7.50 (m, 14H), 8.14 (br-s, 1H)

Example 1 Pro-Ser-Pro-Gly-Asp-Trp-O
Synthesis of H a) Production of Boc-Asp (OBzl) -Trp (Z) -OBzl Boc-Trp (Z) -OBzl 70.00 g obtained by repeating Production Example 1
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 spectrometry (C ₄₂ H ₄₃ N ₃ O ₉ : 733.3) FAB-MS: 7
56 (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, 2
H), 4.95 (dd, J = 4.0,9.2,1H), 5.01 (s, 2H), 5.02 (s,
2H), 5.41 (s, 2H), 7.11-7.52 (m, 19H), 8.15 (br-s, 1H)

B) Preparation of Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl
Concrete a) obtained in 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 (br-s, 1H)

C) Boc-Pro-Gly-Asp (OBzl) -Trp (Z) -OBzl
Manufacturing b) obtained in Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl 4.95g
150 ml of 4N hydrochloric acid / dioxane solution was added to (6.27 mmol), and 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 0.96 g (6.27 mmol), Bo
c-Pro-OH 1.49g (6.90mmol) and water-soluble carbodiimide
1.44 g (7.53 mmol) was added sequentially, and the mixture was stirred in an ice bath for 2 hours,
Further, the mixture was stirred at room temperature for 5 hours and 30 minutes.

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 crude product solidified was purified by column chromatography (Kieselgel 60 manufactured by Merck, elution: chloroform, methanol), and Boc-Pro-Gly-As.
p (OBzl) -Trp (Z) -OBzl was obtained. The yield was 4.35 g, and the yield was 78.27%. The results of mass spectrometry and NMR are as follows.

Mass spectrometry (C ₄₉ H ₅₃ N ₅ O ₁₁ : 887.9) MS: 910
(M + Na) NMR (CDCl ₃ , δ ppm): 1.43 (s, 9H), 1.71-1.96
(m, 2H), 2.03-2.13 (m, 2H), 2.82-2.98 (br, 2H), 3.20
(m, 2H), 3.32-3.52 (m, 2H), 4.17 (t, J = 6.1Hz, 1H), 4.7
6-4.91 (m, 2H), 5.02 (s, 2H), 5.06 (s, 2H), 5.43 (s, 2
H), 7.17-7.51 (m, 19H), 8.08 (br-s, 1H)

D) Boc-Ser (Bzl) -Pro-Gly-Asp (OBzl) -Trp
Preparation of (Z) -OBzl Boc-Pro-Gly-Asp (OBzl) -Trp (Z) -OBzl obtained in c) 1.
50g of 4N hydrochloric acid / dioxane solution was added to 00g (1.13mmol),
After stirring for 3 hours in an ice bath, 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 0.17 g (1.13 mmol),
Boc-Ser (Bzl) -OH 0.83 g (1.24 mmol) and water-soluble carbodiimide 0.26 g (1.35 mmol) were sequentially added, and the mixture was stirred in an ice bath for 2 hours and further at room temperature for 4 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 remove Boc-Ser (Bzl) -Pro-Gly-Asp (OBz
A crude product of l) -Trp (Z) -OBzl was obtained. The yield was 1.51 g. The results of mass spectrometry and NMR are as follows.

Mass spectrometry (C ₅₉ H ₆₄ N ₆ O ₁₃ : 1065.1) MS: 108
7 (M + Na) NMR (CDCl ₃ , δ ppm): 1.33 (s, 9H), 1.78-2.16
(m, 4H), 2.96 (m, 2H), 3.20 (d, J = 6.4,2H), 3.58-3.92
(m, 6H), 4.16 (m, 1H), 4.47 (s, 2H), 4.76-4.92 (m, 2
H), 4.99 (s, 2H), 5.05 (s, 2H), 5.41 (s, 2H), 7.11-7.
62 (m, 24H)

E) Z-Pro-Ser (Bzl) -Pro-Gly-Asp (OBzl) -T
Preparation of rp (Z) -OBzl Boc-Ser (Bzl) -Pro-Gly-Asp (OBzl) -Trp obtained in d)
(Z) -OBzl 1.34 g (1.26 mmol) in 4N hydrochloric acid / dioxane solution 50
After adding ml, the mixture was stirred in an ice bath for 3 hours, and 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 0.19g
(1.26 mmol), Z-Pro-OH 0.36 g (1.38 mmol) and water-soluble carbodiimide 0.29 g (1.51 mmol) were added sequentially, and 2 in an ice bath.
The mixture was stirred for an hour and further at room temperature for 3 hours and 30 minutes.

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 solid product was purified by column chromatography (Kieselgel 60 manufactured by Merck, elution: chloroform, methanol), and Z-Pro-Ser (Bzl).
-Pro-Gly-Asp (OBzl) -Trp (Z) -OBzl was obtained. Yield 1.16g
And the yield was 77.02%. The results of mass spectrometry and NMR are as follows.

Mass spectrometry (C ₆₇ H ₆₉ N ₇ O ₁₄ : 1196.3) MS: 121
8 (M + Na) NMR (CDCl ₃ , δ ppm): 1.70-2.19 (m, 8H), 2.94
(m, 2H), 3.08-3.22 (m, 2H), 3.30-3.90 (m, 6H), 4.04
(m, 1H), 4.42 (s, 2H), 4.75-4.89 (m, 2H), 5.01 (s, 2H)
X2), 5.40 (s, 2H x2), 7.03-7.52 (m, 29H)

F) Production of Pro-Ser-Pro-Gly-Asp-Trp-OH Z-Pro-Ser (Bzl) -P obtained by repeating the steps up to e)
73.0 mg (0.061 mmol) of ro-Gly-Asp (OBzl) -Trp (Z) -OBzl was dissolved in 7 ml of a mixed solution of acetic acid / methanol / water (4: 2: 1), and 16.5 mg of 10% palladium-carbon was added thereto. In addition, the mixture was stirred under a hydrogen stream at room temperature for 19 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). In HPLC, a Bondapak C18 (φ19 × 150 mm) column (Waters) was used at a flow rate of 17 ml / min.
Gradi of 10-25% (20 minutes) acetonitrile in 0.1% TFA
A peak with a retention time of 11.5 minutes was collected by ent elution. HP
As a result of purification by LC, Pro-Ser-Pro-Gly-Asp-Trp-OH was obtained as a trifluoroacetate salt. The yield is 29.5 mg,
The yield was 73.52%. The total yield from a) to f) was 25.67%. The results of mass spectrometry, NMR, amino acid analysis and HPLC analysis are as follows.

Mass spectrometry (C ₃₀ H ₃₉ N ₇ O ₁₀ : 658.3) FAB-MS:
658 (M + H) NMR (DMSO-d ₆ , δ ppm): 1.78-1.94 (m, 8H), 1.94-
2.23 (m, 2H), 2.25 (dd, J = 8.9,18.0,1H), 2.72 (dd, J = 4.
3,18.0,1H), 3.07-3.30 (m, 4H), 4.22 (m, 4H), 4.34-4.
48 (m, 2H), 4.65-4.85 (m, 2H), 7.01 (t, J = 7.6,1H), 7.1
0 (t, J = 7.3,1H), 7.19 (s, 1H), 7.37 (d, J = 7.8,1H),
7.55 (d, J = 7.8,1H), 8.09 (d, J = 8.4,1H), 8.18 (m, 2
H), 8.52 (br-s, 0.5H), 8.66 (d, J = 7.8,1H), 9.27 (br-
s, 0.5H), 10.87 (s, 1H) amino acid analysis (6N HCl ＋ phenol, 24hr, 110 ℃):

This detection method could not be detected because tryptophan was decomposed during acid hydrolysis. Trp − (1) Asp 0.99 (1) Gly 1.13 (1) Pro 2.05 (2) Ser 1.00 (1) HPLC analysis: Cosmosil 5C18-AR (φ4.6 × 200 mm) column (Nacalai Tesque) At 1.0 ml / min, 10-40% (60% 60% acetonitrile in 0.1% TFA.
Min) gradient elution showed a single peak with a retention time of 21.0 min.

Example 2 Pro-Ser-Hyp-Gly-Asp-Trp-O
A peptide represented by the formula: Pro-Ser-Hyp-Gly-Asp-Trp-OH was synthesized in the same manner as in Example 1 except that Boc-Hyp (Bzl) -OH was used in the H synthesis step c). The total yield was 21.11%. The results of amino acid analysis and HPLC analysis are shown below. Amino acid analysis (6N HCl ＋ phenol, 24hr, 110 ℃): Trp − (1) Asp 0.96 (1) Gly 1.10 (1) Hyp 1.04 (1) Ser 1.00 (1) Pro 1.11 (1) HPLC analysis: Cosmosil 5C18-AR ( φ4.6 × 200 mm) column (manufactured by Nacalai Tesque, Inc.) at a flow rate of 1.0 ml / min.
Min) gradient elution showed a single peak with a retention time of 19.7 min.

Example 3 Pro-Ser-Chg-Gly-Asp-Trp-O
A peptide represented by the formula: Pro-Ser-Chg-Gly-Asp-Trp-OH was synthesized in the same manner as in Example 1 except that Boc-Chg-OH was used in the H synthesis step c). The total yield was 29.39%. The results of amino acid analysis and HPLC analysis are shown below. Amino acid analysis (6N HCl ＋ phenol, 24hr, 110 ℃): Trp − (1) Asp 1.01 (1) Gly 0.99 (1) Chg 1.08 (1) Ser 1.00 (1) Pro 1.05 (1) HPLC analysis: Cosmosil 5C18-AR ( φ4.6 × 200 mm) column (manufactured by Nacalai Tesque, Inc.) at a flow rate of 1.0 ml / min.
Min) gradient elution showed a single peak with a retention time of 39.5 minutes.

Example 4 Pro-Ser-Tle-Gly-Asp-Trp-O
A peptide represented by the formula: Pro-Ser-Tle-Gly-Asp-Trp-OH was synthesized in the same manner as in Example 1 except that Boc-Tle-OH was used in the H synthesis step c). The total yield was 22.43%. The results of amino acid analysis and HPLC analysis are shown below. Amino acid analysis (6N HCl ＋ phenol, 24hr, 110 ℃): Trp − (1) Asp 0.99 (1) Gly 1.05 (1) Tle 1.02 (1) Ser 1.00 (1) Pro 1.10 (1) HPLC analysis: Cosmosil 5C18-AR ( (φ4.6 × 200 mm) column (manufactured by Nacalai Tesque, Inc.) at a flow rate of 1.0 ml / min and 10-40% (60%) of acetonitrile in 0.1% TFA.
Min) gradient elution showed a single peak with a retention time of 34.0 minutes.

Test Example 1 Activity Measurement of Synthetic Peptides The peptides obtained in Examples 1 to 4 were assayed for in vitro human platelet aggregation activity using PRP. The subjects were healthy men who had not taken any medication for at least 2 weeks. Blood sampling is 1/10 with needle # 19
Blood was collected from the vein of the inferior sac on an empty stomach using a plastic syringe in which a volume of 3.8% sodium citrate solution had been previously put. Immediately after blood collection, the two solutions were mixed by gently stirring the syringe. This blood was centrifuged at room temperature for 15 minutes (1100 rpm, 250 g), the rotation was stopped without applying a brake, and the supernatant was taken with a Komagome pipette to obtain platelet-rich plasma (P
RP) and stored at room temperature. Centrifuge the remaining blood after centrifugation for 15 minutes at room temperature (3500 rpm, 1500 g), stop without stopping the brake, and take the supernatant.
(PPP). After the PRP was prepared, the number of platelets was counted, and the experiment described below was carried out only when the number of platelets was 2 × 10 ⁸ / ml or more.

Platelet aggregation was performed by an 8-channel platelet aggregometer (Hematracer, Nikoh Bioscience, Tokyo, Jap.
an) was used to measure the change in the light transmittance of PRP.
First, put 200 μl of PPP and PRP in a glass cuvette and incubate at 37 ℃.
Of 100% and PRP of 0%. Next, the physiological saline or the physiological saline containing the sample is PRP.
Add 10 μl to the plate and incubate at 37 ℃ for 1 minute, then
Add 10 μl of 100 μg / ml collagen solution (final concentration 5 μl
(g / ml) Aggregation was induced, and then the permeability was measured for 7 minutes. In the experiment, it was first confirmed that aggregation was caused by using collagen and ADP, and only those having a maximum aggregation rate of collagen of 70% or more were used in the experiment.

The sample was dissolved in physiological saline so as to have a concentration of 2.2 × 10 ^-2 M, and a 2-fold dilution series was prepared based on this and used for the experiment. 10 for samples insoluble in saline
It was dissolved in a physiological saline solution containing dimethyl sulfoxide (%). The result is calculated as follows.

[0056]

[Equation 1]

A diagram was prepared by plotting the aggregation inhibition rate against the concentration of the sample, and from this figure, the concentration at which aggregation was suppressed by 50%
(IC ₅₀ ) was calculated. Table 1 shows the IC ₅₀ of each peptide.
Compared to RGDS (Table 1, Comparative Example 1) which is an amino acid sequence present in a fibrinogen molecule, each peptide having a basic skeleton introduced with an amino acid having a Pro-Ser structure at the N-terminus and having no guanidino group is Platelet aggregation inhibitory activity was greatly increased.

[0058]

[Table 1]

[0059]

According to the present invention, the formula: Pro-Ser-X-Gly-As
The peptide represented by p-Trp-OH can be produced in large quantities by liquid phase synthesis.

Claims (6)

[Claims] 1. A formula (I): Pro-Ser-X-Gly-Asp-Trp-OH (I) (wherein X represents proline, hydroxyproline, cyclohexylglycine or tertiary leucine). A) Boc-Trp (Z) -OBzl (wherein Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl, and
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-X-OH, d) the obtained Boc-X-Gly-Asp (OBzl) -Trp (Z) -OBzl Boc
The group was removed and coupled with Boc-Ser (Bzl) -OH (wherein Bzl represents benzyl), and e) the resulting Boc-Ser (Bzl) -X-Gly-Asp (OBzl)- Trp (Z) -OB
The Boc group of zl was removed and coupled with Z-Pro-OH, f) the resulting Z-Pro-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp (Z)-
A method for producing a peptide, which comprises removing a Bzl group and a Z group from OBzl. 2. The formula: Boc-Asp (OBzl) -Trp (Z) -OBzl (wherein Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl and OBzl represents benzyl ester). The peptide represented. 3. The formula: Boc-Gly-Asp (OBzl) -Trp (Z) -OBzl.
(In the formula, Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl, and OBzl represents benzyl ester). 4. The formula: Boc-X-Gly-Asp (OBzl) -Trp (Z) -OBzl.
(In the formula, X represents proline, hydroxyproline, cyclohexylglycine or tertiary leucine, Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl, and OBzl represents benzyl ester.). . 5. The formula: Boc-Ser (Bzl) -X-Gly-Asp (OBzl) -Trp.
(Z) -OBzl (where X is proline, hydroxyproline,
Represents cyclohexylglycine or tertiary leucine, Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl and OBzl represents benzyl ester. The peptide represented by. 6. The formula: Z-Pro-Ser (Bzl) -X-Gly-Asp (OBzl) -T.
rp (Z) -OBzl (wherein X represents proline, hydroxyproline, cyclohexylglycine or tertiary leucine, Boc represents t-butoxycarbonyl, Z represents benzyloxycarbonyl and OBzl represents benzyl ester. ) The peptide represented by.