HRP920779A2 - IMPROVED PROCEDURE FOR THE PREPARATION OF TRIPEPTIDE ALDEHYDS - Google Patents

Abstract

An improved process for the preparation of a tripeptide aldehyde of formula I RD-Phe-Pro-Arg-H.H2SO4 And wherein R is hydrogen or methyl, Phe is a phenylalanine group Pro is an L-proline group and Arg is an L-arginine group, comprising the release of NG- of protected L-arginine lactams in situ during condensation of NG-protected-L-arginine lactam hydrochloride of formula IV H-Arg (Z) -lactam.HCl IV and dipeptide of formula V ZR (D-Phe) -Pro-OH V into mixed anhydride in to the reaction mixture by adding a base to the mixture of said reaction components, then reducing the thus obtained tripeptide-protected lactam of formula III, ZR (D-Phe) -Pro-Arg (Z) -lactam III decomposition of the thus obtained complex by adding the reaction mixture to an excess of 1N H2SO4 at a temperature of 0 to 5 degrees C, and then performing the hydrogenolysis of the protected tripeptide of formula II ZR- (D-Phe) -Pro-Arg (Z) -H II at a temperature below 10 degrees C in the presence of 1-2 molar equivalents of sulfuric acid.

Description

The field of technology

The invention is from the field of peptide chemistry.

Technical problem

The subject of the invention relates to an improved process for obtaining tripeptide aldehydes of formula I,

R-D-Phe-Pro-Arg-H.H2SO4 I

where

R represents water or methyl,

Phe denotes the phenylalanine group,

Pro is the L-proline group,

Arg is the L-arginine group

in a pure state suitable for pharmaceutical use.

/ Abbreviations of L-proline and L-arginine as Pro and Arg are in accordance with the previous technique, eg Biochem. 7,126,773, 1972), Biochemistry 14, 449 (1975)/.

State of the art

Salts of D-phenylalanine-L-prolyl-L-arginine aldehyde (H-D-PHe-Pro-Arg-H) are known to exhibit antithrombin activity. However, the acetate salt described in Hungarian patent specification no. 169 870 is not suitable for therapeutic use because it loses its activity very quickly whether it is in solid or liquid form. The same phenomenon can be observed in the case of other salts such as hydrochloride, citrate, tartrate, tosylate and NG-carboxy derivatives of tripeptide aldehyde. However, the sulfate salt H-D-Phe-Pro-Arg-H and its N-methyl derivative are very stable even in aqueous solvent (British Patent Specification No. 2 091 270 and Published European Patent Application No. 185 390) and therefore can be suitable for therapeutic use.

The above sulfate salts can be obtained as follows:

1. The sulfate salt H-D-Phe-Pro-Arg-H peptide can be obtained according to British patent specification no. 2 091 270 from t-butoxycarbonyl D-phenylalanyl-L-prolyl-L-arginine aldehyde hemisulfate or from t-butoxycarbonyl-D-phenylalanyl-L-prolyl-NG-carboxy-L-arginine aldehyde in the presence of 1-12 equivalents of 1- 12N aqueous sulfuric acid at a temperature of 40-60ºC. (The preparation of the starting first material is described in Example 1 of the above-cited patent specification, while the preparation of the protected NG-carboxy derivative is given in Belgian Patent Specification No. 880 844).

2. The sulfate salt of the H-D-Phe-Pro-Arg-H peptide can be further obtained according to the aforementioned specification by hydrogenolysis of benzyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde in the presence of a molar equivalent amount of sulfuric acid. (The preparation of the doubly-protected tripeptide aldehyde used as starting material is described in Hungarian Patent Specification No. 169,870).

According to the published European patent application no. 185 390 N-methyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate (H-D-MePhe-Pro-Arg-H-H2SO4) was obtained similarly to the free tripeptide aldehyde sulfate by removing the protecting groups from N-benzyloxycarbonyl N-methyl- D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde by hydrogenolysis in the presence of sulfuric acid. (The preparation of the tripeptide aldehyde used as starting material is also described in the same patent application).

The main disadvantage of the above procedures is, especially if the production is carried out on a larger scale, that it does not give a product of adequate purity.

Thus, for example, the product obtained using procedure no. 1 comprises about 4-6% calcium sulfate. Based on our layer-chromatographic analysis, the product so dibven can contain about 10% of other impurities (e.g. tripeptide alcohol, - acid, -acetal) except calcium yarn 20%. A further disadvantage of the process is that the neutralization of excess sulfuric acid and the filtering of settled calcium sulfate cause further difficulties.

Tripeptide aldehyde sulfate salt prepared by process no. 2 (hydrogenolysis) includes, in addition to 5-10% of pollutants detectable by thin-layer chromatography, a further 10-15% of tripeptide aldehyde sulfate D-L-L-configuration according to HPLC analysis: thus a total of 15-25% of polluting side products are built. The above-mentioned 5-10% of pollutants are composed of the following substances: tripeptide acid awarded during the synthesis of tripeptide aldehyde, tripeptide alcohol, cyclic decomposition product of the structure described in the published European patent application no. 185 390 and a substance of unknown structure awarded in various amounts of spots located below the desired compounds on the chromatogram.

"Pre-hydration" was observed to play an important role in the production of the latter by-product. Namely, if hydrogenolysis is carried out in a known way, i.e. in the presence of 1 molar equivalent of sulfuric acid at room temperature and the procedure continues after the completion of hydrogenolysis, an additional spot (Rf=0.39-0.44) corresponding to the pollutant always appears below product spots (in the case of H-D-Phe-Pro-Arg-H.H2SO4) on a thin-layer chromatogram (absorbent: Kieselgal G, eluent: 25:20:6:11 ethyl acetate/pyridine/acetic acid/water; developer: chlorptolidine ), where the said pollutant, in some cases, represents the main impurity.

In order to eliminate contamination, the end product of the reaction must be detected very accurately and the reaction must be stopped when the end point is reached.

However, since the end point of the reaction is determined by thin-layer chromatographic analysis, which requires 20-30 minutes of work when using the above eluent mixture, the reaction cannot be stopped at the end point, especially under industrial conditions. Thus, a side product with a predefined Rf value is usually obtained in an amount of about 3-4% due to "prehydration". Said by-product is more toxic than the end product, so it must be eliminated well for this reason.

The product is very sensitive to racemization, therefore the polluting substances obtained during the above-described procedures cannot be practically removed. Since the end product cannot be purified, in order to achieve the purity of the end product that is suitable for pharmaceutical use, the production of intermediates of appropriate purity and suppression of the formation of the side product must be solved. This means inhibition of racemization during the process.

Description of the solution to the technical problem with examples of implementation

The aim of the present invention is to develop a process that ensures the suppression of the formation of the above-mentioned side products and the obtaining of the tripeptide aldehyde salt of formula I in a purity of at least 95%.

The invention is based on the knowledge that the formation of D-L-D products, i.e. racemization, is highly dependent on pH. In an environment that has an alkaline pH, and even in an environment with a neutral pH, the final product and intermediates undergo racemization. In an environment with an acidic pH (6pH=2), the final product is stable for months. Therefore, technology must be provided to ensure that the pH of the reaction mixture is not neutral or basic even for a short period of time. In order to achieve this, the arginine lactam is liberated from its salt in situ during the mixed anhydride condensation of NG-benzyloxycarbonyl-L-arginine lactam hydrochloride of formula IV

H.Arg(Z)-lactam-HCl (IV)

where Arg is the same as defined in the introductory part and Z represents the benzyloxycarbonyl of the dipeptide of formula V

ZR(D-Phe)-Pro-OH (V)

where R, Pho and Pro are the same as defined in the introduction, Z is the same as defined above, such that the base is then added to the reaction mixture. Under such conditions, the arginine lactam released in situ reacts at once, in situ nascendi, with the mixed anhydride to form the corresponding protected tripeptide lactam of formula III

ZR (D-Phe)-Pro-Arg(Z)-lactam (III)

wherein Z is the same as defined above and R, Phe, Pro and Arg are the same as defined in the preamble, thus the construction of a racemizing base environment is eliminated.

Further, the invention is based on the knowledge that if the complex awarded with lithium aluminum hydride, obtained during the reduction of the protected tripeptide lactam of formula III obtained as described above, is decomposed in such a way that the reaction medium is added to a dilute, preferably 1N, cold (0 -5ºC) sulfuric acid used in excess against the published European patent application no. 185 390 in which sulfuric acid is added to the reaction mixture, the acidity of the pH of the reaction mixture can be ensured. Therefore, racemization of the protected tripeptide aldehyde can be avoided or greatly reduced.

The invention is based on the further knowledge that the hydrogenolysis of the tripeptide aldehyde of formula II obtained as described above must be carried out in the presence of excess sulfuric acid at a low temperature below 10ºC, preferably at 6-8ºC.

According to our experimental data, if the hydrogenolysis of Z-D-Phe-Pro-Agr(Z)-H is carried out at various temperatures in the presence of 1 molar equivalent of sulfuric acid, the impurities detectable by thin-layer chromatographic analysis of the thus obtained product are as follows: 17% at 40ºC , 10% at room temperature (20-25ºC) 4% at 6-10ºC. The D-L-D isomer contents of the product detectable by HPLC were as follows: 12%, 5% and 2-3%, respectively, ie the total impurity content of the product was 29%, 15% and 6-7%, respectively. This means that if hydrogenolysis is performed at lower temperatures, the amount of side products decreases significantly, while the time required for hydrogenolysis does not increase significantly when the temperature decreases.

If the hydrogenolysis is carried out at lower temperatures in the presence of excess sulfuric acid, a further major advance occurs so that the formation of a by-product having an Rf value of 0.39-0.44 can be greatly suppressed and accurate detection of the end point of the reaction is also eliminated. According to our experiments, if the hydrogenolysis of Z-D-Phe Pro-Arg(Z)-H is carried out in the presence of 1.5-1.7 molar equivalents of sulfuric acid in tetrahydrofuran at a temperature of 6-8ºC, the side product having the upper Rf value is only formed in traces even after 1 hour of "prehydration".

Further, it is preferable to use sulfuric acid during all stages of the process, so if, for example, Hcl is used, the sulfate salt may be contaminated with chloride ions.

As a result of carrying out the reaction as described here, the amount of DLD isomer in the final product of formula I is at most 2-4%, while other contaminants (tripeptide acid and -alcohol) build up in an amount below 1%.

Based on the above, the invention relates to an improved process for obtaining a tripeptide aldehyde of formula I, where R, Phe, Pro and Arg are as defined above, in a pharmaceutically pure form, by condensing a lactam of formula IV, where Arg is as described forward, and Z represents benzyloxycarbonyl, with a dipeptide of formula V, where R, Z, Phe and Pro are as defined above in the introduction and Z is as defined above, via a mixed anhydride, the reduction of a tripeptide lactam of formula III, where R, Z, Phe, Pro and Arg are as defined above, with lithium aluminum hydride and hydrolysis of the thus obtained protected tripeptide aldehyde of formula II, where R, Z, Phe and Arg are saturated as defined above in the presence of sulfuric acid , which includes:

a1) release of the NG-protected L-arginine lactam in situ during the condensation of the NG-protected L-arginine lactam hydrochloride of formula IV, where Z is the same as defined above, and the dipeptide of formula V, where R, Z, Phe and Pro are the same as defined above, to the mixed anhydride in the reaction mixture, then adding a base to the mixture of said components, then reducing the thus obtained protected tripeptide lactam of formula III where R, Z, Phe, Pro and Arg are the same as defined above, decomposition thus of the awarded complex by adding the reaction mixture to an excess of 1N sulfuric acid at a temperature of 0-5ºC, then performing hydrogenolysis of the protected tripeptide of formula II, where R, Z, Phe, Pro and Arg are the same as defined above at a temperature below 10ºC in the presence of 1- 2 molar equivalents of sulfuric acid, or

a2) after the reduction of the thus obtained protected tripeptide lactam of formula III, where R, Z, Phe, Pro and Arg are the same as defined above, decomposition of the thus awarded complex by adding the reaction mixture to an excess of 1N H2SO4 at a temperature of 0-5ºC, then performing hydrogenolysis of the thus obtained protected tripeptide of formula II, wherein R, Z, Phe, Pro and A are as defined above, at a temperature below 10ºC in the presence of 1-2 molar equivalents of H2SO4, or

a3) performing hydrogenolysis of the protected tripeptide of formula II, where R, Phe, Pro and Arg are the same as defined above, at a temperature below 10ºC in the presence of 1-2 molar equivalents of H2SO4.

According to the present invention, the reaction is preferably carried out as follows:

From the t-butoxycarbonyl-NG-benzyloxycarbonyl-L-arginine lactam the t-butoxycarbonyl protecting group is removed in a known manner. The NG-benzylcarbonyl-L-arginine lactam hydrochloride of formula IV thus obtained is kept in a dimethyl formamide solvent at a temperature of -15 to -20ºC. This solution is then added to the mixed anhydride obtained from the dipeptide of formula V (benzyloxycarbonyl-D-denylalanyl-L-proline) and isobutyl ester of chlorocarbonic acid. A base, preferably trimethyl amine is then added to the reaction mixture. Under these conditions, the lactam released in situ due to the base effect reacts simultaneously with the mixing anhydride to form the protected tripeptide lactam, and thus the formation of a base environment that causes racemization can be avoided. If desired, the product thus obtained can be purified by rapid filtration through a layer of silica gel. By choosing the above operation sequence, which is different from that of the prior art, there is no need for expensive purification of the different lactam tripeptide by column chromatography.

The protected tripeptide lactam of formula III thus obtained is reduced with lithium aluminum hydride in dry tetrahydrofuran. The complex thus obtained is analyzed against the published European patent application no. 185 390 by adding the reaction mixture to a dilute solution, preferably 1N H2SO4 used in excess with cooling at a temperature of 2-4ºC. The final pH value of 2-3 is optionally adjusted by adding H2SO4. The reaction mixture is then processed in a known manner. The protected tripeptide aldehyde of formula II thus obtained is purified by filtering through a layer of silica gel.

Hydrogenolysis is preferably carried out in a polar organic solvent, preferably tetrahydrofuran or lower alkanol, eg methanol, ethanol, i-propanol, preferably in ethanol. It is preferable if hydrogenolysis is not performed in a closed system, but by bubbling hydrogen through the reaction mixture. Practically, the conductive gas is bubbled through the system in a finely divided form, which can be ensured by introducing the gas through the G2-filter.

According to a preferred embodiment of the process, the protected tripeptide aldehyde of formula II of suitable purity is dissolved in tetrahydrofuran and the resulting solution is added to a cooled aqueous catalytic suspension comprising 1.5-1.7 molar equivalents of 1N H2SO4. The ratio of the organic solvent to the water fraction is 70-50 : 30-50, preferably 60 : 40. As a catalyst, 10-30% wt. 10% palladium on charcoal or palladium was used. Hydrogenation is carried out under cooling at a temperature of 6-8ºC by bubbling finely divided conductor gas through the system. The progress of the reaction is monitored using thin-layer chromatographic analysis.

According to another preferred embodiment of the process of the invention, the protected tripeptide aldehyde used as starting material is dissolved in tetrahydrofuran and a catalyst comprising 10% palladium deposited on barium sulfate or aluminum oxide as a carrier, preferably barium sulfate, is used.

Hydrogenation is performed at a temperature of 6-8ºC in the presence of 1.5 molar equivalents of H2SO4 by bubbling a finely divided conductor gas through the system. The end of the reaction is monitored by thin-layer chromatographic analysis.

According to a further preferred embodiment of the process of the invention, the protected tripeptide used as starting material is dissolved in ethanol, then the solution is added to a cooled suspension comprising 10% palladium on carbon catalyst and 1 molar equivalent of 1N H2SO4. Hydrogenation is performed at a temperature of 6-8ºC by bubbling a finely divided guide gas through the system and the process is monitored by thin-layer chromatography.

The product can be simply separated from the reaction mixture by filtering the catalyst and managing the organic solvent from the aqueous-organic solvent mixture at a temperature below 40ºC, preferably 25-30ºC, then by freeze-drying the aqueous solvent directly or after adjusting the pH to about 4pH is preferably adjusted using ion- hydroxy-phase exchange resin or by using 0.1 N H2SO4.

When the process according to the invention is carried out on a pilot plant, the maximum pollution of the product thus obtained (D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate and N-methyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate) is detectable using thin-layer chromatographic analysis is at most 1%, while D-L-D content detectable by HPLC is at most 2-4%. Thus, the total amount of impurities can be reduced to about 3-5%, which is in accordance with the requirements given for pharmaceutical substances and contrary to the impurity content of 15-25% in the case of known procedures.

The process of the present invention is further illustrated by the following non-limiting examples.

Rf values were determined by thin-layer chromatography on silica gel (Kieselgel G, Reanal, Budapest) using the eluent mixtures given in the examples. Developer: chlorinated chromatographic plates were treated with 0-tolide solvent (2.5 g of o-tolide and 10 ml of acetic acid dissolved in 500 ml of water) after drying.

Example 1

Preparation of D-phenylalanyl-L-propyl-L-arganine aldehyde sulfate

201 g (0.3 mol) of benzyloxycarbonyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine aldehyde is dissolved in 2 liters of tetrahydrofuran at room temperature. At the same time, 60g of 10% palladium on charcoal is suspended in 0.6 liters of deionized water, then 900ml of H2SO4 (0.45 mol) is added. The suspension comprising the catalyst is cooled to a temperature of 2-50ºC, and the tetrahydrofuran solvent of the protected tripeptide aldehyde and an additional 0.2 liters of deionized water are added with stirring. Then hydrogen is bubbled through the mixture, the temperature of which is maintained at 6-8ºC with stirring.

The progress of the reaction was monitored by thin-layer chromatography using a 60:20:6:11 mixture of ethyl acetate/pyridine/acetic acid/water as eluent. (Rf values of starting materials, intermediates and final product are 0.92-0.96, 0.38-0.44 and 0.0-0.10 respectively).

The reaction ends in 3-4 hours. At the end of the reaction, the catalyst is filtered off and washed 3 times with 100 ml of deionized water each. The filtrate and washing liquid are combined, then the tetrahydrofuran is distilled off in a rotary evaporator under vacuum at a temperature of 25-30ºC.

The remaining aqueous solution is extracted with 2x100 ml of dichloromethane, then the pH of the aqueous phase is controlled. If the pH differs from 4, then the pH is adjusted to 4 by adding 0.1N H2SO4 or OH-cycling ion-exchange resin (eg AG 1x2).

The volume of the solution is made up to 1.5 liters by adding deionized water, then the solution is freeze-dried. Thus, 125g (83.2%) of the desired product is obtained.

Rf=0.52-0.57 (ethyl acetate/pyridine/acetic acid/water=25:20:6:11).

/α/20 D = -117 (c=1, water).

The total content of impurities according to thin-layer chromatographic analysis: 3-5%, while the D-L-D isomer content determined by HPLC is about 2-3%.

Benzyloxycarbonyl-D-phenylalanyl-L-1proline-NG-benzyloxycarbonyl-L-arginine aldehyde used as starting material can be obtained as follows:

Degree 1

Benzyloxycarbonyl-D-phenylalanyl-propyl-NG-benzyloxycarbonyl-arginine lactam (published European Patent Application No. 185 390) was suspended in 1300 ml of dry chloroform. Then, 1300ml of 189g/100ml (about 5N) ethyl acetate hydrochloride is added dropwise with mixing and with cooling with ice water at a speed that ensures the avoidance of temperature rise above 15ºC. Soon the dissolution and precipitation of the crystalline substance begins. The suspension is stirred at room temperature for 3 hours, then diluted with 3000 ml of a dry 1:1 mixture of ether and ethyl acetate. The settled crystalline substance is filtered off, washed with 2x1000 ml of acetone and 500 ml of ether, then dried over solid potassium hydroxide and phosphorus pentoxide in a vacuum desiccator.

After about 1 hour of drying, NG-benzyloxycarbonyl-L-arginine-lactam hydrochloride is dissolved in 1000 ml of dry dimethyl formamide, cooled to a temperature of -15 to -20ºC and added to the mixed anhydride obtained as follows:

306g (1 mol) of N-benzyloxycarbonyl-D-phenylalanyl-L-proline (Nicolaides E. et al: J.Med-.Chem. 11, 74(1968) and published European Patent Application No. 185 396) is dissolved in 1000 ml of anhydrous dimethyl formamide and 111 ml (1 mol) of N-methyl morpholine are added. Then the mixture is cooled to a temperature of -15ºC and 132ml (1 mol) of isobutyl chlorocarbonic acid is added dropwise at the same temperature with stirring for 5-10 minutes. The mixed anhydride is cooled to a temperature of -20°C to -25°C after 10 minutes of stirring and combined with the dimethylformamide solvent of the NG-benzyloxycarbonyl-L-arginine-lactam hydrochloride obtained above. Then 308 ml (2.2 ml) of triethyl amine is added dropwise to the reaction mixture at the same temperature for about 30 minutes, the suspension is then stirred for another hour at a temperature of -20 to -15ºC. The pH of the vapor space is controlled with wet pH paper: if the pH falls below 8, a further amount of thiethylamine is added. Then the mixture is diluted with 2 liters of benzene, the settled salts are filtered off and washed twice with about 500 ml of benzene.

1.5 liters of water are added to the benzene-dimethyl formamide filtrate and the phases are separated. The aqueous dimethyl formamide layer is extracted with 2x500 ml of benzene. The combined benzene solvents are washed with 2x0.5 liters of water, 0.5 liters of 0.1N H2SO4, then 3x0.5 liters of water, dried over anhydrous sodium sulfate and evaporated to 2 liters under vacuum at a temperature of no more than 40ºC.

360g of Kieselgel-G absorbent is suspended in benzene and a filter layer is formed, where the ratio of diameter to height is 8:1 to 4:1.

The benzene solution is sucked through the filter layer under a weak vacuum, then the filter layer is washed with 4.8 liters of benzene in the same way. The diluted benzene solutions are concentrated to a volume of about 600ml under vacuum at a temperature of no more than 40ºC, then 3 liters of diisopropyl ether are added with intensive mixing.

The precipitate is filtered off, washed with 2x600 ml of diisopropyl ether and dried in a vacuum desiccator over P2O5 and paraffin flakes. Thus, 510g (76%) of the desired product is obtained. The total contaminant content of the product determined by thin-layer chromatography is 1-2%.

Rf=0.55 - 0.65 (ethyl acetate)

(Rf value of L-D-L lactam is 0.45-0.50 in ethyl acetate).

/α/20 D = -55.8 (c=1, tetrahydrofuran).

Degree 2

Benzyloxycarbonyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine aldehyde

300 mg (0.45 mol) of the protected tripeptide lactam (Example 1, step 1) was dissolved in 2.5 liters of anhydrous tetrahydrofuran cooled to a temperature of -5ºC. The solution is cooled to a temperature of -20ºC to -25ºC in a dry ice-acetone bath, and with vigorous stirring for 20-25 minutes, a solution of lithium aluminum hydride and tetrahydrofuran is added, which has a concentration of about 0.7 mol/liter, which corresponds to 0.375 moles of lithium aluminum chloride. The progress of the reaction was monitored by thin-layer chromatography using a 240:20:6:11 mixture of ethyl acetate/pyridine/acetic acid/water as eluent. If necessary, a further amount of lithium aluminum hydride solution is added until the spot disappears, having an Rf value of 0.75-0.80, corresponding to the lactam.

When the reaction is finished, the reaction mixture is gradually poured into 3 liters of 1N H2SO4 solution at a temperature of 2-3ºC with stirring. The final pH value is adjusted by optionally adding a further amount of H2SO4. Water is added to the solution until it becomes cloudy, then it is extracted with 3x0.9 liters of n-hexane. Between this extraction, the material is separated as an oil phase, therefore it is homogenized with tetrahydrofuran, then the appearance of milk is obtained by adding some water. The aqueous phase is extracted with 2x2 liters of dichloromethane. The combined dichloromethane solutions are washed with 2x0.5 liters of NaHCO3 solution at 4-8ºC, then with 2x0.5 liters of distilled water at a temperature of 4-8ºC, dried over anhydrous sodium sulfate and evaporated under vacuum at a temperature of no more than 40ºC (about 300 g of oily substance is obtained).

The oily residue is dissolved in a 7:3 mixture of dichloromethane and acetone, by flowing through a filter layer (diameter:height = 5:1) obtained from 900g of Kieselgel G absorbent using a 7:3 mixture of dichloromethane and acetone as eluent without suction, then the filter layer is washed with 6 liters of the same eluent mixture. The procedure may not last longer than 20 minutes. The combined filtrates are evaporated to about 600ml at a temperature of no more than 40ºC, then 1 liter of benzene is added and the solution is evaporated again, then the product is settled by adding 1.6 liters of cyclohexane and dried in a vacuum desiccator at room temperature over paraffin shells. Thus, 240g (79.7%) of the title compounds are obtained.

Rf=0.52-9.62 (ethyl acetate/pyridine/acetic acid/water=240:20:6:11).

The impurity content according to thin-layer chromatographic analysis is 0.5--%.

/α/20 D = -0.8 (c=1, tetrahydrofuran).

Example 2

Obtaining D-phenylalanine-L-arginine-aldehyde sulfate

20.1g (0.03mol) of benzyloxycarbonyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine aldehyde is dissolved in 214ml of tetrahydrofuran and the solution is cooled to a temperature of 0ºC. 4.3g of palladium on carbon catalyst is suspended in 70ml of deionized water and 120ml of 1N H2SO4 is added. The catalyst suspension is cooled to a temperature of 2-5ºC, air is eliminated using nitrogen gas. Then, the above tetrahydrofuran solution is added to the suspension with stirring and the finely divided guide gas is introduced into the system. The temperature is kept between 6 and 8ºC by cooling with ice. The progress of the reaction is monitored by thin-layer chromatography and the sieved eluent mixture is used as described in example 1. At the end of the reaction (about 3 hours), the reaction mixture thus obtained is processed according to example 1. Yield: 12.5g (83.2 %).

Rf=0.52-0.57 (ethyl acetate/pyridine/acetic acid/water=25:20:6:11)

/α/20 D = -117 C (c=1, water)

The content of impurities based on thin-layer chromatographic analysis is 3.5%.

The content of the D-L-D isomer based on HPLC analysis is 1.5%.

Example 3

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

201 g (0.3 mol) of benzyloxycarbonyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde is dissolved in 2 liters of ethanol at room temperature. 60g of catalyst comprising 10% palladium on barium sulfate as carrier is not suspended in 0.6 liters of deionized water and 600m 1N H2SO4 (0.3 mol) is added. The suspension is cooled to a temperature of 2-5ºC and an ethanol solution of the protected tripeptide aldehyde is added. Deionized water is then added in an amount corresponding to a 6:4 ratio of solvent to aqueous phase.

Then the guide gas is bubbled through the mixture with mixing and cooling at a temperature of 6-8ºC. The progress of hydrogenation is monitored by thin-layer chromatography according to example 1. At the end of the reaction (about 3-4 hours), the catalyst is filtered off and washed with deionized water several times. The filtrates are combined and the alcohol is evaporated on a rotary evaporator at a temperature of 25-30ºC. The aqueous solution, which has a pH of about 3-4, is directly freeze-dried. Thus, 101g (65%) of the desired product is obtained in the form of monohydrate, which has the same quality as the product obtained in example 1.

Example 4

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

The procedure from example 1 was followed, except that 43g of 10% palladium on carbon catalyst was previously washed until neutralization and then used as a catalyst.

Thus, 125.1g (83.2%) of the desired product is obtained, which has the same quality as the product from example 1.

Example 5

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

20.5g (0.03 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine aldehyde is dissolved in 214ml of ethanol, then 110ml of deionized water, 60ml of 1N H2SO4 ( 0.03 mol) and 3 g of 10% palladium on charcoal as a catalyst. Then the finely divided gas is introduced into the mixture with cooling at a temperature of 6-8ºC. The progress of the reaction is monitored by thin-layer chromatography using a mixture of 25:20:6:1 ethyl acetate/pyridine/acetic acid/water as eluent. On the chromatogram, the Rf values of the starting material, intermediate, and end product are about 0.9, 0.7, and 0.4, respectively. The reaction ends in about 2 hours. At the end of the reaction, the catalyst is filtered off and washed with 3x30 ml of deionized water. The filtrate and the washing liquid are combined, then evaporated on a rotary evaporator to about 100ml at a temperature of 25ºC, the residue is then extracted with 2x50ml of dichloromethane.

Traces of organic solvent are removed from the aqueous solution by distillation and the residue is diluted with deionized water to a volume of about 200 ml. The pH was further checked at 4. If necessary, the pH was adjusted by adding 0.1 N H2SO4 or an OH-phase ion exchange resin (eg AG 1xB), then the solvent was frozen and freeze-dried. Yield 12.5g (75%).

Rf=0.35-0.45 methylacetate/pyridine/acetic acid/water=25:20:6:1)

/α/20 D = -126.0+5±5 (c=1, water)

The total impurity content based on thin-layer chromatographic analysis is 2-3%.

Impurity content based on HPLC is 3-5%.

N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine-lactam

42.95g (0.11mol) of t-butoxycarbonyl-NG-benzyloxycarbonyl-L-arginine lactam was suspended in 110ml of dry chloroform and 275ml of ethyl acetate hydrochloride was added with constant stirring. The reaction mixture was stirred for about 3 hours at room temperature, the end point of the reaction was determined by thin-layer chromatography using a mixture of 60:20:6:11 ethyl acetate/pyridine/acetic acid/water as eluent. (The Rf values of the starting material and the final product are about 0.9 and 0.3 respectively). At the end of the reaction, the mixture is diluted with 400ml of diethyl ether, the settled crystalline substance is filtered off, washed with 2x100ml of diethyl ether and 2x50ml of acetone, then dried on a vacuum desiccator over phosphorus pentoxide and potassium hydroxide. After drying for 1-2 hours, the lactam salt thus obtained (about 0.1 mol) is dissolved in 100 ml of anhydrous dimethyl formamide and stored at -15ºC until its use by adding the mixed anhydride obtained as follows:

Release of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-proline:

50.9g (0.1 mole) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-proline cyclohexyl ammonium salt was dissolved in 200ml of diethyl ether and 120ml of 1N H2SO4. The ethyl ether phase was washed with 2x50ml of water, dried over anhydrous sodium sulfate and evaporated under reduced pressure at a temperature of no more than 35ºC until a thick oil was obtained.

The residue (about 0.1 mol) was dissolved in 170 ml of anhydrous dimethyl formamide, cooled to a temperature of -15ºC and 11.1 ml (0.1 mol) of N-methyl-morpholine was added with stirring. Then 13.2 ml of i-butoxycarbonyl chloride was added dropwise over 3-5 minutes while maintaining the same temperature. After a further 5-10 minutes of mixing, the above dimethyl formamide suspension of the amino component was added by mixing to the anhydride thus obtained, then 35 ml of triethyl amine was added to the reaction mixture with strong mixing at a temperature of -20 to -25ºC. The pH of the vapor space is checked (pH must be above 8) and, if necessary, a further amount of thiethyl amine is added. The reaction mixture is stirred for 1 hour at a temperature of -15ºC, 1 hour at 0ºC and then diluted with 200ml of benzene and 150ml of water is added.

The phases are separated and the aqueous phase is extracted with 3x30 ml of benzene. The combined benzene solvents are washed with 2x50ml of water, dried over anhydrous sodium sulfate and evaporated under reduced pressure at a temperature of no more than 40ºC until a thick oil is obtained. The oily residue is dissolved in 75 ml of a 2:1 mixture of ether and tetrahydrofuran and filtered through a filter layer as follows:

300g (5-fold amount) of Kieselgel-60 absorbent is suspended in ether and a filter layer is prepared with a diameter:height ratio of 3:1. The contaminating upper spot was eluted with a mixture of 95:5 ether and tetrahydrofuran, while the desired product was eluted with pure tetrahydrofuran. The substance content of the fractions was controlled by thin-layer chromatography using a mixture of 480:20:6:11 ethyl acetate/pyridine/acetic acid/water as eluent. The Rf value of the desired product is 0.71-0.75. The combined fractions were evaporated under vacuum at 35ºC to a thick residue and the product was consumed at once or stored in dry ice.

Yield: 58.8g (85%).

Rf=0.71-0.75 ethyl acetate/pyridine/acetic acid/water=480:20:6:11)

/α/20 D = +13.5 (c=1, tetrahydrofuran)

Degree 2

N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine-aldehyde

34.14 g (0.05 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-propyl-NG-benzyloxycarbonyl-L-arginine lactam (Example 5, step 1) is dissolved in 150 ml of dry tetrahydrofuran cooled to temperature from 0ºC. The solution was cooled to -20ºC and 0.0375 mol of lithium aluminum hydride was dissolved in tetrahydrofuran and added with stirring over 5-10 minutes. The progress of the reaction was monitored by thin-layer chromatography using a mixture of 240:20:6:11 ethyl acetate/pyridine/acetic acid and water. Rf values of lactams used as starting material and aldehydes are 0.8 and 0.5 respectively).

If necessary, a further amount of lithium aluminum hydride is added and at the end of the reaction, the mixture is gradually poured with continuous stirring with 320ml of 1N H2SO4 previously cooled to a temperature of 2-4ºC. The final pH should be between 2 and 3, this pH is adjusted by adding more H2SO3 if necessary. If a precipitate forms, it is filtered off, the solution is diluted with water until cloudy and then extracted with 3x100ml of n-hexane.

The aqueous tetrahydrofuran phase was extracted with 250ml of dichloromethane 2 or 3 times and the combined dichloromethane solvents were washed with 2x50ml of water, 2x50ml of cold 5% NaHCO3 solution, then with 2x50ml of water. Then the solution was dried over anhydrous sodium sulfate and evaporated under vacuum at a temperature of no more than 40ºC.

The evaporation residue was dissolved in a 7:3 mixture of dichloromethane and acetone (120 ml) and passed without suction through a filter layer obtained from 180 g of Kieselgel-60 absorbent using 200 ml of a 7:3 mixture of dichloromethane and acetone, then the filter layer was washed with 600 ml of the same eluent mixture. The procedure does not last longer than 20 minutes.

The combined filtrates are evaporated to 50-60ml under reduced pressure at a temperature of no more than 40ºC, then this operation is repeated after adding 10ml of benzene to the residue. The product is precipitated from a solution of about 50-60 ml by adding 100 ml of cyclohexane.

The resulting precipitate was washed with 2 x 30 ml of cyclohexane and dried over paraffin shells in a vacuum desiccator. The yield is 18g (53%).

Rf=0.55-0.59 ethyl acetate/pyridine/acetic acid/water=240:20:6:11)

/α/20 D = +14.1 (c=1, tetrahydrofuran).

The total content of impurities in the product is a maximum of 2-3%.

Claims (5)

1. Improved process for obtaining tripeptide aldehyde of formula I R-D-Phe-Pro-Arg-H.H2SO4 where R represents hydrogen or methyl, Phe is a phenylalanine group Pro is the L-proline group and Arg denotes the L-arginine group in a pharmaceutically pure form, by condensation of the NG-protected-L-arginine lactam of formula IV, H-Arg(Z)-lactam.HCl IV where Arg has the same meaning as above and Z denotes benzyloxycarbonyl with the dipeptide of formula V ZR(D-Phe)-Pro-OG V wherein R, Z, Phe and Pro are the same as defined above, via anhydride mixing, reduction of the tripeptide lactam of formula III, ZR(D-Phe)-Pro-Arg(Z)-H II wherein R, Z, Phe and Arg are as defined above in the presence of H 2 SO 4 , which includes a1) release of NG-protected L-arginine lactam in situ during condensation of NG-protected-L-arginine lactam hydrochloride formula IV, H-Arg(Z)-lactam, Hcl IV where Z is the same as defined above and dipeptide of formula V, ZR(D-Phe)-Pro-OH V where R, Z, Phe and Pro are the same as defined above, to the mixed anhydride in the reaction mixture and then adding a base to the mixture of the mentioned reaction components, then after reduction of the thus obtained protected tripeptide lactam of formula III, ZR(D-Phe)-Pro-Arg(Z)-lactam III wherein R, Z, Phe, Pro and Arg are the same as defined above, decomposing the complex thus obtained by adding the reaction mixture to an excess of 1N H2SO4 at a temperature of 0 to 5ºC and then performing hydrogenolysis of the protected tripeptide of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II where R, Z, Phe, Pro and Arg are saturated as defined above, at a temperature below 10ºC in the presence of 1-2 molar equivalents of H2SO4 or a2 after reduction of the thus obtained protected tripeptide lactam of formula III, ZR-(D-Phe)-Pro-Arg(Z)-lactam III where R, Z, Phe, Pro and Arg are the same as defined above, decomposing the thus awarded complex by adding the reaction mixture to an excess of 1N H2SO4 at a temperature of 0 to 5ºC, then performing hydrogenolysis of the thus obtained protected tripeptide of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II where R, Z, Phe, Pro and Arg are as defined above, at a temperature below 10ºC in the presence of 1-2 molar equivalents of H2SO4. 2. The process according to claim 1, characterized in that it comprises the release of the NG-protected-L-arginine lactam from the lactam hydrochloride of formula IV with triethyl amine. 3. The method according to claim 1, characterized in that it comprises the performance of hydrogenolysis at a temperature of 6-8ºC. 4. The method according to one of the claims 1-3, characterized in that it comprises the performance of hydrogenolysis by bubbling the guide gas through the system. 5. The method according to one of claims 1-3, characterized in that the hydrogenolysis is carried out in ethanol or tetrahydrofuran.