SI9011225A - Improved production procedure of tripeptide aldehydes - Google Patents

Description

IMPROVED PROCEDURE FOR THE PREPARATION OF TRIPEPTID ALDEHYDS

TECHNICAL FIELD

The invention is in the field of peptide chemistry.

TECHNICAL PROBLEM

The present invention relates to an improved process for the preparation of tripeptide aldehydes of formula I,

R-D-Phe-Pro-Arg-H.H2SO4 And where

R is hydrogen or methyl,

Phe stands for the phenylalanine group,

Pro is an L-proline group,

Arg is a pure L-arginine group suitable for pharmaceutical use.

/ The abbreviations of L-proline and L-arginine such as Pro and Arg are in accordance with the prior art, e.g. Biochem.7,126,77311972), Biochemistry 14, 449 (1975) /.

BACKGROUND OF THE INVENTION

The known D-phenylalanine-L-prolyl-L-arqinin aldehydes (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 is losing its own

2.

activity very quickly whether in solid or liquid form. The same phenomenon can be observed in the case of other schools such as hydrochloride, citrate, tartrate, tosylate and N-carboxy derivatives of the tripeptide aldehyde. However, sulphate with H-D-Phe-Pro-Arg-H and hers. .N-methyl derivative, very stable even in the form of an aqueous solution (British Patent Specification No. 2 091 270 and European Patent Application Publication No. 185 390) and may therefore be suitable for therapeutic use.

The above sulfate salts may be obtained as follows:

1. The sulphate salt of the H-D-Phe-Pro-Arg-H peptide is obtainable according to British Patent Specification no. 2 091 270 from t-butoxycarbonylD-phenylalanil-L-prolyl-L-arginine aldehyde hemisulfate or from t-butoxycarbonyl-D-phenylalanyl-L-prolyl-N-carboxy-L-arginine aldehyde in the presence of 1-12 equivalents 1- 12N aqueous sulfuric acid at 40-60 ° C. (Obtaining the starting first material is described in Example 1 of the previously cited patent specification, while 0 obtaining the protected N-carboxy derivative is given in Belgian Patent Spec. No. 880 844).

2. The sulphate salt of the H-D-Phe-Pro-Arg-H peptide can be further prepared according to the above specification by hydrogenolysis of benzyloxycarbonyl-D0 phenylalanyl-L-prolyl-N-benzyloxycarbonyl-L-arginine. of aldehydes in the presence of a molar equivalent amount of sulfuric acid. (Preparation of the double-protected tripeptide aldehyde used as starting material is described in the Hungarian Patent. Spec. No. 169 870).

3. According to published European patent application no. 185 390

N-methyl-D-phenylalanil-L-prolyl-L-arginine aldehyde sulfate (HD-MePhe-Pro-Arg-HH ₂ SO ₄ ) was obtained similar to free tripeptide aldehyde sulfate by removing the protecting groups from N-benzyloxycarbonyl 0

N-Methyl-D-phenylalanyl-L-prolyl-N-benzyloxycarbonyl-L-arginine aldehyde by hydrogenolysis in the presence of sulfuric acid. (Preparation of a tripeptide aldehyde used as a starting material is also described in the same patent application).

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

3.

So e.g. the product obtained by method No. 1 comprises about 4-6% calcium sulfate. Based on our layer-chromatographic analysis, the product thus obtained may comprise up to about 10% of other impurities (e.g., tripeptide alcohol, -acid, -acetal) other than calcium salt; this means that the total amount of contaminants can exceed 20%. A further disadvantage of this process is that the neutralization of excess sulfuric acid and the filtration of calcium sulfate precipitates further difficulties.

The tripeptide aldehyde sulfate salt obtained by process no. 2 (hydrogenolysis) comprises, except 5-10% of detellers, using thin layer chromatography, further 10-15% of the tripeptide aldehyde sulfate DLD configuration according to HPLC analysis: thus, a total of 15-25% of contaminants are constructed. product. The aforementioned 5-10% of contaminants are composed of the following substances: tripeptide acid rewarded during the synthesis of tripeptide aldehyde, tripetide alcohol, a cyclic decomposition product of the structure described in published European Patent Application no.

185 39u and a substance of unknown structure rewarded in varying amounts of stain contained.under the desired compound on the chromatogram.

It is noted that dahydration plays an important role in the production of the last by-product. Namely, if the hydrogenolysis is performed in a known manner, that is. in the presence of lmolar equivalent of sulfuric acid at room temperature and the process continues after complete hydrogenolysis, an additional stain (R ^ = 0.39-0.44) corresponding to the pollutant always occurs below the stain of the product (in the case of HD-Phe-Pro-Arg -H.H2SO4) on a thin-layer chromatogram (adsorbent: Kieselgel G, eluent: 25: 20: 6: 11 ethyl acetate / pyridine / acetic acid / water mixture · developer: chlorptolidine), in which cases the pollutant is, in some cases, the major impurity.

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

However, as the reaction endpoint is determined by thin-layer chromatographic analysis, which requires 20-30 minutes of operation using the above eluent mixture, the reaction cannot be stopped at the final

4.

point especially in industrial conditions. Thus a by-product with a predefined value is usually obtained in an amount of about

3-4% due to overgrowth. The by-product mentioned is more toxic than the end product, so it must be eliminated well for this reason

The product is highly sensitive to racemization, and therefore pollutants rewarded during the procedures described above cannot be practically removed. As the end product cannot be purified, in order to achieve purity of the end product which is suitable for pharmaceutical use, the production of intermediates of adequate purity and the suppression of construction of the by-product must be solved. This means to inhibit racemization during the process.

DESCRIPTION OF THE TECHNICAL PROBLEM SOLUTION WITH EXAMPLES

The object of the present invention is to develop a process that provides for the suppression of the construction of the above-mentioned by-products and the preparation of the tripeptide aldehyde salt of formula I in a purity of at least 95%.

The invention is based on the knowledge that the construction of D-L-D products, ie. racemization is highly pH dependent. In an environment that has an alkaline pH, and even in an environment with a neutral pH, the end product and intermediates are subject to racemization. In an environment that has an acidic pH of 6pH = 2) the end product is stable for months. Therefore, technology must be provided to ensure that the pH of the reaction mixture is not neutral or basic for even a short time. To achieve this, arginine lactam is released from its salt in situ during mixed anhydrous condensation G

N-benzyloxycarbonyl-L-arginine lactam hydrochloride of formula IV

H. Arg (Z) -lactam.HCl (IV) wherein Arg is as defined in the introductory part and Z denotes benzyloxycarbonyl and dipeptides of formula V

ZR (D-Phe) -Pro-OH (V) where R, Phe and Pro are the same as defined in the introductory part, Z is

5.

the same as defined above, in such a way that the base is then added to the reaction mixture. Under such conditions, arginine lactam released in situ reacts at once, in a statue nascendl, with mixed anhydride to a suitable protector. . tripeptide by the elbow of formula III

ZR (D-Phe) -Pro-Arg (Z) -lactam (III) where Z is the same as defined above, and R, Phe, Pro and Arg are the same as defined in the introductory part, so building a base environment which gives racemization is eliminated.

Further, the invention is based on the knowledge that if a complex rewarded with lithium aluminum hydride, obtained during the reduction of the protected tripeptide elbows of formula III obtained as described above, is decomposed in such a way that the reaction medium is added to the diluted, preferably 1 N, cold (0 -5 ° C) sulfuric acid used in excess in contrast to 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 prdpeptide aldehyde can be avoided or significantly reduced.

v

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

According to our experimental data, if hydrogenolysis. ZD-Phe-PrO-Arg (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 product thus obtained are as follows: 17% at 40 ° C, 10% at room temperature (20-25 ° C 4% at 6-10 ° C. DLD isomer contents of HPLC detectable product are as follows: 12%, 5% and 2-3%, respectively, ie the total impurity content of the product is This means that if the hydrogenolysis is carried out at lower temperatures, the amount of by-products decreases significantly while the time required for the hydrogenolysis is not significantly increased as the temperature decreases.

6.

If hydrogenolysis is carried out at lower temperatures in the presence of excess sulfuric acid, further great progress is made so that the construction of a by-product having a value of 0.39-0.44 can be substantially suppressed and accurate detection of the reaction endpoint is also eliminated. According to our experiments, if the hydrogenolysis of ZD-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 6-8 ° C, a by-product having an upper R ^ value builds only in traces even after 1 hour of overtaking.

Further, it is desirable to use sulfuric acid throughout all stages of the process so that e.g. uses HCl, the sulfate salt can be contaminated with chloride ions.

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

Based on the above, the invention relates to an improved process for the preparation of a tripeptide aldehyde of formula I, wherein R, Phe, Pro and Arg are the same as defined above, in pharmaceutically pure form, by condensation of the elbows of formula IV, wherein Arg is as is described above and Z denotes benzyloxycarbonyl, with a dipeptide of formula V, wherein R, Z, Phe and Pro are as defined above in the introductory part and Z is the same as defined above, via mixed anhydride, reduction of the tripeptide lactams of formula III, where R, Z, Phe, Pro and Arg are the same 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 the same as defined above in the presence of sulfuric acid, which includes

Mr

a ..) release of N-protected L-araginin lactam in situ during * - G condensation of N-protected L-arginine lactam hydrochloride of the formula

IV, wherein Z is the same as defined above, of the dipeptide of the 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, featim reduction of the thus obtained protected tripeptide elbows of formula III where R, Z, Phe, Pro and Arg are the same as defined above, decomposing the complex thus rewarded by adding the reaction mixture to excess IN sulfuric acid at a temperature of 0-5 ° C, then performing the 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 10 ° C in the presence of 1-2 molar equivalent of sulfuric acid, or a ₂ ) after reduction of the thus obtained protected tripeptide elbows of formula III, where R, Z, Phe, Pro and Arg are the same as which is defined by decomposing the complex thus rewarded by adding the reaction mixture to excess INH ₂ SO 4 at a temperature of 0-5 ° C, then performing the hydrogenolysis of the thus obtained protected tripeptide of formula II, where R, Z, Phe, Pro and Ar are the same as what is the def initiated at a temperature below 10 ° C in the presence of 1-2 molar equivalents of H ~ SO. or z 4 a ^) performing the hydrogenolysis of a protected tripeptide of formula II, wherein R, Phe, Pro and Arg are the same as defined above, at temperature. below 10 ° C in the presence of l-2molar equivalent of H ₂ SO ^.

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

Mr

From t-butoxycarbonyl-N-benzyloxycarbonyl-L-arginine lattam

The G_ t-butoxycarbonyl protecting group is removed in a known manner. N benzylcarbonyl-L-arginine lactam hydrochloride: Formula IV thus obtained is held in dimethyl formamide solution at a temperature of -1b to -20 ° C. Then this solution is added to a mixed anhydride rewarded with a dipeptide of formula V (benzyloxycarbonyl-D-phenylalanil- L-proline or N-methyl-N-benzyloxycarbonyl-D-phenylalanyl-L-proline) and chlorocarboxylic acid isobutyl ester. The base, preferably trimethyl amine, is then added to the reaction mixture. Under these sulums, the lactam os swan is in situ due to the effect of the base, reacts at once with mixed anhydride to build a protected tripeptide. Elbows, and thus the construction of a base environment that causes racemization can be avoided. If desired, the product thus obtained can be purified by rapid filtration through a pad of silica gel. By choosing the above sequence of operations, which is

8.

different from the BC from the prior art, there is no need for expensive purification by tripeptide elbow protected by column chromatography.

The protected lactam protected tripeptide of formula III thus obtained is reduced with Jitium aluminum hydride in dry tetrahydrofuran. The complex thus obtained is decomposed-> against .. published European Patent Application no. 185 390 by adding the reaction mixture to the diluted solution, preferably IN Η ₂ θ ° 4 k ° ^r and ^ en in excess with cooling at 2-4 ° C. The final pH value of 2-3 is optionally adjusted by the addition of I ^ SO ^. The reaction mixture is then processed in a known manner. The protected tripeptide aldehyde of formula II thus obtained is purified by filtration through a layer of silica gel.

Hydrogenolysis is. preferably carried out in a polar organic solvent, preferably tetrahydrofuran or lower alkanol, e.g. methanol, ethanol, i-propanol, preferably in ethanol. It is preferable that hydrogenolysis is not carried out in a closed system,_kby bubbling hydrogen through the reaction mixture. Practically hydrogen gas is bubbled through the system in a finely divided form which can be provided by introducing gas through Gj filter.

According to a preferred embodiment of the process, the protected tripeptide.-Aldehyde of formula II of suitable purity is thus dissolved in tetrahydrofuran and the solution thus obtained is added to the cooled aqueous catalytic suspension comprising 1.5 -1.7 molar equivalents IN I ^ SO ^ · Organic ratio of the solvent to the aqueous fraction is 70-50: 30-50, preferably 60:40. As a catalyst, it burns? 10-30% wt. 10% of palladium on coal or palladium is used. Hydrogenation is carried out under cooling at 6-8 ° C by bubbling fine hydrogen gas through the system. The progress of the reaction is monitored by thin-layer toromatographic analysis.,

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

The hydrogenation is carried out at a temperature of 6-8 ° C in the presence of 1.5 molar equivalent of H2SO4 by bubbling fine hydrogen gas through the system. The end of the reaction is monitored by thin layer chromatographic analysis.

According to a further preferred embodiment of the method 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 coal catalyst and 1 molar equivalent of IN H-SO ..

+4

Hydrogenation is carried out at 6-8 C by bubbling fine hydrogen gas through the system and the process is followed by thin layer chromatography.

v

The product can be simply separated from the reaction mixture by filtering the catalyst and evaporating the organic solvent from the aqueous-organic solvent mixture at a temperature below 40 ° C, preferably 25-30 ° C, then freezing the aqueous solution directly or after adjusting the pH to about 4. pH is preferably adjusted by the use of a hydroxy-phase ion exchange resin or by the use of O, 1N H2SO4.

When the process according to the invention is carried out on a pilot plant, maximum contamination of the product thus obtained (D-phenylalanil-L-propyl-L-raginine aldehyde sulfate and N-methyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde sulphate) is detectable by thin-layer chromatogarf analysis is at most l%, while DLD has its 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 for pharmaceutical suspensions and contrary to the impurity content of 15-25% in the case of lean processes.

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

R _f values were determined by flash silica chromatography on silica gel (Kieselgel G, Reanal, Budapest) using the eluent mixtures given in the examples. Developer: Chlorinated chromatographic plates were treated with a 0-tolide solution (2.5 g o-tolidine and 10 ml acetic acid dissolved in 500 ml water) after drying.

EXAMPLE 1

Preparation of D-Phenylalanil-L-prolyl-L-arganine aldehyde sulfate

Mr

201g (0.3 mol) of benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N-benzyloxycarbonyl-L-arginine aldehyde was dissolved in 2 liters of tetrahydrofuran at room temperature. At the same time, 60 g of 10% palladium-on-coal are suspended in 0.6 liters of deionized water, then 900 ml of 1 SO 2 (0.45 mol) is added. The suspension comprising the catalyst was cooled to 2-50 ° C, and a tetrahydrofuran solution of the protected tripeptide aldehyde and additional about 21 liters of deionized water was added with stirring. The hydrogen is then bubbled through a mixture whose temperature is maintained at 6-8 ° C with stirring.

The progress of the reaction is followed by thin layer chromatographic analysis using a 60: 20: 6: 11 ethyl acetate / pyridine / acetic acid / water mixture as eluent. (R ^ values of starting materials, intermediate and end product are 0.92-0.96, 0.38-0.44 and 0.0-0.10, respectively).

v

The reaction is complete for 3-8 hours. At the end of the reaction, the catalyst was filtered off and washed 3 times with 100 ml deionized water each. The filtrate and the rinsing liquid are combined, then distilled off with tetrahydrofuran in a rotary evaporator under vacuum at a temperature of 25-30 ° C.

The remaining aqueous solution was extracted with 2x100 ml of dichloromethane, then the pH of the aqueous phase was controlled. If the pH differs from 4, then the pH is adjusted to 4 by the addition of 0, 1 N H 2 SO 4 or OH-cyclic ion exchange resins (eg AG 1x2).

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

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

/ a /, 20 ^{/ A /} d = -117 ° (c = 1, water).

Total impurity content by thin-layer chromatography

11.

analysis: 3-5%, while the D-L-D isomeric content determined by HPLC is about 2-3%.

Mr

Benzyloxycarbonyl-D-phenylalanyl-L-lproline-N-benzyloxycarbonyl-Larginine aldehyde used as starting material can be obtained as follows:

Level 1

Mr

Benzylocarbonyl-D-phenylalanyl-prolyl-N-benzyloxycarbonylL-arginine lactam

Mr

429.5g (1.1 mol) of t-butoxycarbonyl-N-benzyloxycarbonyl-arginine actam (European Patent Application Publication No. 185 390, suspended in 1300ml of dry chloroform) Then l300ml of lb9g / 100ml (about 5N) was added dropwise. ethyl acetate hydrochloride and cooled with ice water at a rate that avoids temperature rise above 15 ° C. The dissolution and precipitation of the crystalline substance begins shortly. The suspension is stirred at room temperature for 3 hours, then diluted with 3000 ml of dry 1: 1 ether mixture and The crystalline solids were filtered off, washed with 2x1000 ml acetone and 5 [mu] l ether, then dried over solid potassium hydroxide and phosphorus pentoxide in a vacuum desiccator.

Mr

After about 1 hour of drying, the N-benzyloxycarbonyl-L-arginine-lactam hydrochloride was dissolved in 100 ml of dry dimethyl formamide, cooled to -15 ° C and -20 ° C, and added to the mixed anhydride obtained as in ED:

306g (1 mol) of N-benzyloxycarbonyl-D-phenylalanil-L-proline (Nicolaides E. et al: J.Med.Chem. 11, 74 (1968) and European Patent Application Publication No. 185 396) are dissolved in 1000 ml of anhydrous dimethyl foraamide and 11 ml (1 mol) of N-methyl morpholine are added. The mixture was then cooled to -15 [deg.] C. and 132 ml (1mol) of isobutyl ester of chlorocarboxylic acid were added dropwise at the same temperature with stirring for 5-10 minutes. The mixed anhydride was cooled to -20 C to -2b ° C after 10 minutes of stirring and combined with dimethyl

12.

Q foramide solution of N -benzyloxycarbonyl-L-arginine lactam nitrochloride obtained above. Then 308ml (2.2ml) of triethyl amine was added dropwise to the reaction mixture at the same temperature for about 3 minutes, the suspension was then stirred for a further hour at -20 to -15 ° C. The pH of the vapor space is controlled by wet pH paper: if the pH drops below 8, a further amount of triethyl amine is added. The mixture is then diluted with 2 liters of benzene, the precipitated salts are filtered off and washed twice with 500 ml of benzene each.

To the benzene dimethyl formamide filtrate was added 1.5 liters of water and the phases were separated. The aqueous dimethyl formamide layer was extracted with 2x500ml of benzene. The combined benzene solutions were washed with 2x0.5 liters of water, 0.5, 11 tra, IN-H ₂ SO ₄ , then with 3x0.5 liters of water, dried over anhydrous sodium sulfate and evaporated to 2 liters under vacuum at maximum temperature of 40 ° C.

360g of Kieselgel_G adsorbent is suspended in benzene and a filter layer is formed, where the diameter to height ratio is 8: 1 to 4: 1. The benzene solution is aspirated through a flatter layer under low vacuum, then the filter layer is washed with 4.8 liters of benzene in the same way, the combined benzene solutions are concentrated to about 600ml volume under vacuum at a temperature of up to 40 ° C, then with vigorous stirring added 31 liters of diisopropyl ether.

The precipitate was filtered off, washed with 2x600 ml of diisopropyl ether and dried in a vacuum desiccator above Ρ ₂ ^ο ς ¹ paraffin flakes. In this way 5g (76%) of the desired product is obtained. The total content of the contaminant of the product is determined by the Stanko-sebaceous Chromatography © is 1-2%.

Rf = 0.55-0.65 (ethyl acetate) (Rf value of L-D-L lactams is 0.45-0.50 in ethyl acetate).

(0H) - -55.8 ° (c = 1, tetrahydrofuran).

D

Level 2

Mr

Benzyloxycarbonyl-D-phenylalanyl-L-propyl-N-benzyloxycarbonyl-L-arginine aldehyde.

300 mg (0.45 mol) of protected tripeptide lactam (Example 1, step 1) was dissolved in 2.5 liters of anhydrous tetrahydrofuran cooled to -5 ° C. The solution was cooled to -20 ° C to -25 ° C in a dry ice / acetone bath, and stirred vigorously for 20-25 min + and a solution of lithium aluminum hydride and tetrahydrofuran was added, having a concentration of about 0.7 moles / liter, equivalent to 0.375 moles of lithium aluminum hydride. The progress of the reaction was monitored by thin layer chromatography using 240: 20: 6: 11 ethyl acetate / pyridine / acetic acid / water mixture as eluent. If necessary, a further amount of lithium aluminum hydride solution is added until a stain having an R _f value of 0.75-0.80 corresponding to the lactam disappears.

When the reaction is complete, the reaction mixture is gradually poured into 3 liters of IN H2 ^S ° 4 ^solution at 2-3 ° C with stirring. The final pH value is adjusted to dcdavaa.4era.p0 to select a further amount and ^ SO ^.

The water is added to the solution until it is cloudy, then extracted with 3x0.9 liters of n-hexane. Between the two extractions, the material is separated as an oily phase and is therefore homogenized to tetrahydrofuran, then obtained as milk by the addition of some water. The aqueous phase is extracted with 2x21 liters of dichloromethane. The combined dichloromethane solutions were washed with 2x0, b liters of 5% NaHCO4 solution at 4-8 ° C, then with 2x0.5 liters of distilled water at 4-8 ° C, dried over anhydrous sodium sulfate and evaporated in vacuo at a temperature of not more than 40 ° C (about 300g of oily substance is obtained)

The oily residue was dissolved in a 7: 3 mixture of dichloromethane and acetone, filtered through a filter layer (diameter: height = 5: 1) obtained from 900g of Kieselgel G adsorbent using a 7: 3 mixture of dichloromethane and acetone as eluent without suction, then the filter layer was washed with 6 liters of the same eluent mixture. The process may take more than 20 minutes. The combined filtrates were evaporated to about 600 ml at a temperature of up to 40 ° C, then 1 liter of benzene was added and the solution was re-evaporated, then the product was precipitated by adding 1.6 liters of LkJLhexane with stirring. The precipitate was filtered off, washed with 2x0.3 liters of cyclohexane and dried in a vacuum desiccator at room temperature above paraffin flakes. This gives 240g (79.7%) of the title compound.

14.

R _f = 0.52-0.62 (ethyl acetate / pyridine / acetic acid / water =

240: 20: 6: 11,.

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

Δ = -0.8 ° (c = 1, tetrahydrofuran).

D

EXAMPLE 2 k

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

Q

20. The Ig (0.03 mol) benzyloxycarbonyl-D-phenylalanyl-1-prolyl-N benzyloxycarbonyl-L-arginine aldehyde is dissolved in 214 ml of tetrahydrofuran and the solution is cooled to a temperature of 0 ° C. 4.3g of palladium on coal catalyst is suspended in 70ml of deionized water and 102ml IN I ^ SO ^ is added. The catalyst suspension was cooled to 2-5 ° C, the air was eliminated with the help of gaseous nitrogen. The top tetrahydrofuran solution is then added to the suspension with stirring and fine hydrogen gas is introduced into the system. The temperature is kept between 6 and 8 ° C by cooling with ice. The progress of the reaction was monitored by thin layer chromatography and the eluent sieve mixture was crucible as described in Example 1. At the end of the reaction (about 3 hours), the reaction mixture thus obtained was treated according to Example 1. Yield: 12.5 g (83.2 %).

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

-, / 20 = -117 ° C (c = 1, water)

The impurity content of thin-layer chromatographic analysis is 3.5%. The content of D-L-D isomers based on HPLC analysis is 1.5%.

EXAMPLE 3

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

15.

Mr

201g (0.3 mol) of benzyloxycarbonyl-D-phenylalanyl-L-prolyl-N benzyloxycarbonyl-L-arginine aldehyde was dissolved in 2 liters of ethanol at room temperature. 60g of catalyst comprising 10% palladium on barium sulphate as a carrier was suspended in 0.6 liters of deionized water and 600m INH SO (0.3 mol) was added. The suspension was cooled to 2-5 ° C and ethanolic solution of the protected tripeptide aldehyde was added. Deionized water is then added in an amount corresponding to a 6: 4 ratio of solvent to aqueous phase.

Hydrogen gas is then bubbled through the mixture with stirring and cooling at 6-8 ° C. The progress of hydrogenation is followed 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 filters were combined and the alcohol was evaporated on a rotary evaporator at a temperature of 25-30 ° C. An aqueous solution having a pH of about 3-4 is directly freeze-dried. 10g (65%) of the desired product is obtained in the form of monohydrate, which has the same qualietfet as the product obtained in example 1.

s

EXAMPLE 4

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

The procedure of Example 3 was followed except that 43cj was 10% palladium on coal catalyst, previously rinsed to neutralization and then used as catalyst.

This gives 125, lg (83.2%) of the desired product having the same quality as the product of Example 1.

EXAMPLE 5

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

2u, 5g (0.03 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-LG prolyl-N-benzyloxycarbonyl-L-arginine aldehyde was dissolved in 214ml of ethanol, then HOml of deionized water was added, 60ml of IN ^υ 4.

(0.03 mol) and 3g of 10% palladium on coal as catalyst. Subsequently, the finely made gas is introduced into the mixture with cooling at 6-8 ° C. The progress of the reaction was monitored by thin layer chromatography using a mixture of 25: 20: 6: 1 ethyl acetate / pyridine / acetic acid / water <RTI ID = 0.0> eluent. On the chromatogram, the values of the starting material, intermediate and end product are about 0.9, 0.7 and 0.4, respectively. The reaction is completed in about 2 hours. At the end of the reaction, the catalyst was filtered off and washed with 3x30ml deionized water. The filtrate and the pouring liquid are combined, then evaporated on a rotary evaporator to about 100 ml at a temperature of not more than 25 ° C, the residue is then extracted with 2x50 ml of dichloromethane.

Traces of the organic solvent are removed from the aqueous solution by distillation and the dilute is diluted with deionized water to a volume of about 200ml.Checked jeidalic.is pH 4. If necessary, the pH is adjusted by adding 0.1 NH ₂ SO ₄ or ion exchange resin OH phase (e.g. . AG 1ΧΒ), then the solution is frozen ~ and_frozen is dried. Yield 12.5g (75%)

Rf = 0.35-0.45 lethal acetate / pyridine / acetic acid / water = 25: 20: 6: 1) / (// ² ° = -126.0 + 5 ° (c = l, water)

D

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

The impurity content based on HPLC analysis is 3-5%.

N-Benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolll-N-benzyloxycarbonyl-L-arginine aldehyde was used as starting material and can be obtained as follows:

Level 1

Mr

N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N benzyloxycarbonyl-L-arginine lactam

Mr

42.95 g (0, 10 mol) of t-butoxycarbonyl-N-benzyloxycarbonyl-L-aroinine lactam was suspended in HOml of dry chloroform and 275ml of ethyl acetate hydrochloride, was added with constant stirring. The reaction mixture is

17.

stirred for about 3 hours at room temperature, the reaction end point 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 end product are about 0.9 and 0.3 respectively). At the end of the reaction, the mixture was diluted with 400 ml of diethyl ether, the crystalline solid filtered off, washed with 2x100 ml of diethyl ether and 2x50 ml of acetone, then dried on a vacuum ejector over phosphorus pentoxide and potassium hydroxide. After 1-2 hours of drying, the lactam salt thus obtained (about 0.1 mol) was dissolved in 100 ml of anhydrous dimethyl formamide and magnified at -15 [deg.] C. to its used anhydrous anhydride / obtained as follows:

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

50.9g (0.1 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-Lproin cyclohexyl ammonium salt was dissolved in-2U0ml diethyl ether and l20ml IN H ₂ SO ₄ · Ethyl ether fa-3 was washed with 2x50ml of water, dried over anhydrous sodium alfafac and evaporated under reduced pressure at a temperature of not more than 35 ° C to give a thick oil.

The residue (about 0, 1 mol) was dissolved in .-: and 170 ml of anhydrous dimethyl formamide, cooled to -15 [deg.] C. and 1, 2 ml (0, 1 mol) of N-methyl-morpholine were added with stirring. Then, 13.2ml of i-butoxycarbonyl chloride was added dropwise over 3-5 minutes while maintaining the same temperature. After further 5-10 minutes of stirring, the above dimethyl formamide suspension of amino kw.ngS3Mente was * added to the mixed anhydride thus obtained, then 35 ml of triethyl amine was added to the reaction mixture with vigorous stirring at -20 to -25 ° C. The pH of the vapor space was checked (the pH should be above 8) and a further amount of triethyl amine was added if necessary. The reaction mixture was stirred for 1 hour at -15 ° C, then at 0 ° C and then diluted with 200ml benzene and 150ml water was added.

The phases were separated and the aqueous phase was extracted with 3x30ml benzene.

The combined benzene solutions were washed with 2x50ml of water, dried over anhydrous sodium sulfate and evaporated under reduced pressure at a temperature of up to 40 ° C to obtain a thick oil. The oily residue was dissolved in 75 ml of a 2: 1 mixture of ether and tetrahydrofuran

18.

and filter through a filter layer as follows:

300g (5-fold amount, Kieselgel-60 adsorbents are sundried in ether and the filter layer is prepared with a diameter: 3: 1 ratio. The contaminating top spot is eluted with a mixture of 95: 5 ether and tetrahydrofuran, while the desired product is eluted with The 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 eluebt. The desired product was 0.71-0.75. The combined fractions were evaporated under ¹ vacuum. at 35 [deg.] C. to give a thick residue and the product was used at one time or m &apos; s in dry ice Yield: 58.8g (85%).

Rf = 0.7l-0.75 (ethyl acetate / pyridine / acetic acid / water = 480: 20: 6:11.

= + 13.5 ° (c = l, tetrahydrofuran)

D

Level 2

Mr

N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-N benzyloxycarbonyl-L-arginine aldehyde

34.14g (0.05 mol) of N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-LG propyl-N-benzyloxycarbonyl-L-argin lactam (example 5, step 1) was dissolved in 150 ml of dry tetrahydrofuran cooled to 0 ° C. The solution was cooled to -20 ° C and 0.0375 mol of lithium aluminum hydride was dissolved in tetrahydrofuran and added with stirring for 5-10 minutes. The progress of the reduction was followed by thin layer chromatography using a mixture of 240: 20: 6: 11 ethyl acetate / pyridine / acetic acid and water. (The R ^ values of the elbows 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 mixed with continuous stirring.

320ml IN H 'SO pre-cooled to 2-4 ° C. The final z 4 pH should be between 2 and 3, this pH is adjusted by adding further

19.

quantities I ^ SO ^ .if necessary. If a precipitate is formed, this is filtered off, the solution is diluted with water until turbid and then extracted with 3xl00ml n-hexane.

The aqueous tetrahydrofuran phase was extracted with 250 ml of dichloromethane 2 or 3 times and the combined dichloromethane solutions were washed with 2x50ml of water, 2x50ml of cold 5% NaHCO 3 solution, then with 2x50ml of water. The solution was then dried over anhydrous sodium sulfate and evaporated in vacuo to a temperature of not more than 40 ° C.

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

The combined filtrates were evaporated to 50-60ml under reduced pressure at a temperature of more than 40 ° C, then this operation was repeated after the addition of 100ml of benzene to the residue. The product was precipitated from a solution of about 50-60ml by the addition of 100ml of cyclohexane.

The precipitate thus obtained was washed with 2x30ml cyclohexane and dried over paraffin flakes in a vacuum desiccator. Yield is 18g (53%).

Rf = 0.55-0.59 (ethyl acetate / pyridine / acetic acid / water =

240: 20: 6: 11).

/<λ./ ² θ = + 14.1 ° (c = l, tetrahydrofuran).

D

The total impurity content of the product is 2-3% by weight.

Claims (5)

Patent claims An improved process for the preparation of tripeptide aldehydes of formula I RD-Phe-Pro-Arg-HH ₂ SO ₄ I where R represents hydrogen or methyl, Phe is a phenylalanine group, is a Pro L-proline group, and Arg denotes the L-arginine group in pharmaceutically pure form, by condensation of the N ^G -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 a dipeptide of formula V, ZR (D-Phe) -Pro-OH V where R, Z, Phe and Pro are the same as defined previously, via mixed anhydride, reduction of the tripeptide lactam of formula III, ZR (D-Phe) -Pro-Arg (Z) -lactam III wherein R, Z, Phe, Pro and Arg are the same as previously defined with lithium aluminum hydride and hydrogenolysis of the protected tripeptide aldehyde of formula II thus obtained ZR- (D-Phe) -Pro-Arg (Z) -H II wherein R, Z, Phe and Arg are the same as defined previously in the presence of H ₂ SO ₄ , characterized in that af comprises the release of Unprotected L -arginin lactam in situ during the condensation of N ^G protected L-arginine lactam hydrochloride of the formula IV, H-Arg (Z) -lactam.HCl IV wherein Z is the same as previously defined and a dipeptide of formula V ZR (D-Phe) -Pro-OH V wherein R, Z, Phe and Pro are the same as previously defined in the mixed anhydride, in the reaction mixture and then adding the base to the mixture of said reaction components, then, 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 previously, decomposing the complex thus obtained by adding the reaction mixture to excess AND H ₂ SO ₄ at temperature from 0 to 5 ° C and then carrying out the hydrogenolysis of the protected tripeptide of formula II, ZR- (D-Phe) -Pro-Arg (Z) -H II where R, Z, Phe, Pro and Arg are the same as previously defined at a temperature below 10 ° C in the presence of 1-2 molar equivalents of H ₂ SO ₄ or a ₂ ) after reduction of the protected tripeptide lactam of formula III thus obtained, ZR (D-Phe) -Pro-Arg (Z) -lactam III wherein R, Z, Phe, Pro and Arg are the same as defined previously, decomposing the complex thus formed by adding the reaction mixture to excess AND H ₂ SO ₄ at a temperature from 0 ° C to 5 ° C, followed by the hydrogenolysis of the protected tripeptide of formula II thus obtained, ZR- (D-Phe) -Pro-Arg (Z) -H II where R, Z, Phe, Pro and Arg are the same as previously defined at a temperature below 10 ° C in the presence of 1-2 molar equivalents of H ₂ SO ₄ , or a ₃ ) performing a hydrogenolysis of a protected tripeptide of formula II, ZR- (D-Phe) -Pro-Arg- (Z) -H II 22 where R, Z, Phe, Pro and Arg are the same as previously defined at a temperature below 10 ° C in the presence of 1-2 molar equivalents of H ₂ SO ₄ . The process of claim 1, wherein the release comprises Νθ-protected-Larginine lactam from lactam hydrochloride of formula IV with triethylamine. Process according to claim 1, characterized in that it comprises carrying out hydrogenolysis at a temperature of 6 - 8 ° C. Process according to one of Claims 1-3, characterized in that it comprises performing hydrogenolysis by blowing hydrogen gas through the system. Process according to one of Claims 1-3, characterized in that the hydrogenolysis is carried out in ethanol or tetrahydrofuran.