KR0147817B1 - Method for producing tripeptide aldehydes - Google Patents

Abstract

None.

Description

Method for preparing tripeptide aldehyde

The present invention relates to a method for preparing tripeptide aldehyde, and more particularly, to a method for preparing tripeptide aldehyde represented by the following formula (I) having a high purity suitable for use in the pharmaceutical field.

In which R is hydrogen or a methyl group,

Phe is a phenylalanine group,

Pro is an L-proline group,

Arg is an L-arginine group.

(Pro and Arg are abbreviations for L-proline and L-arginine, respectively, as indicated by Biochem. 7, 126, 773 (1972) and Biochemistry 14, 449 (1975).)

D-phenylalanyl-L-prolyl-L-arginine aldehyde salt (unsuitable for therapeutic use because it loses antithrombin activity very rapidly in HD-Phe-form, and is also unsuitable for use in hydrochloride, citrate, tartarate, The same phenomenon occurred with the N ^G -carboxy derivatives of tosylate and tripeptide aldehyde.

However, the sulfates of HD-Phe-Arg-H and its N-methyl derivatives are very stable even in aqueous solution (British Patent No. 2,091,270 and European Patent No. 185,390), which are suitable for therapeutic use. The preparation method is as follows: (1) According to British Patent No. 2,091,270, t-butoxycarbonyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate or t-butoxycarbonyl Sulfate of HD-Phe-Arg-H by reacting -D-phenylalanyl-L-prolyl-N ^G -carboxy-L-arginine aldehyde with 1-12 equivalents of 1-12N sulfuric acid solution at a temperature of 40-60 ° C. (The manufacturing method of the first starting material is described in Example 1 of the patent application specification, and the manufacturing method of the N ^G -carboxy derivative is described in Belgian Patent No. 880,844).

(2) Further, according to the patent, benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde (ZD-Phe-Pro-Arg / Z /- H) may be hydrolyzed in the presence of sulfuric acid in the same amount as above to prepare sulfate of HD-Phe-Arg-H (how to prepare double-protected tripeptide aldehyde used as starting material is 169,870).

(3) Further, according to EP 185,390, N-methyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate (HD-Phe-Arg-HH ₂ SO ₄ ) is N-benzyl Tripeptide aldehyde sulfate freed by hydrolysis of oxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde in the presence of sulfuric acid to remove the protecting group It can be prepared by carrying out a method similar to the preparation of (the method for preparing the starting material tripeptide aldehyde is also described in the patent).

However, when the compound is prepared by such a manufacturing method, there is a disadvantage in that the target compound cannot be obtained with a high degree of purity desired, especially when a large amount is to be produced.

That is, the product obtained according to the method of (1) will contain about 4 to 6% by weight of calcium sulfate, which is analyzed by thin layer chromatography. In addition to the calcium salt, other impurities (eg, tripeptide alcohol, -Acid, -acetal) is contained in about 10% by weight or more. That is, not only is more than 20% by weight of the total product contaminated with impurities, but also hassles of neutralizing excess sulfuric acid and filtering precipitated calcium sulfate.

In addition, the tripeptide aldehyde sulfate prepared according to the method (2) above (hydrolysis of hydrogen), in addition to 5 to 10% by weight of contaminants which can be detected by thin layer chromatography analysis, is additionally detected by HPLC analysis. 10-15% by weight of the tripeptide aldehyde sulfate in an array is included. That is, 15 to 25% by weight of the total product will be contaminated. At this time, the 5 to 10% by weight of the contaminant is composed of the following, that is, the tripeptide acid produced in the synthesis of tripeptide aldehyde, tripeptide alcohol, the known structure and the unknown structure described in EP 185,370 It consists of different amounts of cyclic degradation products with and compounds that are located below the spots that appear due to the target compound in the chromatogram.

This overhydration has a significant influence on the formation of side reaction products in the latter part of the manufacturing process. If the hydrogenolysis is carried out in a known manner, i.e. in the presence of 1 mole of sulfuric acid at room temperature, hydrogenolysis Even after the reaction was completed, the reaction proceeded continuously and the target product was obtained in thin layer chromatography (adsorbent: kigel gel G, eluent: ethyl acetate / pyridine / acetic acid / water = 25: 20: 6: 11 mixture, developer: chlorotolidine). (in the case of _{HD-Phe-Arg-HH 2} SO 4 R f = 0.52 to 0.57) to the resulting spot additional spot (R _f = 0.39 to 0.44) in the lower part than the position which corresponds to the contaminants is always displayed, the In this case, the pollutants are mainly regarded as impurities.

In order to remove these contaminants, the end point of the reaction must be able to be detected very accurately, and once the reaction reaches the end point, the reaction must stop and not stop.

However, when the end point of the reaction is determined by thin layer chromatography analysis, when the eluent is used, the analysis time takes 20 to 30 minutes, and thus the reaction cannot be easily stopped when the end point is reached. Not applicable

In general, the perhydration reaction produces about 3 to 4% by weight of a side reaction product having the R _f value defined above, which must be removed because it is more toxic than the target compound obtained at the end point of the reaction.

Since the compound is very prone to racemization, contaminants produced during the processes cannot be substantially removed. In this way, the target compound produced at the end point cannot be purified, so in order to obtain it in high purity enough for pharmaceutical use, the intermediate compound having the desired purity must be produced and the formation of the side reaction product must be suppressed. This means that the racemization reaction must be suppressed during the process.

Accordingly, it is an object of the present invention to provide a preparation method that suppresses the formation of the side reaction product and obtains at least 95% of the tripeptide aldehyde salt represented by the general formula (I).

Therefore, the present invention is based on the formation of the DLD product, that is, the racemization reaction affects the pH, whereby the target compound and the intermediate in the solvent having a basic pH and the solvent having a neutral pH cause a racemization reaction, In solvents with acidic pH (pH = 2), the target compound can remain stable for several months. Therefore, it is very important that the case where the pH of the reaction mixture does not change to a neutral or basic pH even for a while does not occur. In order to achieve this object, N ^G -benzyloxycarbonyl-L-arginine lactam hydrochloride represented by the following general formula (IV) is mixed with a compound represented by the following general formula (V), according to the above-described method. Condensation by addition of a base to the mixed anhydride releases the arginine lactams from their salts. Under these conditions, the free arginine lactam immediately reacts with the mixed anhydride to form a protected tripeptide lactam represented by the following general formula (III), and then the racemization reaction generated due to the basic solvent can be removed.

In the above formula, Z is a benzyloxycarbonyl group,

Arg, R, Phe and Pro are as described above.

Moreover, in the present invention, if the complex compound obtained during the reduction of the protected protected tripeptide lactam represented by the above general formula (III) prepared as described above with lithium aluminum hydride is decomposed according to the above method, European Patent No. 185,390 In contrast to adding sulfuric acid to the reaction mixture as described above to make the pH of the reaction mixture acidic, the reaction mixture is added to dilute sulfuric acid, preferably 1 N cold (0-5 ° C.) sulfuric acid solution. . It was thus based on being able to stop or significantly reduce the racemization of the protected tripeptide aldehyde.

In addition, the present invention is the excess of the hydrogenolysis of the tripeptide aldehyde represented by the general formula (II) prepared by the above method at a low temperature, that is, the temperature of 10 ℃ or less, preferably 6 to 8 ℃ It should be based in the presence of sulfuric acid.

According to the experimental data, if the hydrogenolysis of ZD-Phe-Pro-Arg proceeds in the presence of 1 mole of sulfuric acid at a range different from the above temperature, the impurity content detected by thin layer chromatography of the obtained compound is 40 ° C. 17%, 10% at room temperature (20-25 ° C.), 4% at 6-10 ° C., and the content of DLD isomers detected by HPLC analysis was 12%, 5% at 40 ° C., room temperature and 10-6 ° C., respectively. And from 2 to 3%, the total impurity content of the product is 29%, 15% and 6-7%, respectively, at this temperature.

Thus, if the hydrocracking reaction takes place at low temperatures, the time required for the hydrocracking reaction does not increase significantly with decreasing temperature, while the amount of side reaction products can be significantly reduced.

If the hydrogenolysis reaction is carried out at low temperature in the presence of excess sulfuric acid, the formation of a side reaction product having an R _f value of 0.39 to 0.44 can be significantly suppressed, and also the need for accurate detection of the end point of the reaction is not required. There is this. According to the experiments, if held in ZD-Phe-Pro-Arg ( Z) the temperature of 6 to 8 ℃ -H of the hydrogenolysis reaction in the presence of 1.5 to 1.7 mole of sulfuric acid in tetrahydrofuran and the R _f value The side reaction product possesses only a small amount even after the perhydration reaction is carried out for 1 hour.

Moreover, it is preferred to use sulfuric acid in all steps of the reaction, if hydrochloric acid is used the sulfate may be contaminated with chloride ions.

As a result of proceeding the reaction according to the method described above, the amount of DLD isomers in the final target compound represented by the following general formula (I) is at most 2 to 4% by weight, the content of other contaminants (tripeptide acid and -alcohol) is 1 It will be reduced to less than or equal to weight percent.

Hereinafter, the present invention will be described in detail.

The present invention is a mixed anhydride obtained by condensation of an N ^G -protected-L-arginine lactam represented by the following general formula (IV) with a dipeptide represented by the following general formula (V): Reduced tripeptide lactam to lithium aluminum hydride to prepare a protected tripeptide aldehyde represented by the following general formula (II), and then hydrolyzed it in the presence of sulfuric acid to give the tree represented by the following general formula (I) In preparing the peptide aldehyde, a condensation reaction was carried out by adding a base to a mixture of N ^G -protected-L-arginine lactam hydrochloride represented by the following general formula (IV) and the dipeptide represented by the following structural formula (V). After obtaining the protected tripeptide lactam represented by the following general formula (III), which is a mixed anhydride, and adding the reaction mixture to the excess 1N sulfuric acid at a temperature of 0 to 5 ℃ To prepare a protected tripeptide represented by the following general formula (II) by dissolution, and then to the hydrogenated reaction of the protected tripeptide at a temperature of 10 ℃ or less in the presence of 1 to 2 moles of sulfuric acid It is a method for producing tripeptide aldehyde having a purity suitable for use in pharmaceutical use represented by general formula (I).

Where Arg is an L-arginine group,

Z is a benzyloxycarbonyl group,

R is hydrogen or a methyl group,

Phe is a phenylalanine group,

Pro is an L-proline group.

The present invention will be described in more detail as follows.

t- butoxycarbonyl -N ^{G-benzyloxycarbonyl} -L- arginine lactam N ^G represented by the general formula (IV) the t- butoxycarbonyl protecting group obtained by removing by a known method from the-benzyl Oxycarbonyl-L-argininelactam hydrochloride is added to the dimethylformamide solution at a temperature of -15 to -20 ° C, and then benzyloxycarbonyl-D-phenylalanyl-L-proline or N-methyl-N The reaction mixture is added to a mixed anhydride formed from a dipeptide represented by formula (V) prepared from -benzyloxycarbonyl-D-phenylalanyl-L-proline and chlorocarbonic acid isobutyl ester. . Subsequently, a base, preferably trimethylamine, is added thereto, and under these conditions, the lactam liberated by the base is immediately reacted with the mixed anhydride to produce a protected tripeptide lactam, which leads to the formation of a basic solvent which causes a racemization reaction. You can prevent it. If necessary, the product obtained at this time may be rapidly filtered through a silica gel plate for purification. Unlike known techniques, the selection of these steps eliminates the need for expensive purification of the protected tripeptide lactam by column chromatography.

The protected tripeptide lactam represented by the general formula (III) thus obtained is reduced by lithium aluminum hydride in dry tetrahydrofuran to form a complex compound, and the complex compound is decomposed. European Patent No. 185,390 In contrast to the above, the reaction mixture was added to sulfuric acid diluted with cooling at a temperature of 2 to 4 ° C., preferably an excess of 1N sulfuric acid solution, and then additional sulfuric acid was added to adjust the final pH to 2-3. Next, it is performed according to a well-known method. The resulting protected tripeptide aldehyde represented by the following general formula (II) was purified by filtration through a silica gel plate, and further subjected to a hydrogenolysis reaction.

The hydrocracking reaction is preferably a polar organic solvent, preferably tetrahydrofuran, or an alkanol, ie, lower alkanols such as methanol, ethanol, i-propanol, and particularly preferably ethanol. If the hydrocracking reaction is carried out in a closed system, hydrogen is not bubbled in the reaction mixture. However, substantially the hydrogen gas is bubbled through the system in a finely pulverized form, ie it becomes a gas through the G ₂ -filter.

The protected tripeptide aldehyde represented by the following general formula (II) having a suitable purity prepared in the present invention is dissolved in tetrahydrofuran and this solution is added to a cooled aqueous catalyst suspension containing 1.5 to 1.7 moles of 1N sulfuric acid. . At this time, the ratio of the oil solvent and the aqueous fraction is 70 to 50: 30 to 50, preferably 60: 40, and 10 to 30% by weight of 10% Pd / C or Pt as a catalyst, The hydrogenolysis reaction is carried out by bubbling hydrogen pulverized hydrogen gas through the reaction system while cooling at a temperature of 6 to 8 ° C., followed by thin layer chromatography analysis.

The protected tripeptide aldehyde, starting material, is dissolved in tetrahitfuran, and 10% Pd precipitated in a barium sulfate or aluminum oxide catalyst carrier, preferably a barium sulfate catalyst, is used as a catalyst.

The hydrogenolysis reaction is carried out by bubbling the finely divided hydrogen gas by passing through the reaction system in the presence of a temperature of 6 to 8 ℃ and 1.5 mol of 1N sulfuric acid. When the reaction is complete, it is analyzed by thin layer chromatography.

The protected tripeptide used as starting material is dissolved in ethanol and then the reaction mixture is added to a cold aqueous suspension containing 10% Pd / C catalyst and 1 mole of 1N sulfuric acid. The hydrogenolysis reaction is carried out by evaporating the finely divided hydrogen gas through a reaction system at a temperature of 6 to 8 ℃, followed by thin layer chromatography.

The desired compound can be easily recovered from the reaction mixture by filtration of the catalyst and evaporation of the organic solvent in an aqueous-oil solvent mixture at a temperature below 40 ° C. The pH is then adjusted directly or later to about 4, followed by lyophilization of the lyophilized aqueous solvent, which pH may be adjusted using ion exchange resins on hydroxy or 0.1 N sulfuric acid.

When the reaction proceeds as planned, the maximum contaminants obtained (D-phenylalanyl-L-proline-L-arginine aldehyde sulfate and N-methyl-D-phenylalanyl-L-propyl-L-arginine aldehyde sulfate) are thin layer chromatography. At most 1% and as detectable by HPLC analysis, at most 2-4% of the DLD isomers are detectable by chromatography analysis. Therefore, the total amount of impurities can be reduced to the level of about 3 to 5% required for pharmaceutical substances, which is in contrast to conventional impurities content of 15 to 25%.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The R _f value was determined by thin layer chromatography on silica gel (Kieselgel G, Rialal, Budapest) using the eluent mixture in each example, and the developer was chlorinated chromatography plate, which was dried and then 0-tolidine. The solution was treated with 2.5 g of 0-tolidine and 10 ml of acetic acid in 500 ml of water.

Example 1

Preparation of D-phenylalanyl-L-prolyl-L-arginine Aldehyde Sulfate

Benzyloxycarbonyl-D-phenylalanyl-L-propyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde 201 g (0.3 mole) was dissolved in 2 L of tetrahydrofuran at room temperature and then similarly 10% Pd 60 g / C was suspended in 0.6 L of non-ionized water and 900 mL (0.45 mol) of 1N sulfuric acid was added thereto. The suspension containing the catalyst was cooled to a temperature of 2 to 5 ° C., and then a tetrahydrofuran solution of protected tripeptide aldehyde and additionally 0.2 L of non-ionized water were added to the reaction mixture with stirring. Hydrogen was then bubbled through the reaction mixture and stirring continued at a temperature of 6-8 ° C.

The reaction was analyzed by thin layer chromatography, but a mixture of acetate / pyridine / acetic acid / water = 60: 20: 6: 11 was used as the eluent (R _f value: starting material: 0.92 to 0.96, intermediate: 0.38 to 0.44, purpose). Compound 0.0 to 0.10).

The reaction took 3 to 4 hours to complete and the catalyst was filtered off at the end of the reaction and the residue was washed with non-ionized water (3x100 mL). The filtrate and wash were collected and the temperature of 25 to 30 ° C. in a rotary evaporator. And tetrahydrofuran was distilled off under vacuum.

Subsequently, the remaining aqueous solution was extracted with dichloromethane (2x300 mL) and the pH of the aqueous phase was adjusted. If the pH was not 4, the pH was adjusted using 0.1 N sulfuric acid or OH-cyclic ion exchange resin (ie AG 1x2). Adjusted to.

Next, the reaction mixture was freeze-dried after supplementing non-ionized water until the volume of the reaction solution became 1.5 L, thereby obtaining 125 g of the target compound (yield: 83.2%).

R _f = 0.52 to 0.57 (ethylacetate / pyridine / acetic acid / water = 25: 20: 6: 11).

α _D ²⁰ = − 117 ° (C = 1, water).

Total impurity content according to thin layer chromatography analysis: 3 to 5%,

Content of D-L-D isomer according to HPLC: about 2 to 3%.

Benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde used as starting material was prepared by the following process.

First step: preparation of benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

429.5 g (1.1 mol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam (European Patent No. 185,390) was suspended in 1300 ml of dry chloroform, and the temperature of the reactant was 15 ° C. 1300 ml of hyclochlorethyl ethyl acetate (189 g / 100 ml: about 5 N) was added dropwise to the reaction mixture while stirring and cooling with ice water at a rate to prevent the increase from exceeding crystalline material. it started.

The suspension was stirred at room temperature for 3 hours, and then diluted by adding 3000 ml of an ether and ethyl acetate (1: 1) dry mixture. The precipitated crystalline material was then filtered off and washed with acetone (2 × 1000 mL) and ether (500 mL), respectively, and then dried with solid potassium hydroxide and phosphate pentoxide in a vacuum drying paper.

In water dried for 1 hour, N ^G -benzyloxycarbonyl-L-arginine-lactam hydrochloride was dissolved in 1000 ml of dimethylformamide and cooled to a temperature of -15 to -20 ° C, which was then To the prepared mixed anhydride was added:

396 g (1 mole) of N-benzyloxycarbonyl-D-phenylalanyl-L-propyl (Nicolaides, E. et al: J. Med. Chem. 11, 74 (1968) and European Patent No. 185,396) After dissolving in 1000 ml of formamide, the reaction mixture to which 111 ml (1 mol) of N-methylmorpholine was added was cooled to a temperature of -15 ° C, and 132 ml (1 mol) of chlorocarbonic acid isobutyl ester. Was added dropwise over 5-10 minutes with stirring at the same temperature as above. Anhydride and the reaction was stirred for 10 minutes, then N ^G obtained in the above was cooled to a temperature of -20 to -25 ℃ - was collected with a dimethylformamide solution of benzyloxycarbonyl -L- arginine hydrochloride rakcham yeogieda 308 mL (2.2 moles) of triethylamine was added dropwise over about 30 minutes at the same temperature as above. The suspension was then further stirred for 1 hour at a temperature of about −20 to −15 ° C., and then the pH of the vapor phase was adjusted to wet pH, but further triethylamine was added if the pH was 8 or less. After diluting the reaction mixture with 2 L of benzene, the precipitated salt was filtered off and washed with benzene (2x500 mL).

1.5 liters of water was added to the benzene-dimethylformamide filtrate, and each phase was separated. The lower aqueous dimethylformamide solution was extracted with benzene (2x500ml), and the benzene solution was collected to collect water (2x0.5l). , 0.1N sulfuric acid (1 × 0.5 L) and water (3 × 0.5 L), respectively, were washed with anhydrous sodium sulfate and dried to 2 L at a temperature below 40 ° C. and vacuum.

360 g of Kieselgel-G adsorbents were suspended in benzene to form filter plates with a diameter to height ratio of 8: 1 to 4: 1.

The benzene solution was sucked by passing through a filter plate under a slight vacuum, washed with 4.8 L of benzene in the same manner as above, and then collecting the collected benzene solution at a temperature of 40 ° C. or lower and about 600 mL in volume. After concentrating until 3 L of diisopropyl ether was added thereto with vigorous stirring.

Subsequently, the precipitate was filtered off and washed with diisopropyl ether (2 × 600 mL), and then dried in a vacuum evaporator with phosphate pentoxide paraffin chip to obtain 510 g of the target compound (yield: 76%).

Impurity content according to thin layer chromatography: 1-2%.

R _f = 0.55 to 0.65 (ethyl acetate)

( _Rf = 0.45 to 0.50 of DLD lactam in ethyl acetate).

[α] _D ²⁰ = -55.8 ° (C = 1, tetrahydrofuran)

Second Step: Preparation of benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

300 ml (0.45 mole) of the protected tripeptide lactam (first step of Example 1) were dissolved in 2.5 l of tetrahydrofuran cooled to a temperature of -5 ° C, and then -20 to-in a dry ice / acetone bath. Cool to a temperature of 25 ° C. and concentrate the concentrated solution of lithium aluminum hydride and tetrahydrofuran containing 0.375 mol of lithium aluminum hydride to 0.7 mol / l with vigorous stirring (over 20 to 25 minutes). It was added to the reaction mixture. Subsequently, the reaction mixture was subjected to thin layer chromatography using an eluent.

If necessary, a lithium aluminum hydride solution was added until the spot of 0.75 to 0.80, which is the R _f value corresponding to the lactam, disappeared.

Upon completion of the reaction, a portion of the reaction mixture was added to 3 L of 1N aqueous sulfuric acid solution while stirring at a temperature of 2 to 3 ° C., followed by further addition of sulfuric acid to adjust the final pH value to an optimum, and the reaction mixture until turbid. Water was added to the mixture, and extracted with n-hexane (3x0.9 L). Since the material was separated as an oil phase between the two extracts, they were homogenized with tetrahydrofuhan and then turbid by adding water. At this time, the aqueous phase was extracted with dichloromethane (2x2L), and the collected dichloromethane solution was washed with 5% sodium hydrogen carbonate (2x0.5L) at a temperature of 4 to 8 ℃. It was then dried over anhydrous sodium sulfate and evaporated in vacuo at a temperature below 40 ° C. (about 300 g of oily material was obtained).

The oily residue was dissolved in a mixture of dichloromethane and acetone 7: 3 and passed through a filter plate (diameter: height = 5: 1) prepared from 900 g of Kegel gel 600 adsorbent, but not inhaled, whereby dichloromethane was used as the eluent. A mixture of methane and acetone 7: 3 was used. The filter plate was washed with 6 liters of the same eluent mixture as above, which took less than 20 minutes. The collected filtrate was evaporated at a temperature of 40 ° C. or lower until it became about 600 ml, and then evaporated again by adding 1 L of benzene thereto, and additionally, 1.6 L of cyclohexane was added thereto, and the product precipitated. It became. The precipitate was filtered off, washed with cyclohexane (2 × 0.3 L), and dried in a vacuum evaporator at room temperature and in a paraffin chip to obtain 240 g of the target compound (yield: 79.7%).

R _f = 0.52 to 0.62 (ethylacetate / pyridine / acetic acid / water = 240: 20: 6: 11).

Impurity content according to thin layer chromatography: 0.5 to 2%.

[α] _D ²⁰ = -0.8 ° C. (C = 1, tetrahydrofuran)

Example 2

Preparation of D-phenylalanyl-L-prolyl-L-arginine-aldehyde sulfate

Benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde 20.1 g (0.03 mol) was dissolved in 214 ml of tetrahydrofuran, and then to a temperature of 0 ° C. After cooling, 4.3 g of Pd / C catalyst was dissolved in 70 ml of non-ionized water and 102 ml (0.051 mol) of 1N sulfuric acid, and then added to the reaction mixture. After cooling the catalyst suspension to a temperature of 2 to 5 ° C. and removing the air by nitrogen gas, the tetrahydrofuran solution was added to the suspension while stirring the tetrahydrofuran solution and finely divided hydrogen gas was added to the system, followed by cooling. The reaction temperature was maintained at 6-8 ° C. Subsequently, 12.5 g (yield: 83.2%) of the reaction mixture obtained at the end point of the reaction (about 3 hours) by thin layer chromatography using the same eluent mixture as described above was carried out as in Example 1.

R _f = 0.52 to 0.57 (ethylacetate / pyridine / acetic acid / water = 25: 20: 6: 11).

α _D ²⁰ = − 117 ° C. (C = 1, water).

Total impurity content according to thin layer chromatography analysis: 3.5%,

Content of D-L-D isomer according to HPLC: 1.5%.

Example 3

Preparation of D-phenylalanyl-L-prolyl-L-arginine-aldehyde sulfate

Benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde 20.1 g (0.03 mol) was dissolved in 2 L of ethanol at room temperature, and then 10% in barium sulfate carrier. 60 g of catalyst containing palladium were suspended in 0.6 L of non-ionized water, followed by addition of 600 mL (0.3 mol) of 1N sulfuric acid. The reaction suspension was cooled to a temperature of 2-5 [deg.] C. and an ethanol solution of the protected tripeptide aldehyde was added, followed by addition of non-ionic water at a ratio of 6: 4 to the aqueous phase. The hydrogen gas was bubbled by passing through the reaction mixture while stirring and cooling at a temperature of 6 to 8 ° C, and analyzed by thin layer chromatography according to Example 1 above. At the end of the reaction (after about 3 to 4 hours), the resulting catalyst is filtered off and washed several times with non-ionized water, the filtrate is collected and the alcohol is evaporated in a rotary evaporator to a temperature of 25 to 30 ° C. and the pH is about 3 As a result of direct lyophilization of the aqueous solution of 4 to 4, the target compound in monohydrate form was obtained in the same amount as in Example 1.

Example 4

Preparation of D-phenylalanyl-L-prolyl-L-arginine-aldehyde sulfate

125.1 g (yield 83.2%) of the target compound was obtained in the same amount as in Example 1, except that 43 g of Pd / C catalyst washed beforehand as a catalyst was used. .

Example 5

Preparation of N-methyl-D-phenylalanyl-L-prolyl-L-arginine-aldehyde sulfate

20.5 g (0.03 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-propyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde was dissolved in 214 ml of ethanol, and then 110 mL of non-ionized water, 60 mL (0.03 mol) of 1N sulfuric acid and 3 g of 10% Pd / C catalyst were added. Finely pulverized hydrogen gas was added to the mixture at a temperature of 6 to 8 ° C., followed by thin layer chromatography using a 25: 20: 6: 1 mixture of ethyl acetate / pyridine / acetic acid / water as eluent. At this time, R _f values of starting material, intermediate and target compound were 0.9, 0.7 and 0.4, respectively, and it took about 2 hours to complete the reaction. At the end of the reaction, the catalyst was filtered off and washed with non-ionized water (3x30ml). . Subsequently, the filtrate and washings were collected and evaporated in a rotary evaporator at a temperature below 25 ° C. until it became about 100 ml, and the residue was extracted with dichloromethane (2 × 50 ml).

The aqueous solution was distilled off to remove a small amount of organic solvent, the residue was distilled with non-ionic water until it became about 200 ml and the pH was adjusted to about 4. If necessary, pH was adjusted by addition of 0.1 N sulfuric acid or ion exchange resin on OH '(ie, AG 1x8), and the solution was freeze-dried and freeze-dried.

Yield: 12.5 g (75%)

R _f = 0.35 to 0.45 (ethylacetate / pyridine / acetic acid / water = 25: 20: 6: 1).

[a] _D ² ° = -126.0 ± 5 ° C (C = 1, water).

Total impurity content according to thin layer chromatography analysis: 2-3%,

Content of D-L-D isomer according to HPLC: 3 to 5%.

N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde used as starting material was prepared as follows:

First Step: Preparation of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam

42.95 g (0.11 mol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam was suspended in 110 ml of dry chloroform, and then hydrochloric ethyl acetate (11 to 15 g / 100 ml) was added thereto. 275 ml was added with stirring. The reaction mixture was stirred at room temperature for 3 hours, and ethyl acetate / pyridine / acetic acid / water (60: 20: 6: 11) mixture was thin-layer chromatographed with eluent to determine the end point (Rf of starting material and target compound was determined. 0.9 and 0.3 respectively). After diluting the reaction mixture to 400 ml with diethyl ether at the end point, the precipitated crystalline material was filtered off, and the crystalline material was filtered out and washed with diethyl ether (2x100 ml) and acetone (2x50 ml), respectively. Then, the resultant was dried with solid potassium hydroxide and phosphate pentoxide in a vacuum dryer.

After drying for 1-2 hours, the lactam salt (about 1 mole) was dissolved in 100 ml of anhydrous dimethylformamide and cooled to a temperature of -15 and then added to the mixed anhydride prepared as follows:

Free of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-proline

50.9 g (0.1 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-proline cyclohexyl ammonium salt was dissolved in 200 ml of diethyl ether and 120 ml of 1N sulfuric acid, and then the diethyl ether layer was Washed with water (2x50 mL), dried over anhydrous sodium sulfate and evaporated at a temperature below 35 ° C. and reduced pressure until a cloudy oil formed. The residue (about 0.1 mol) was dissolved in 170 ml of anhydrous dimethylformamide, cooled to a temperature of -15 ° C, and then 11.2 ml (0.1 mol) of N-methyl-morpholine was added thereto, and then I-butoxycarbonyl 13.2 mL of chloride was added dropwise over 3 to 5 minutes at the same temperature as above. Subsequently, the mixed anhydride obtained by throwing the azo component into the dimethylformamide suspension was stirred, and then 35 ml of triethylamine was added thereto while stirring at a temperature of -20 to -25 ° C and vigorously. Adjust the pH of the vapor phase (should be at least pH = 8), add more triethylamine as needed, stir for 1 h at -15 ° C, and stir for 1 h at 0 ° C It was then diluted with 200 ml benzene and 150 ml water.

The phases were separated and the aqueous phase was extracted with benzene (3 × 30 mL), then the benzene solution was collected, washed with water (2 × 50 mL), dried over anhydrous sodium sulfate and turbid under oil at a temperature below 40 ° C. and reduced pressure. Dried. The oily residue was dissolved in 75 ml of a mixture of ether and tetrahydrofuran 2: 1 and filtered through a simple filter plate as follows:

300 g of Kiegel-Gel-60 adsorbents (used in 5-times divisions) were suspended in ether to form a filter plate with a 3: 1 ratio of diameter to height. Contaminant lower spots were detected using an ether and tetrahydrfuran 95: 5 mixture as eluent, and the target compound was eluted with pure tetrahydrofuran. When the mixture of ethyl acetate / pyridine / acetic acid / water 480: 20: 6: 11 was used as the eluent, the R _f value of the target compound was 0.71 to 0.75, and the collected fractions were clouded at 35 ° C. or lower. Vacuum evaporation until produced. The target compound thus obtained was immediately stored in dry ice.

Yield: 58.5 g (85%)

R _f = 0.71 to 0.75 (ethylacetate / pyridine / acetic acid / water = 480: 20: 6: 11).

α _D ²⁰ = + 13.5 ° C. (C = 1, tetrahydrofuran).

Second Step: Preparation of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine aldehyde

34.14 g (0.05 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N ^G -benzyloxycarbonyl-L-arginine lactam (first step of Example 5) After dissolving in 150 ml of dry tetrahydrofuran at a temperature of 0 ° C., it was cooled to a temperature of −20 ° C. and 0.0375 mol of lithium aluminum hydride dissolved in tetrahydrofuran was added to the reaction mixture. Stir vigorously for minutes. This reaction mixture was then chromatographed with an ethyl acetate / pyridine / acetic acid / water mixture of 240: 20: 6: 11 as eluent (R _f value: starting material lactam: 0.8, aldehyde: 0.5).

Lithium aluminum hydride is further added, if necessary, and when the reaction is completed, a portion of the reaction mixture is added to 320 ml of a pre-cooled 1N sulfuric acid solution with stirring at a temperature of 2 to 4 ° C, and then the pH is 2-3. But further sulfuric acid was added and filtered if necessary. Subsequently, a precipitate was formed, which was filtered off. Water was added to the reaction mixture until the reaction mass became turbid, diluted, and extracted with n-hexane (3 × 0.9 L).

The tetrahydrofuran aqueous solution was extracted twice or three times with 250 mL of dechloromethane, and the collected dichloromethane solution was washed with water (2 × 50 mL), 5% sodium hydrogencarbonate (2 × 50 mL), and water (2 × 50 mL). It was then dried over anhydrous sodium sulfate and evaporated in vacuo at a temperature of 40 ° C.

The evaporated residue was dissolved in a mixture of dichloromethane and acetone 7: 3 (120 ml) and passed through a filter plate prepared from 108 g of Kieselgel 600 adsorbent, where a mixture of dichloromethane and acetone 7: 3 was used as the eluent. Used and did not rot. The filter plate was washed with 600 ml of the same eluent mixture as above, which took less than 20 minutes.

The collected filtrate was evaporated under reduced pressure until the temperature was below 40 ° C. until it became about 50 to 60 ml. Then, 100 ml of benzene was added thereto, and this process was repeated, and 100 ml of cyclohexane was added while stirring. About 50-60 ml of product precipitated out. The precipitate was washed with cyclohexane (2 × 30 mL) and dried over paraffin chips on an evaporator to yield 18 g of the target compound (yield: 53%).

R _f = 0.52 to 0.59 (ethylacetate / pyridine / acetic acid / water = 240: 20: 6: 11).

α _D ²⁰ = + 14.1 ° C. (C = 1, tetrahydrofuran).

Total impurity content according to thin layer chromatography analysis: 2-3%,

Claims (5)

The tripeptide represented by the following general formula (III), which is a mixed anhydride obtained by condensation of an N ^G -protected-L-arginine lactam represented by the following general formula (IV) with a dipeptide represented by the following general formula (V) The lactam is reduced to lithium aluminum hydride to prepare a protected tripeptide aldehyde represented by the following general formula (II), which is then hydrolyzed in the presence of sulfuric acid to obtain the tripeptide aldehyde represented by the following general formula (I). In preparing, a mixed anhydride obtained by adding a base to a mixture of N ^G -protected-L-arginine lactam hydrochloride represented by the following general formula (IV) and a dipeptide represented by the following structural formula (V) After obtaining the protected tripeptide lactam represented by the general formula (III), the reaction mixture was introduced into the excess 1N sulfuric acid at a temperature of 0 to 5 ° C to decompose the complex formed. After preparing a protected tripeptide represented by the general formula (II), the protected tripeptide is subjected to a hydrogenolysis reaction at a temperature of 10 ° C. or less in the presence of 1 to 2 moles of sulfuric acid. A method for producing tripeptide aldehyde having a purity suitable for use in the pharmaceutical use represented by (I).

Wherein Arg is an L-arginine group, Z is a benzyloxycarbonyl group, R is hydrogen or a methyl group, Phe is a phenylalanine group, and Pro is an L-proline group. The method according to claim 1, wherein the N ^G -protected -L-arginine lactam is liberated by adding triethylamine to the lactam hydrochloride of the following general formula (IV). The method of claim 1, wherein the hydrogenolysis reaction is carried out at a temperature of 6 to 8 ℃. The method according to any one of claims 1 to 3, wherein the hydrogenolysis reaction is carried out by bubbling hydrogen gas through the entire reaction system. The method according to claim 1, wherein the hydrogenolysis reaction is carried out in ethanol or tetrahydrofuran.