JP2662998B2 - Proline-containing peptide - Google Patents

Description

The present invention relates to a novel proline-containing peptide having anticoagulant activity.

[Conventional technology]

Conventionally, as anticoagulants, heparin which forms a complex with antithrombin III and inhibits the action of coagulation factors, or warfarin which inhibits the production of vitamin K-dependent coagulation factors is known. Recently, fibrin α-chain, N-terminal peptide glycylprolylarginyl valine and its derivative glycidylprolylarginylproline have been reported as peptides that inhibit the polymerization of fibrin, which is the final stage of blood coagulation. (Biochemistry, 19, 1013, 1980).

[Problems to be solved by the invention]

However, the effect is not always satisfactory, and development of a drug having an even better anticoagulant effect has been desired.

[Means for solving the problem]

Under such circumstances, the present inventors have conducted intensive studies on peptide compounds that exhibit an anticoagulant effect by suppressing the polymerization of fibrin, and as a result, completed the present invention.

That is, the present invention provides the following general formula (I): [In the formula, Z represents a proline residue or a prolylproline residue, and R ¹ and R ² are each independently a hydrogen atom, a linear or branched lower alkyl group, a cyclic alkyl group, a paranitrophenyl group or Represents an amino group, or R ¹
R ² together form an alkylene group or group (Where R ³ and R ⁴ each independently represent a hydrogen atom, a lower alkyl group, a hydroxyl group, a carboxyl group, a lower alkyloxycarbonyl group, a carbamoyl group or a lower alkylaminocarbonyl group, and m and n are simultaneously 0) Which is an integer from 0 to 5).] And a salt thereof.

In the formula (I), examples of the lower alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a secondary butyl group, a tertiary butyl group, a pentyl group and a neopentyl group. Linear or branched ones can be exemplified. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.

The compound of the formula (I) may be in a free form or a salt. When R ³ and / or R ^{4 in the} formula (I) is a carboxyl group, the salts of these carboxylic acids include, for example, alkali metal salts such as lithium salt, sodium salt and potassium salt, and alkali salts such as magnesium salt and calcium salt. Inorganic salts such as earth metal salts, ammonium salts, triethylamine salts, N-methylglucamine salts, and tris- (hydroxylmethyl) aminomethane salts, and organic salts. Arginine, lysine, or a basic portion of an amino acid such as ornithine can be an acid addition salt, as the acid addition salt, hydrochloride, sulfate, nitrate, hydrobromide, hydroiodide, Inorganic salts such as phosphates, or acetates,
Organic acid salts such as methanesulfonate, benzenesulfonate, toluenesulfonate, citrate, maleate, fumarate and lactate can be mentioned.

These free products, acid addition salts, and salts of carboxylic acids may exist as hydrates.

Further, the proline-containing peptide (I) of the present invention includes both optical isomers and racemates.

The proline-containing peptide (I) of the present invention can be produced by a chemical synthesis method using a known means.
For the peptide synthesis, either a liquid phase method or a solid phase method can be adopted.

The peptide chain extension method in peptide synthesis includes a stepwise elongation method in which amino acids are sequentially extended and a fragment condensation method in which a fragment consisting of several amino acids is synthesized in advance and then coupled between fragments. There is,
The proline-containing peptide of the present invention can be produced by any method.

Examples of the condensation method include an azide method, a mixed acid anhydride method, a dicyclohexylcarbodiimide (DCC) method, an active ester method, a carbodiimidazole method, a redox method, a diphenylphosphate azide (DPPA) method, and a DCC + additive (1-hydroxy Benzotriazole, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide) method, a method using Woodward reagent K, and the like.

The solvent can be appropriately selected from those known to be usable for the peptide condensation reaction. For example, dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide, dioxane, tetrahydrofuran, ethyl acetate, chloroform, dichloroethane or a mixture thereof can be mentioned.

The reaction temperature is not particularly limited and can be appropriately selected within a wide range, but it is generally preferable to carry out the reaction at a temperature of about -30 ° C to 30 ° C.

In producing the proline-containing peptide of the present invention, a carboxyl group of an amino acid or a peptide that does not participate in the reaction is generally esterified, that is,
Lower alkyl esters (methyl ester, ethyl ester, tertiary butyl ester, etc.), aralkyl esters (benzyl ester, p-methoxybenzyl ester,
(p-nitrobenzyl ester, etc.). As a protecting group for an amino group not involved in the reaction, a benzyloxycarbonyl group, a p-methoxybenzyloxycarbonyl group, a formyl group, a tertiary butyloxycarbonyl group, a trifluoroacetyl group and the like are usually used in the field of peptide synthetic chemistry. The protecting groups used are used.

Further, among the amino acids having a functional group in the side chain, as a protecting group for the guanidino group of arginine, for example, nitro group, tosyl group, p-methoxybenzenesulfonyl group, mesitylene-2-sulfonyl group, benzyloxycarbonyl group, An isobornyloxycarbonyl group, an adamantyloxycarbonyl group and the like are used.

Deprotection of a protective group-containing amino acid, a peptide fragment, and a proline-containing peptide having a protective group,
Usually, methods used in this field include a catalytic reduction method using palladium black, palladium carbon, platinum or the like as a catalyst, a liquid sodium method, a trifluoroacetic acid method, a hydrogen bromide method, a hydrogen chloride method, and a fluoride method. It is performed by a hydrogen method, an acetic acid method, a formic acid method, a methanesulfonic acid method, a trifluoromethanesulfonic acid method, or the like. In the method using an acid, it is effective to add a cation scavenger such as anisole or thioanisole.

Amino acids having a protecting group, peptide fragments, proline-containing peptides having a protecting group, and further a proline-containing peptide of interest are usually used to separate peptides.
It is easily isolated and purified by the means employed, for example, extraction, reprecipitation, silica gel chromatography, ion exchange chromatography, countercurrent distribution, gel chromatography and the like.

〔The invention's effect〕

The proline-containing peptide of formula (I) of the present invention has a novel structure and action mechanism different from conventional anticoagulants, and has a selected anticoagulant effect. It is also characterized by high activity compared to recently known fibrin α-chain and N-terminal peptide derivatives. Hereinafter, the anticoagulant effect of the proline-containing peptide of the present invention will be specifically described.

That is, fibrinogen thrombin time was measured by the following method. Sample Tris buffer 100μ
Was added to 100 μl of a bovine fibrinogen solution dissolved at a concentration of 6 mg / ml in Tris buffer, followed by incubation at 37 ° C. for 2 minutes. A bovine thrombin solution dissolved at a concentration of 2 to 3 U in Tris buffer was added thereto, and the coagulation time was measured using Crottech (manufactured by Sanko Junyaku). From the standard curve of thrombin amount-clotting time, the clotting time of the sample was converted to the amount of thrombin, and the inhibition rate (%) was calculated. A 50% inhibitory concentration (IC ₅₀ ) was determined from the sample concentration and the inhibition rate (%), and this was used as an index of the coagulation action. The relative intensity at the time of the IC ₅₀ of glycyl prolyl Al vinyl proline and 1 are shown in Table 1.

Since the compound of the present invention has a strong anticoagulant effect, it can be used as a human medicine.

When the compound of the present invention is used as a human medicine, the dose varies depending on the administration method, but in the case of intravenous administration,
10 mg to 250 mg, preferably 30 mg to 5 mg / day for an adult
The range is 0 mg. This dose may be intravenous or intravenous drip. In the case of oral administration, the dose is 60 mg to 1 g, preferably 300 mg to 600 mg per day for an adult. This dose is orally administered in two to four divided doses. Also, the daily doses indicated above may optionally exceed the above-mentioned amounts.

The compounds of the present invention are effective against diseases caused by abnormalities in the coagulation system, and can treat, prevent or reduce diseases caused by abnormalities in the coagulation system.

Diseases caused by abnormalities in the coagulation system include:
For thromboembolism such as venous thrombosis, myocardial infarction, pulmonary embolism, cerebral embolism, limb thromboembolism, thromboembolism during and after surgery, and pandemic intravascular coagulation syndrome (DIC) is there.

The anticoagulant containing the compound of the present invention can be prepared by selecting an appropriate preparation according to the administration method, and preparing various commonly used preparations. As oral preparations, for example, tablets,
Examples include powders, granules, capsules, solutions, syrups, elixirs, oily or aqueous suspensions, and the like.
Injectables may contain stabilizers, preservatives, and solubilizing agents in the formulation, and may contain a solution that may contain these adjuvants in a container, and then be prepared as a solid formulation by freeze-drying etc. May be prepared as a preparation. One dose may be stored in a container, or multiple doses may be stored in the same container.

Solid preparations include pharmaceutically acceptable additives together with the active compound, such as fillers, extenders, binders, disintegrants, dissolution enhancers, wetting agents, lubricants and the like. They can be selected and mixed as needed to form a formulation.

Liquid preparations include solutions, suspensions, emulsions and the like, but may also contain suspending agents, emulsifiers and the like as additives.

As an example of an injection preparation, a lyophilized preparation vial containing 50 mg of the compound of Example 1, 25 mg of mannitol, and 45 mg of sodium chloride was reconstituted with 5 ml of sterile water for injection, and then physiological saline or 5% dextrose injection. Can be mixed.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

The abbreviations used below are the abbreviations used in ordinary amino acid chemistry, some of which are as shown below.

Z; benzyloxycarbonyl group Z (OMe); p-methoxybenzyloxycarbonyl group Arg (NO ₂ ); ^NG- nitroarginine Boc; tertiary butoxycarbonyl group Arg (Mts); ^NG -mesitylenesulfonylarginine and ROHM・ Resin manufactured by And Haas Company, ABERLITE IRF-400 (AMBERLITE IRF-400), YMC-p chromatographic column for high performance liquid chromatography manufactured by YMC
ack D-ODS and the like were used for purification.

Each Rf value in the thin layer chromatography is a value when the following solvent is used.

Rf ¹ ; Rf value when the lower layer of n-butanol: acetic acid: water = 4: 1: 5 is used as a developing solvent.

Rf ² ; Rf value using n-butanol: pyridine: acetic acid: water = 4: 1: 1: 2 as a developing solvent Rf ³ ; chloroform: methanol: water = 8: 3: 1 as a developing solvent Rf value at the time.

Rf ⁴ ; R when benzene: ethyl acetate = 1: 1 is used as a developing solvent
f value.

Rf ⁵ ; Rf value when chloroform: acetic acid: methanol = 90: 2: 8 is used as a developing solvent.

The 1) H-Pro-N ( C 2 H 5) 2 · AcOH Z-Pro-OH5.00g: Example 1 H-Gly-Pro-Arg -Pro-N (C 2 H 5) Synthesis of ₂ · 2HCl After dissolving in 30 ml of N, N-dimethylformamide (DMF) and cooling to below -10 ° C with ice-salt,
2.8 ml of triethylamine and isobutyl chloroformate
2.6 ml was added. After about 10 minutes, cooled diethylamine 4.1
ml was added and stirred overnight. The solvent was distilled off, and the residue was dissolved in ethyl acetate.
The mixture was extracted and washed in the order of a% citric acid aqueous solution and water, and the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was dissolved in methanol (20 ml), acetic acid (1 ml) and palladium black were added, and the mixture was subjected to catalytic reduction at room temperature for 1 hour. After removing the catalyst, the solvent was distilled off and the residue was dried.

Yield: 1.43 g, oil, [α] _D : -57.3 ゜ (c = 1.3, methanol), Rf ³ : 0.58 2) Z (OMe) -Arg (NO ₂ ) -Pro-N (C ₂ H ₂ ) ₂ Dissolve 2.82 g of Z (OMe) -Arg (NO ₂ ) -OH in 20 ml of DMF, cool to −10 ° C. or lower with ice-brine, and add triethylamine.
1.0ml, 1-hydroxybenztriazole (HOBT)
1.00 g and 1,3-dicyclohexylcarbodiimide (DC
C) 1.50 g was added. About 10 minutes later, AcOH.H-Pro-N (C) neutralized with 0.7 ml of triethylamine under ice-cooling in 5 ml of DMF
₂ H _{5) 2} 0.83g was added and stirred overnight. The residue was dissolved in ethyl acetate, extracted and washed in the order of a 10% aqueous sodium carbonate solution, water, a 5% aqueous citric acid solution and water. The ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was purified using a silica gel column (φ1.6 × 45 cm) and a solvent (chloroform, 1% methanol / chloroform). 1%
The solvent of the fraction eluted with methanol / chloroform was distilled off, and the residue was dried.

Yield 2.20 g, amorphous, [α] _D : -46.6 ゜ (c = 1.0,
Methanol), Rf ³ : 0.73 Elemental analysis: Calculated as C ₂₄ H ₃₇ N ₇ O ₅ C 53.82, H 6.96, N 18.31 Analytical values C 53.98, H 7.11, N 17.97 3) Boc-Gly-Pru-Arg (NO ₂ ) -Pro-N (C ₂ H ₅ ) ₂ Z (OMe) -Arg (NO ₂ ) -Pro-N (C ₂ H ₅ ) ₂ 1.00 g was added to anisole 2.0 ml, and trifluoroacetic acid ( TFA)
3.0 ml was added and the mixture was stirred at room temperature for 1 hour to remove the Z (OMe) group. Then, ether was added thereto under ice cooling to precipitate the precipitate, which was collected by filtration. 0.51 g of Boc-Gly-Pro-OH was dissolved in 8 ml of DMF, cooled to −10 ° C. or less with ice-salt, 0.26 ml of triethylamine and 0.25 ml of isobutylchloroformate were added, and the mixture was stirred. about
After 10 minutes, H-Arg (NO ₂ ) -Pro-N (C ₂ H ₅ ) ₂ neutralized with 0.26 ml of triethylamine in 10 ml of DMF was added and stirred overnight. The solvent was distilled off, the residue was dissolved in n-butanol, and then extracted and washed in the order of 10% aqueous sodium carbonate solution, water, 5% aqueous citric acid solution and water, and the solvent of the n-butanol layer was distilled off. Ether was added to the residue for crystallization.

Yield 0.96 g, mp 118-122 ° C, [α] _D : -89.7 ゜ (c = 1.
0, methanol), Rf ² : 0.77 Elemental analysis: C ₂₇ H ₄₇ N ₉ O ₈ · 1 / 2H ₂ O Calculated C 51.09, H 7.62, N 19.86 Analytical values C 51.04, H 7.60, N 19.72 4) H -Gly-Pro-Arg-Pro- N (C 2 H 5) 2 · 2HCl Boc-Gly-Pro-Arg (NO 2) -Pro-N (C 2 H 5) 2 0.50g
Was dissolved in methanol (10 ml), palladium was added, and the mixture was cooled to room temperature.
The catalytic reduction was performed for 18 hours to remove the nitro group. The solvent was distilled off, and 2.0 ml of 6N hydrogen chloride / dioxane was added to the residue under ice cooling, followed by stirring at room temperature for 1 hour to remove the Boc group. Ether was added under ice cooling to precipitate, decanted several times with cold ether, and dried. Water was added to the residue and lyophilized.

Yield 0.44 g, amorphous, [α] _D : -119.3 ゜ (c = 1.0,
H ₂ O) Amino acid ratio after 20 hours acid hydrolysis: Gly1.00, Pro2.05, A
Rg0.98, recovery rate (92%) Elemental _{analysis: C 22 H 40 N 8 O} 4 · 2HCl · 2H 2 O Calculated C 44.82, H 7.86, N 19.01 analytical values C 44.92, H 7.98, N 18.53 Example 2 and 3 In the same manner as in Example 1, Was synthesized. The physical properties of these and the intermediates for synthesizing them are as follows.

Melting point: 97-98 ° C [α] _D : −21.5 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₁₈ H ₂₄ N ₂ O ₃ C 68.33 H 7.65 N 8.85 Analytical value C 68.25 H 7.65 N 8.70 Rf ⁴ : 0.27 [Α] _D : -48.2 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₂₅ H ₃₇ N ₇ O ₇ C 54.83 H 6.81 N 17.91 Analytical value C 54.66 H 6.70 N 17.66 Rf ³ : 0.77 [Α] _D : -90.1 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₃₁ H ₄₅ N ₉ O ₈ C 55.43 H 6.75 N 18.77 Analytical value C 55.19 H 6.80 N 18.48 Rf ³ : 0.72, Rf ⁵ ; 0.32 [Α] _D: -144.6 DEG (c = 1.0, water) Elemental _{analysis: C 23 H 42 N 8 O} 5 · 2AcOH · 3H 2 O Calculated C 47.36 H 8.24 N 16.36 analytical values C 47.71 H 8.44 N 16.69 Rf ² : 0.46 amino acid ratio by acid degradation: Gly, 1.00; Pro, 1.04; Arg, 0.
96 (recovery rate; 77%) Melting point: 55-58 ° C [α] _D : -33.0 ゜ (c = 1.0, methanol) Rf ³ ; 0.68 Melting point: 91-95 ° C [α] _D : −22.4 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₃₃ H ₅₀ N ₆ O ₆ S C 58.65 H 7.94 N 13.24 Analytical value C 58.38 H 7.83 N 12.90 Rf ⁵ : 0.60 Melting point: 108-111 ° C [α] _D : −72.1 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₄₁ H ₅₉ N ₈ O ₈ S · 1 / 2H ₂ O C 59.11 H 7.26 N 13.45 Analytical value C 59.07 H 7.04 N 13.24 Rf ⁵ : 0.58 [Α] _D : -104.3 ゜ (c = 1.0, water) Amino acid ratio by acid decomposition: Gly, 1.00; Pro, 1.91; Arg, 1.
07 (recovery rate; 80%) ・ Z-Gly-Pro-OH P.Bruckner et al., Helv. Chim. Acta., 58, 1276 (197
Synthesized according to 5).

Example 4 ・ Synthesis of 2HCl: 1) 1.10 g of Z-Pro-pNA p-nitroaniline was dissolved in 6.0 ml of dry pyridine, cooled to −10 ° C. or less with ice-saline, and then mixed with phosphorus trichloride (0.35 g).
ml) of dry pyridine (5.0 ml) was added dropwise. After about 15 minutes, the temperature was returned to room temperature, 2.00 g of Z-Pro-OH was added, and the mixture was heated to 60 to 70 ° C. After about 3 hours, the mixture was returned to room temperature and stirred overnight. The solvent was distilled off, the residue was dissolved in ethyl acetate, extracted and washed with 10% aqueous sodium carbonate, water, 5% aqueous citric acid and water in this order, and the ethyl acetate layer was dried over anhydrous sodium sulfate.
The solvent was distilled off. Ethyl acetate was added to the residue for crystallization.

Yield 1.76 g, mp 167-169 ° C., [α] _b : -107.5 ° (c = 1.
0, methanol), Rf ⁵ : 0.53 Elemental analysis: C ₁₉ H ₁₉ N ₃ O ₅ Calculated value C 61.78, H 5.18, N 11.38 Analysis value C 61.86, H 5.19, N 11.45 2) Boc-Arg (Mts) − Pro-pNA Z-Pro-pNA 1.00 g was added to a hydrogen bromide acetic acid solution (30
%, HBr / AcoH) and stirred at room temperature for 1 hour to remove the Z group. 1.50 g of Boc-Arg (Mts) -OH was dissolved in 10 ml of DMF, cooled to −10 ° C. or lower with ice-salt, 0.45 ml of triethylamine and 0.42 ml of isobutylchloroformate were added. After about 10 minutes, H-Pro-pNA neutralized with 0.40 ml of triethylamine in DMF under ice cooling was added, and the mixture was stirred overnight. The residue was subjected to a silica gel column (φ1.5 × 27 cm), solvent (chloroform, 1% methanol / chloroform, 2% methanol /
(Chloroform). The solvent of the fraction eluted with 2% methanol / chloroform was distilled off, and ether was added to the residue.
Crystallized.

Yield 1.40 g, mp 134-137 ° C., [α] _D : -72.0 ゜ (c = 1.
0, methanol), Rf ^3: 0.71 ³⁾ under ice-cooling anisole Z-Gly-Pro-Arg ( Mts) -Pro-pNA Boc-Arg (Mts) -Pro-pNA1.00g
0.2 ml and 3.0 ml of TFA were added, and the mixture was stirred at room temperature for 1 hour to remove the Boc group. Dissolve 0.55 g of Z-Gly-Pro-OH in 8 ml of DMF, cool to −10 ° C. or less with ice-salt, and then add 0.25 ml of triethylamine.
And 0.24 ml of isobutyl chloroformate. About 10 minutes later, H was neutralized with 0.21 ml of triethylamine in ice-cooled DMF.
-Arg (Mts) -Pro-pNA was added and stirred overnight. The solvent is distilled off, the residue is dissolved in ethyl acetate, and the extract is washed with 10% aqueous sodium carbonate solution, water, 5% aqueous citric acid solution and water in this order, and the ethyl acetate layer is dried over anhydrous sodium sulfate. did. Ethyl acetate and ether were added to the residue,
Crystallized.

Yield 1.05 g, mp 131 ° -134 ° C., [α] _D : -106.3 ゜ (c = 1.
0, methanol), Rf ³ : 0.67 Elemental analysis: C ₄₁ H ₅₁ O ₁₀ N ₉ S 1/2 H ₂ O Calculated value C 56.54, H 6.02, N 14.47 Analysis value C 56.36, H 6.06, N 14.22 4) H-Gly-Pro-Arg-Pro-pNA · 2HCl Z-Gly-Pro-Arg (Mts) -Pro-pNA 0.50 g was added with thioanisole 0.68 ml and TFA 9.89 ml under ice cooling and dissolved, and trifluoromethanesulfonic acid ( (TFNSA) 1.03 ml was added and stirred. The mixture was stirred for 1 hour under ice cooling and then for 1 hour at room temperature to perform deprotection. After stirring for a total of 2 hours, 250 ml of ether cooled to −10 ° C. or less with water-salt was added to precipitate, decanted several times with cold ether, and dried. The residue was dissolved in a 5% aqueous acetic acid solution, washed with ether, and the 5% acetic acid layer was removed from Amberlite IR.
The mixture was treated with A-400 (AMBER LITE IRA-400) at room temperature for 20 minutes to obtain acetate, and then freeze-dried. CDS column (YMC
-Pack D-CDS-5 φ20 × 250 mm), and purified by prep. To make the hydrochloride, an equimolar 1N HCl was added and lyophilized.

Yield 0.18 g, amorphous, [α] _D : -152.1 ゜ (c = 1.0,
Water), amino acid ratio after acid hydrolysis for 24 hours: Gly1.20, Pro
1.99, Arg1.00, recovery rate (85%) ・ Synthesis of 2HCl: Dissolve 5.00 g of Boc-Pro-OH in 30 ml of DMF, and ice-salt-
After cooling to 10 ° C or less, triethylamine 3.5 ml, HOBt 6.20 g
And 7.20 g of DCC were added. About 10 minutes later, 2.6 ml of cyclohexylamine cooled under ice cooling was added, and the mixture was stirred overnight. After dicyclohexylurea (DC-Urea) was removed, the solvent was distilled off, the residue was dissolved in ethyl acetate, extracted and washed with 10% aqueous sodium carbonate solution and water in this order, and the ethyl acetate layer was dried over anhydrous sodium sulfate. The solvent was distilled off. Ethyl acetate was added to the residue for crystallization.

Yield 5.42 g, mp 142-144 ° C., [α] _D : -48.1 ゜ (c = 1.
0, MeOH), Rf ⁵ : 0.72 Elemental analysis: C ₁₆ H ₂₈ O ₃ N ₂ Calculated C 64.83, H 9.52, N 9.45 Analytical value C 65.02, H 9.80, N 9.45 Add 0.9 ml of anisole and 2.0 ml of TFA to 0.95 g under ice-cooling.
The mixture was stirred for 1.5 hours to remove the Boc group. Then, ether was added thereto under ice-cooling to cause precipitation, decanted with cold ether several times, and dried. Dissolve 2.00 g of Boc-Arg (Mts) -OH in 10 ml of DMF, cool to −10 ° C. or less with ice-salt, and then add 0.67 ml of triethylamine.
And 1.20 g of HOBt and 1.40 g of DCC were added. After about 10 minutes, DMF10ml
Neutralized with 0.6 ml of triethylamine under ice cooling in medium Was added and stirred overnight.

After removing DC-Urea, the residue was dissolved in ethyl acetate,
The extract was washed with 10% sodium carbonate, water, 5% citric acid aqueous solution and water in this order, the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue is purified on a silica gel column (φ
1.2 × 19 cm) and a solvent (chloroform, 1% methanol / chloroform). The solvent of the fraction eluted with 1% methanol / chloroform was distilled off, and ether was added to the residue for crystallization.

Yield 1.88 g, mp 115-118 ° C., [α] _D : −30.8 ° (c = 1.
0, methanol), Rf ⁵ : 0.51 Elemental analysis: Calculated as C ₃₁ H ₅₀ O ₆ N ₆ S Calculated C 58.65, H 7.94, N 13.24 Analytical values C 58.93, H 8.18, N 12.91 0.1 ml of anisole and 2.0 ml of TFA were added to 1.00 g under ice cooling,
The mixture was stirred for 1.5 hours to remove the Boc group. Z-Gly-Pro-OH 1.
50 g was dissolved in 10 ml of DMF, cooled to −10 ° C. or less with ice-salt, and 0.7 ml of triethylamine, 0.50 g of HOBt, and 0.50 g of DCC were added. About 10 minutes later, H-Ar neutralized with 0.2 ml of ice in DMF under ice cooling.
g (Mts) − Was added and stirred overnight. After removing DC-Urea, the solvent was distilled off, the residue was dissolved in ethyl acetate, and the extract was washed with 10% aqueous sodium carbonate solution, water, 5% aqueous citric acid solution and water in that order, and the ethyl acetate layer was dried over anhydrous sodium sulfate And the solvent was distilled off. The residue is purified on a silica gel column (φ2.1 × 23c
m) and a solvent (chloroform, 1% methanol / chloroform). The solvent of the fraction eluted with 1% methanol / chloroform was distilled off, and petroleum ether was added to the residue for crystallization.

Yield 0.95 g, mp 122-125 ° C., [α] _D : -76.0 ゜ (c = 1.
0, methanol), Rf ⁵ : 0.66 Elemental analysis: C ₄₁ H ₅₈ O ₈ N ₈ S Calculated value C 59.83, H 7.10, N 13.61 Analysis value C 59.79, H 7.15, N 13.47 Under ice-cooling, 0.86 ml of thioanisole and 12.43 ml of TFA were added and dissolved, and 1.29 ml of TFMSA was added and stirred. The mixture was stirred for 1 hour under ice cooling and then for 1 hour at room temperature to perform deprotection. After stirring for a total of 2 hours, 250 ml of ether cooled to −10 ° C. or lower with ice-salt was added to precipitate, decanted several times with cold ether, and dried.
The residue was dissolved in 5% acetic acid, washed with ether, and the 5% acetic acid layer was treated with Amberlite IRA-400 at room temperature for 20 minutes.
After being made into acetate, it was freeze-dried. To make it a hydrochloride,
Equimolar 1N HCl was added and lyophilized.

Yield 0.40 g, amorphous, [α] _D : -105.2 ゜ (c = 1.0,
H ₂ O) Amino acid ratio after 24 hours acid hydrolysis: Gly1.00, Pro2.11, A
Rg1.03, recovery rate (87.7%) Elementary _{analysis: C 24 H 42 O 4 N} 8 · 2HCl · 2H 2 O Calculated C 46.82, H 7.86, N 18.20 analytical values C 46.64, H 7.56, N 17.91 Example 6 and 7 As Example 5 Was synthesized. The physical properties of the intermediates for synthesizing these are as follows.

Melting point: 148-150 ° C [α] _D ; -45.3 ° (c = 1.0, methanol) Elemental analysis: Calculated for C ₁₇ H ₃₀ N ₂ O ₃ C 65.77, H 9.74, N 9.02 Analytical values C 65.71, H 9.97 , N 9.10 Rf ⁵ : 0.75 Melting point: 112-116 ° C [α] _D : −30.6 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₃₂ H ₅₃ N ₆ O ₆ S Calculated value C 59.14, H 8.22, N 12.93 Analytical value C 59.27, H 8.37, N 12.63 Rf ⁵ : 0.78 Melting point: 118-121 ° C [α] _D : -71.1 (c = 1.0, methanol) Elemental analysis: Calculated as C ₄₂ H ₆₀ N ₈ O ₈ S · H ₂ O C 59.00 H 7.31 N 13.10 Analytical value C 59.26 H 7.27 N 13.09 Rf ⁵ : 0.50 ・ 2HCl [α] _D : -117.3 ゜ (c = 1.0, water) Elemental analysis: Calculated as C ₂₅ H ₄₄ N ₈ O ₄ 2HCl / 5 / 4H ₂ O C 48.74 H 7.93 N 18.18 Analytical value C 49.10 H 7.94 N 17.82 Amino acid ratio by acidolysis: Gly, 1.00; Pro, 2.08; Arg, 0.
94 (recovery rate; 89%) Melting point: 135-138 ° C [α] _D : -50.1 ゜ (c = 1.0, methanol) Elemental analysis: Calculated as C ₁₈ H ₃₂ N ₂ O ₃ C 66.63 H 9.94 N 8.63 Analytical value C 66.50 H 10.17 N 8.72 Rf ⁵ : 0.83 Melting point: 101-103 ° C [α] _D : −30.7 ゜ (c = 1.1, methanol) Elemental analysis: Calculated as C ₃₃ H ₅₄ N ₆ O ₆ S C 59.74 H 8.21 N 12.68 Analytical value C 59.76 H 8.51 N 12.38 Rf ⁵ : 0.80 Melting point: 113-116 ° C [α] _D ; -73.0 ゜ (c = 1.0, methanol) Elemental analysis: Calculated for C ₄₃ H ₆₂ N ₈ O ₈ S C 60.68 H 7.34 N 13.17 Analytical value C 60.41 H 7.26 N 12.90 Rf ³ : 0.67 ・ 2HCl [α] _D : -116.1 ゜ (c = 1.0, water) Elemental analysis: Calculated as C ₂₆ H ₄₆ N ₈ O ₄ 2HCl 7 / 4H ₂ O C 48.86 H 8.12 N 17.53 Analytical value C 48.86 H 7.99 N 17.53 Amino acid ratio by acid degradation: Gly, 1.00; Pro, 2.13; Arg, 1.
06 (recovery rate: 83%) Synthesis Example 10 H-Gly-Pro-Arg -Pro-Pro-NH 2 · 2AcOH: Applied Biosystems (Applied Biosystem
s) Model 430A peptide automatic synthesizer (MODEL430A PEPTIDE S
YNTHESIZER). The resin used was p-methoxybenzhydrylamine resin (0.61 mmol / g), and each protected amino acid was Boc-Gly-OH, Boc-Pro-OH,
Boc-Arg (Tos) -OH was used. All synthetic procedures followed the Applied Biosystems program. The final deprotection was performed using hydrofluoric acid (H
F) The mixture was treated with 10 ml under ice cooling for 1 hour. HF is distilled off,
The residue was dissolved in 5% AcOH, washed with ether, and washed with 5% AcOH.
The layer was treated with Amberlite IRA-400 at room temperature for 20 minutes to obtain acetate, and then lyophilized. The residue was subjected to ion exchange chromatography using carboxymethylcellulose (φ2.0 × 15 cm), solvent (water, 10 mM ammonium acetate, 2
(5 mM ammonium acetate, 50 mM ammonium acetate). The solvent of the fraction eluted with 25 mM ammonium acetate was distilled off,
Water was added and lyophilized.

Yield 0.14 g, amorphous, [α] _D : -152.3 ゜ (c = 1.0,
Water) Elemental _{analysis: C 23 H 39 O 5 N} 9 · 2CH 3 COOH · 5 / 2H 2 O Calculated C 47.22, H 7.63, N 18.36 analytical values C 47.50, H 7.35, N 18.37

Claims (5)

(57) [Claims] 1. The compound of the general formula (I) [In the formula, Z represents a proline residue or a prolylproline residue, and R ¹ and R ² are each independently a hydrogen atom, a linear or branched lower alkyl group, a cyclic alkyl group, a paranitrophenyl group or Represents an amino group, or R ¹
R ² together form an alkylene group or group (Where R ³ and R ⁴ each independently represent a hydrogen atom, a lower alkyl group, a hydroxyl group, a carboxyl group, a lower alkyloxycarbonyl group, a carbamoyl group or a lower alkylaminocarbonyl group, and m and n are simultaneously 0) A proline-containing peptide and a salt thereof. 2. The proline-containing peptide according to claim 1, which is H-Gly-Pro-Arg-Pro-N (C ₂ H ₅ ) ₂ and a salt thereof. (3) The proline-containing peptide according to claim 1, which is or a salt thereof. (4) The proline-containing peptide according to claim 1, which is or a salt thereof. 5. H-Gly-Pro-Arg- Pro-Pro-NH 2 a proline-containing peptides and salts thereof according to claim 1, wherein.