GB2244994A - Preparation of tripeptide aldehydes - Google Patents

Abstract

Improved process for the preparation of tripeptide aldehydes of formula I, R-D-Phe-Pro-Arg-H.H2SO4 I wherein R represents hydrogen or methyl, Phe stands for phenylalanine group, Pro is L-proline group and Arg stands for L-arginine group, which comprises liberating the N<G>-protected L-arginine lactam (III) in situ in the course of condensation of N<G>-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 a mixed anhydride in the reaction mixture by subsequently adding a base to the mixture of the said reaction components, then reducing the thus-obtained protected tripeptide lactam of formula III, ZR(D-Phe)-Pro-Arg(Z)-lactam III decomposing the complex thus formed by adding the reaction mixture to an excess of 1 N sulfuric acid at a temperature of 0 to 5 DEG C, then carrying out the hydrogenolysis of the protected tripeptide of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II at a temperature below 10 DEG C in the presence of 1 to 2 molar equivalents of sulfuric acid.

Description

I ROVED PROCESS FOR THE PREPARATION OF TRIPEPTIDE ALDEHYDES The present invention relates to an improved process for the preparation of tripeptide aldehydes of formula I, R-O-Phe-Pro-Arg-H. H2 SO4 I wherein R represents hydrogen or methyl, Phe stands for phenylalanine group, Pro is L-proline group and Arg stands for L-arginine group, in a purity suitable for pharmaceutical use.

{The abbreviation of L-proline and L-arginine as Pro and Arg is in accordance with the prior art, e.g. Biochem. 7, 126, 773 (1972), Biochemistry 14, 449 (1975) It is known that the salts of D-phenylalanyl-Lprolyl-L-arginine aldehyde (H-D-PHe-Pro-Arg-H) exhibit significant antithrombine activity. However, the acetate salt described in Hungarian patent specification No. 169,87û is not suitable for therapeutic use as it looses its activity very quickly either in solid form or in the form of an aqueous solution. The same phenomenon can be observed in case of the hydrochloric, citrate, tartarate, tosylate salts and the NG-carboxy derivative of the tripeptide aldehyde.However, the sulfate salt of H-D-Phe-Pro-Arg-H and the N-methyl derivative thereof are highly stable even in the form of aqueous solution (British patent specification No. 2,091,270 and published European patent as?lication Rlo.

185,39û), and therefore they can be suitable for therapeutic use.

The above sulfate salts can be prepared as follows: 1. The sulfate salt ofH-D-Phe-Pro-Arg-H can be prepared according to British patent specification No.

2,091,270 from t-butoxyzarbonyl-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 to 12 equivalents of 1 to 12 N aqueous sulfuric acid at a temperature of 40 to 600C. (The preparation of the first starting material is described by Example 1 of the above-cited patent specification, while the preparation of the protected NG-carboxy derivative is taught by Belgian patent specification No. 880,844.) 2.The sulfate salt of H-D-Phe-Pro-Arg-H can further be prepared according to the above-cited specification by hydrogenolysis of benzyloxycarbonyl-D- phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine alde hyde (Z-D-Phe-Pro-Arg/Z/-H) in the presence of, a molar equivalent amount of sulfuric acid. (The preparation of the double-protected tripeptide aldehyde used as starting material is described by Hungarian patent specification No.

169,870.) 3. According to published European patent application No. 185,390 N-methyl-D-phenylalanyl-L-prolyl-Larginine aldehyde sulfate (H-D-MePhe-Pro-Arg-H.H2S04) is prepared similarly to the free tripeptide aldehyde sulfate by removing the protective 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 by the same patent application.) The main disadvantage of the above processes is, especially if the production is carried out on large scale, that they do not result in a product of appropriate purity.

Thus e.g. the product obtained by process No. 1 comprises about 4 to 6 % of calcium sulfate. On the basis of our thin-layer chromatographic analysis the product thus obtained may comprise more than about 10 % of other impurities (e.g. tripeptide alcohol, -acid, -acetal) in addition to the calcium salt; this means that the total amount of contaminating substances may exceed 20 %. A further disadvantage of this process resides in that the neutralization of the excess of sulfuric acid and the filtration of the precipitated calcium sulfate cause further difficulties.

The tripeptide aldehyde sulfate salt prepared by process No. 2 (hydrogenolysis) comprises, in addition to the 5 to 10 % of contaminations detectable by thin-layer chromatographic analysis, further 10 to 15 % of tripeptide aldehyde sulfate of D-L-D configuration according to HPLC analysis; thus totally 15 to 25 % of contaminating sideproduct can be formed. The above-mentioned 5 to 10 % of contaminations are composed of the following substances: tripeptide acid formed during the synthesis of tripeptide aldehyde, tripeptide alcohol, a cyclic decomposititon product the structure of which is described in the published European patent application No. 185,390 and a substance of unknown structure being formed in different quantities, the spot of which is situated below the target compound on the chromatogram.

It has been observed that the "overhydration" plays an important role in the production of the latter sideproduct. Namely, if the hydrogenolysis is carried out in a manner known per se, i.e. in the presence of 1 molar equivalent of sulfuric acid at room temperature and the process is continued after the hydrogenolysis is completed, an additional spot (Rf = 0.39 - 0.44) corresponding to a contamination always appears below the spot of the product (in case of H-D-Phe-Pro-Arg-H.H2S04 Rf = 0.52 - 0.57) on the thin-layer chromatogram (adsorbent: Kieselgel G, eluent: a 25:20:6:11 mixture of ethylacetate/pyridine/acetic acid/water; developer: chlorotolidine), wherein the said contamination, in some cases, represents the main impurity.

In order to eliminate this contamination, the endpoint of the reaction has to be detected very exactly and the reaction has to be stopped when the end-point is reached.

However, as the end-point of the reaction is determined by thin-layer chromatographic analysis, and the analysis demands 20 to 30 minutes if the above eluent mixture is used, the reaction cannot be stopped at the endpoint especially under industrial conditions. Thus the sideproduct with the above-defined Rf value is generally produced in about 3 to 4 % due to "overhydration". The said side-product is more toxic than the end-product, therefore it has to be eliminated for this reason as well.

The product is highly susceptible to racemization, therefore the contaminating substances formed during the above-described processes cannot practically be removed As the end-product cannot be purified, in order to achieve an end-product in a purity which is suitable for pharmaceutical use, the production of the intermediates in appropriate purity and the suppression of the formation of the side-product had to be solved. This means the inhibition of racemization during the orocess.

The aim of the present invention is the working out of a process which enables the suppression of the formation of the above-mentioned side-products and the preparation of tripeptide aldehyde salts of formula I in a purity of at least 95 %.

The invention is based on the recognition that the formation of D-L-D product, i.e. the racemization, is highly influenced by the pH. In a medium having an alkaline pH, and even in a medium having a neutral pH, the end-product and the intermediates undergo racemization. In a medium having acidic pH (pH = 2) the end-product is stable for months.

Therefore a technology had to be elaborated which ensures that the pH of the reaction mixture does not turn neutral or basic even for a short time. In order to achieve this aim arginine lactame is liberated from its salt in situ during the mixed anhydride condensation of the NG- benzyloxycarbonyl-L-arginine lactam hydrochloride of formula IV H.Arg(Z)-lactam . HC1 IV - wherein Arg is the same as defined in the introductory part and Z stands for benzyloxycarbonyl - and the dipeptide of formula V ZR(D-Phe)-Pro-OH V - wherein R, Phe and Pro are the same as defined in the introductory part, Z is the same as defined hereinabove in such a manner that the base is subsequently added to the reaction mixture.Under such conditions the arginine lactam liberated in situ reacts at once, in statu nascendi, with the mixed anhydride to the corresponding protected tripeptide lactam of formula III ZR(D-Phe)-Pro-Arg(Z)-lactam III - wherein Z is the same as defined hereinabove and R, Phe, Pro and Arg are the same as defined in the introductory part -, thus the formation of a basic medium resulting in racemization can be eliminated.

Further the invention is based on the recognition that if the complex formed with lithium aluminium hydride, obtained during the reduction of the protected tripeptide lactame of formula III obtained as described hereinabove, is decomposed in such a manner that the reaction medium is added to the diluted, preferably 1.N, cold (0-50C) sulfuric acid solution used in an excess, contrary to published European patent application No. 185,390 in which the sulfuric acid is added to the reaction mixture, the acidic pH of the reaction mixture can be assured. Thereby the racemization of the protected tripeptide aldehyde can be avoided or significantly reduced.

The invention is based on the further recognition that the hydrogenolysis of the tripeptide aldehyde of formula II prepared as described hereinabove has to be carried out in the presence of excess of sulfuric acid at low temperature, below 10 OC, preferably at 6 to 8 OC.

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

If the hydrogenolysis is carried out at lower temperatures in the presence of excess of sulfuric acid, the further great advantage arises that the formation of the side-product having an Rf value of 0.39 to 0.44 can significantly be suppressed and the exact detection of the end-point of the reaction is also not needed. According to our experiments if the hydrogenolysis of Z-D-Phe-Pro-Arg(Z)- H is carried out in the presence of 1.5 to 1.7 molar equivalents of sulfuric acid in tetrahydrofuran at a temperature of 6 to 8 OC, the side-product having the above Rf value is formed in only trace amounts even after 1 our of "overhydration".

Further, it is preferable to use sulfuric acid in the course of all stages of the process as if e.g.

hydrochloric acid is used, the sulfate salt may be contaminated with chloride ions.

As a result of the reactions carried out as described hereinabove, the amount of DLD isomer in ;the endproduct of formula I is at most 2 to 4 %, while the other contaminations (tripeptide acid and -alcohol) are formed in an amount below 1 Based on the above, the invention relates to an improved process for the preparation of tripeptide aldehydes of formula I, wherein R, Phe, Pro and Arg are the same as defined hereinabove, in pharmaceutically pure form, by condensing a lactam of formula IV, wherein Arg is the same as defined hereinabove and Z stands for benzyloxycarbonyl, with a dipeptide of formula V, wherein R, Z, Phe and Pro are the same as defined in the introductory part and Z is the same as defined hereinabove, through a mixed anhydride, reducing the tripeptide lactam of formula III, wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, with lithium aluminium hydride, and hydrogenolysing the thusobtained protected tripeptide aldehyde of formula II, wherein R, Z, Phe and Arg are the same as defined hereinabove, in the presence of sulfuric acid, which comprises al) liberating the N-protected L-arginine lactame in situ in the course of condensation of I IG protected L-arginine lactam hydrochloride of formula IV wherein Z is the same as defined hereinabove, and dipeptide of formula V, wherein R, Z, Phe and Pro are the same as defined hereinabove, into a mixed anhydride in the reaction mixture by subsequently adding a base to the mixture of the said reaction components, then reducing the thus-obtained protected tripeptide lactam of formula III, wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, decomposing the complex thus formed by adding the reaction mixture to an excess of 1 N sulfuric acid at a temperature of O to 5 OC, then carrying out the hydrogenolysis of the protected tripeptide of formula II, wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, at a temperature below 100C in the presence of 1 to 2 molar equivalents of sulfuric acid or a2) after reduction of the thus-obtained protected tripeptide lactam of formula III, wherein R. Z, Phe, Pro and Arg are the same as defined hereinabove, decomposing the complex thus formed by adding the reaction mixture to an excess of 1 N sulfuric acid at a temperature of O to 5 OC, then carrying out the hydrogenolysis of the thus-obtained protected tripeptide of formula II, wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, at a temperature below 1000 in the presence of 1 to 2 molar equivalents of sulfuric acid or a3) carrying out the hydrogenolysis of the protected tripeptide- of formula II, wherein R, Z. Phe, Pro and Arg are the same as defined hereinabove, at a temperature below 1000 in the presence of 1 to 2 molar equivalents of sulfuric acid.

According to the present invention the reaction is preferably carried out as follows: From t-butoxycarbonyl-NG-benzyloxycarbonyl-l- arginine lactam the t-butoxycarbonyl protecting group is removed in a manner known per se. The NG-benzyloxycarbonyl L-arginine lactam hydrochloride of formula IV thus obtained is kept in dimethyl formamide solution at a temperature of -150C to -20 OC, then this solution is added to the mixed anhydride formed from the dipeptide of formula V (benzyloxycarbonyl-D-phenylalanyl-L-proline or N-methyl-N benzyloxycarbonyl-D-phenylalanyl-L-proline) and chlorocarbonic acid isobutyl ester. The base, preferably trimethyl amine, is subsequently added to the reaction mixture.Under these conditions the lactam, liberating in situ due to the effect of the base, reacts at once with the mixed anhydride to form a protected tripeptide lactam, and thus the formation of the basic medium causing racemization can be avoided. If desired, the product thus obtained can be purified by a quick filtration through a silica gel bed. By choosing the above sequence of operations, which is different from the prior art, there is no need for the expensive purification of the protected tripeptide lactam by column chromatography.

The protected tripeptide lactam of formula III thus obtained is reduced with lithium aluminium hydride in dry tetrahydrofuran. The complex thus obtained is decomposed, contrary to published European patent application No.

185,390, by adding the reaction mixture to diluted, preferably 1 N sulfuric acid solution used in an excess under cooling at a temperature of 2 to 4 OC. The final pH value of 2-3 is adjusted by optional addition of sulfuric acid. Then the reaction mixture is worked up in a manner known per se. The protected tripeptide aldehyde qf formula II thus obtained is purified by filtering through silica gel bed.

The 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 preferred if the hydrogenolysis is not carried out in a closed system, but by bubbling hydrogen through the reaction mixture Practically hydrogen gas is bubbled through the system in finely divided form which can be assured by e g leading the gas through a G2-filter.

According to a preferred embodiment of the proces.

the protected tripeptide aldehyde of formula II of suitable purity thus prepared is dissolved in tetrahydrofuran and the thus-obtained solution is added to a cooled aqueous catalyst suspension comprising 1.5 to 1.7 molar equivalents of 1 N sulfuric acid. The ratio of the organic solvent to the aqueous fraction is 70-50 : 30-50, preferably 60:40 As catalyst preferably 10 to 30 % by weight of 10 % palladiumon-charcoal or platinum are used Hydrogenation is carried out under cooling at a temperature of 6-8 OC by bubbling finely divided hydrogen gas through the system. The progress of the reaction is followed by thin-layer chromatographic analysis.

According to an other 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 % of palladium precipitated onto barium sulfate or aluminium oxide carrier, preferably to barium sulfate carrier, is used as catalyst.

The hydrogenation is carried out at a temperature of 6 to 8 OC in the presence of 1.5 molar equivalents of 1 N sulfuric acid by bubbling finely divided hydrogen gas through the system. The completition of the reaction is followed 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 aqueous suspension comprising 10 % of palladium-on-charcoal catalyst and 1 molar equivalent of 1 N sulfuric acid. The hydrogenation is carried out at a temperature of 6 to 8 OC by bubbling finely divided hydrogen gas through the system and the process is followed by thinlayer chromatography.

The product can simply be recovered from the reaction mixture by filtering the catalyst and evaporating the organic solvent from the aqueous-organic solvent mixture at a temperature below 40 OC, preferably at 25-30 OC, then freeze-drying the aqueous solution either directly or after adjusting the pH to about 4. The pH is preferably adjusted by using an ion-exchange resin of hydroxy-phase or by using 0.1 N sulfuric acid.

When the process according to the invention is carried out in pilot plant, the maximum contamination of the product thus obtained (D-phenylalanyl-L-prolyl-L-arginine- aldehyde sulfate and N-methyl-O-phenylalanyl-L-prolyl-L- arginine aldehyde sulfate) detectable by thin-layer chromatographic analysis is at most 1 %, while the D-L-D isomer content thereof detectable by HPLC is at most 2 to 4 %. Thus the total amount of impurities can be reduced to about 3 to 5 %, which is in accordance with the requirements set against pharmaceutical substances, in contradistinction to the contents in impurities of 15 to 25 % in case of the known processes.

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

The Rf values were determined by thin-layer chromatography on silica gel (Kieselgel G, Reanal, Budapest) by using the eluent mixtures given in the examples.

Development: the chlorinated chromatographic plates were treated with o-tolidine solution (2.5 g of o-tolidine and 10 ml of acetic acid dissolved in 500 ml of water) after drying.

Example 1 Preparation of D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate 201 g (0.3 mole) of benzyloxycarbonyl-Dphenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde are dissolved in 2 litres of tetrahydrofuran at room temperature. Simultaneously 60 g of 10 % palladium-oncharcoal suspended in 0.6 litre of ion-free water, then 900 ml of 1 N sulfuric acid (0.45 mole) are added. The suspension comprising the catalyst is cooled to a temperature of 2 to 5 OC, and the tetrahydrofuran solution of the protected tripeptide aldehyde and additipnal 0.2 litre of ion-free water are added under stirring. Then hydrogen is bubbled through the mixture, the temperature of which is kept at 6-8 OC, while stirring is maintained, The progress of the reaction is monitored by thinlayer chromatographic analysis using a 60:20:6:11 mixture of ethyl acetate/pyridine/acetic acid/water as eluent. (The Rf values of the starting material, the intermediate and the end-product are 0.92-0.96, 0.38-0.44 and 0.0-0.10, respectively.) The reaction is completed within 3-4 hours. At the end of the reaction the catalyst is filtered off and washed with 3xl00 ml of ion-free water. The filtrate and the washing liquor are combined, then tetrahydrofuran is distilled off in a rotating evaporator under vacuum at a temperature of 25-30 OC.

The residual aqueous solution is extracted with 2x300 ml of dichloromethane, then the pH of the aqueous phase is controlled. If the pH is different from 4, then the pH is adjusted to 4 by 0.1 N sulfuric acid or by an OH-cycle ion-exchange resin (e,g. AG 1x2).

The volume of the solution is supplemented to 1.5 litres by adding ion-free water, then the solution is freeze-dried. Thus 125 g (83.2 %) of aimed product are obtained.

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

L |D = -117 (c = 1, water).

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

The benzyloxycarbonyl-D-phenylalanyl-L-prolyl-NG- benzyloxycarbonyl-L-arginine aldehyde used as starting material can be prepared as follows: Step 1 Benzyloxycarbonyl-D-phenylalanyl-l-proly NG- benzyloxycarbonyl-L-arginine lactam 429.5 g (1.1 moles) of t-butoxycabonyl to benzyloxycarbonyl-L-arginine lactam (publishes European patent application No. 185,390) are suspended in 1300 ml of dry chloroform.Then 1300 ml of 189 9/100 ml (about 5 N) hydrochloric ethyl acetate are added dropwise under stirring and cooling with icy water at a rate which enables to avoid the increase of the temperature to above 15 OC. within a short time, dissolution and precipitation of a crystalline substance begins. The suspension is stirred at room temperature for 3 hours, then diluted with 3000 ml of a dried 1:1 mixture of ether and ethyl acetate. The precipiteted crystalline substance is filtered off, washed with 2x1000 ml of acetone and 500 ml of ether, then dried over solid potassium hydroxide and phosphorous pentoxide in a vacuum desiccator.

After about 1 hour drying, the NG- benzyloxycarbonyl-L-arginine-lactam hydrochloride is dissolved in 1000 ml of dry dimethyl formamide, cooled to a temperature of -15 oC to -20 OC and added to a mixed anhydride prepared as follows: 396 g (1 mole) 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) are dissolved in 1000 ml of anhydrous dimethyl formamide and 111 ml (1 mole) of N-methyl morpholine are added. Then the mixture is cooled to a temperature of -15 oC and 132 ml (1 mole) of chlorocarbonic acid isobutyl ester are added dropwise at the same temperature under stirring within 5-10 minutes. The mixed anhydride is cooled to a temperature of -20 OC to -25 OC after 10 minute stirring and combined with the dimethyl formamide solution of NGbenzyloxycarbonyl-L-arginine lactam hydrochloride obtained hereinabove.Then 308 ml (2.2 ml) of triethyl amine are added dropwise to the reaction mixture at the same temperature within about 30 minutes, then the suspension is stirred for a further hour at a temperature of -20 OC - -15 OC. The pH of the vapour space is controlled with wet pHpaper: if the pH falls under 8, further amount of triethyl amine is added. Then the mixture is diluted with 2 litres of benzene, the salts precipitated are filtered off and washed with 2x500 ml of benzene, To the benzene-dimethyl formamide filtrate 1.5 litres of water are added and the phases are separated. The aqueous dimethyl formamide lower layer is extracted with 2x500 ml of benzene.The combined benzene solutions are washed with 2x0.5 litre of water, 1x0.5 litre of 0.1 N sulfuric acid, then 3x0.5 litre of water, dried over anhydrous sodium sulfate and evaporated to 2 litres under vacuum at a temperature of at most 40 OC.

360 g of Kieselgel-G adsorbent are suspended in benzene and a filtering bed is formed, wherein the ratio of diameter to height is 8:1 to 4:1. The benzene solution is sucked through the filtering bed under slight vacuum, then the filtering bed is washed with 4.8 litres of benzene in the same manner. The combined benzene solutions are concentrated to about 600 ml volume under vacuum at a temperature of at most 40 OC, then 3 litres of diisopropyl ether are added under intensive stirring.

The precipitate is filtered off, washed with 2x600 ml of diisopropyl ether and dried in a vacuum desiccator over phosphorous pentoxide and paraffin chips Thus 510 g (76 %) of the aimed product are obtained. The total contamination content of the product determined by thinlayer chromatography is 1-2 %.

Rf = 0.55 - 0.65 (ethyl acetate) (The Rf value of the D-L-D lactam is 0.45-0.50 in ethyl acetate.) 20 kiD = 55.8o (c = 1, tetrahydrofuran).

Step 2 Benzyloxycarbonyl-D-phenylalanyl-L-prolyl-t4G- benzyloxycarbonyl-L-arginine aldehyde 300 mg (0.45 mole) of protected tripeptide lactam (Example 1 step 1) are dissolved in 2.5 litres of anhydrous tetrahydrofuran cooled to a temperature of -50C. The solution is cooled to a temperature of -200C to -250C in a dry-ice/acetone bath, and a solution of lithium aluminium hydride and tetrahydrofuran, having a concentration of about 0.7 mole/litre, corresponding to 0.375 mole of lithium aluminium hydride, are added under vigorous stirring within 20-25 minutes. The progress of reduction is followed by thin-layer chromatography by using a 240:20:6:11 mixture of ethyl acetate/pyridine/acetic acid/water as eluent.In case of necessity, further amount of lithium aluminium hydride solution is added until the spot, having an Rf value of 0.75-0.80, corresponding to the lactam, disappears.

When the reaction is completed, the reaction mixture is portionally poured into 3 litres of 1 N sulfuric acid solution of a temperature of 2-30C under stirring. The final pH value is adjusted by the optional addition of further amount of sulfuric acid. Water is added to the solution until it becomes turbid, then it is extracted with 3x0.9 litres of n-hexane. Betaleen two extractions the material separates as an oily phase, therefore it is homogenized with tetrahydrofuran, then opalized by adding some water The aqueous phase is extracted with 2x2 litres of dichloromethane.The combined dichloromethane solutions are washed with 2x0.5 litre of 5 % sodium hydrogen carbonate solution of 4 to 80C, then with 2x0.5 litre of distilled water of a temperature of 4 to 8 OC, dried over anhydrous sodium sulfate and evaporated under vacuum at a temperature of at most 400C. (About 300 g of oily substance are obtained.) The oily residue is dissolved in a 7:3 mixture of dichloromethane and acetone, made flow through a filter bed (diameter : height = 5:1) prepared from 900 g of Kieselgel60 adsorbent by using a 7:3 mixture of dichloromethane and acetone as eluent without suction, then the filter bed is washed with 6 litres of the same eluent mixture. The process may not last longer than 20 minutes. The combined filtrates are evaporated to about 600 ml at a temperature of at most 40 OC, then 1 litre of benzene is added and the solution is evaporated again, then the product is precipitated by the addition of 1.6 litres of cyclohexane under stirring. The precipitate is filtered off. washed with 2x0.3 litre of cyclohexane and dried in a vacuum desiccator at room temperature over paraffine chips. Thus 240 g (79.7 %) of title product are obtained.

Rf = 0.52-0.62 (eThyl acetate/pyridine/acetic acid/ater = 240:20:6:11) Impurity content according to thin-layer chromatographic analysis: 0.5 - 2 %.

[)2D0= -0.80 (c=l, tetrahydrofuran)* Example 2 Preparation of D-phenylalanyl-L-prolyl-L-arginine-aldehyde sulfate 20.1 g (0.03 moles) of benzyloxycarbonyl-D- phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde are dissolved in 214 ml of tetrahydrofuran and the solution is cooled to a temperature of OOC. 4.3 g of palladium-on-charcoal catalyst are suspended in 70 ml of ion-free water and 102 ml of 1 k sulfuric acid (0.051 mole) are added. The catalyst suspension is cooled to a temperature of 2-50C, air is eliminated by nitrogen gas.

Then the above tetrahydrofuran solution is added to the suspension under stirring and finely divided hydrogen gas is led into the system. The temperature is kept betwen 6 and 80C by cooling with ice. The progress of the reaction is controlled by thin-layer chromatography and the same eluent mixture is used as described in Example. At the end of the reaction (about 3 hours) the reaction mixture thus obtained is worked up 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 [&alpha;]D = - 117 (c =1, water) Impurity content on the basis of th.ln-layer chromatographic analysis: 3.5 %.

D-L-D isomer content on the basis of HPLC analysis: 1.5 %.

Example 3 Preparation of D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate 201 g (0.3 mole) of benzyloxycarbonyl-D phenylalanyl-L-prolyl-NG-benzyloxyzarbonyl-L-arginine aldehyde are dissolved in 2 litres of ethanol at room temperature. 60 g of catalyst comprising 10 % of palladium on barium sulfate carrier are suspended in 0.6 litre of ionfree water and 600 ml of 1 N sulfuric acid (0.3 mole) are added. The suspension is cooled to a temperature of 2-5 OC and the ethanolic solution of the protected tripeptide aldehyde is added. Then ion-free water is added in such an amount which corresponds to a 6:4 ratio of the solvent to the aqueous phase.

Then hydrogen gas is bubbled through the mixture under stirring and cooling at a temperature of 6-80C. The progress of the 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 ion-free water several times. The filtrates are combined and alcohol is evaporated in a rotating evaporator at a temperature of 25-300C. The aqueous solution having a pH of about 3-4, is directly freeze-dried. Thus 101 g (65 %) of The aimed product are 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-prolyl-L-arginine aldehyde sulfate the process of Example 3 is followed except that 43 g of 10 % palladium-on-charcoal catalyst, previously washed to neutral, are used as catalyst.

Thus 125.1 g (83.2 %) of the aimed product are obtained which has the same quality as the product of Example 1.

Example 5 Preparation of N-methyl-D-phenylalanyl-L-prolyl-L-arginine aldehyde sulfate 20.5 g (0.03 mole) of N-benzyloxycarbonyl-N-methyl D-phenylalanyl-L-prolyl-NG-benzyloxyzarbonyl-L-arginine aldehyde are dissolved in 214 ml of ethanol, then 110 ml of ion-free water, 60 ml of 1 N sulfuric acid (0 03 moles) and 3 g of 10 % palladium-on-charcoal catalyst are added.

Thereafter finely divided hydrogen gas is introduced into the mixture under cooling at a temperature of 6-80C. The progress of the reaction is hollowed by thin-layer chromatography by using a 25:20:6:1 mixture of ethyl acetate/pyridine/acetic acid/water as eluent. On the chromatogram the Rf values of the starting material, intermediate and the end-product are about 0.9, 0.7 and 0.4, respectively. The reaction is completed within about 2 hours. At the end of reaction the catalyst is filtered off and washed with 3x30 ml of ion-free water. The filtrate and the washing liquor are combined, then evaporated in a rotating evaporator to about 100 ml at a temperature of at most 250C, then the residue is extracted with 2x50 ml of dichloromethane.

The traces of the organic solvent are removed from the aqueous solution by distillation and the residue is diluted with ion-free water to a volume of about 200 ml. The pH is controlled whether it is about 4. In case of necessity the pH is adjusted by adding 0.1 N sulfuric acid or ion exchange resin of 0H phase (e.g. AG 1X8), then the solution is frozen and freeze-dried. Yield: 12.5 g (75 %).

Rf = 0.35-0.45 (ethyl acetate/pyridine/acetic acid/water = 25:20:6:1) = = -126.0 + 50 (c=l, water) Total impurity content on the basis of thin-layer chromatographic analysis: 2-3 %.

Impurity content on the basis of HPLC analysis: 3-5 %.

N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L prolyl-NG-benzyloxycarbonyl-L-arginine aldehyde used as starting material can be prepared as follows: Step 1 N-benzyloxycarbonyl-N-methyl-D-phenylalanyl-L-prolyl-NG- benzyloxycarbonyl-L-arginine lactam 42.95 g (0.11 mole) of t-butoxycarbonyl-Nb- benzyloxycarbonyl-L-arginine lactam are suspended in 110 ml of dry chloroform and 275 ml of hydrochloric ethyl acetate (11-15 9/100 ml) are added under constant stirring. Te reaction mixture is stirred for about 3 hours at room temperature, the end-point of the reaction is determined by thin-layer chromatography by using a 60:20:6::11 mixture of 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 reduction the mixture is diluted with 400 ml of diethyl ether, the precipitated crystalline substance is filtered off, washed with 2x100 ml of diethyl ether and 2x50 ml of acetone, then dried in a vacuum desiccator over phosphorous pentoxide and potassium hydroxide. After about 1-2 hour drying the lactam salt thus obtained (about 0.1 mole) is dissolved in 100 ml of anhydrous dimethyl formamide and stored at -150C until it is used up by adding to a mixed anhydride prepared as follows: Liberation of N-benzyloxycarbonyl-N-methyl-D phenylalanyl-L-proline: 50.9 g (0.1 moles) of N-benzyloxycarbonyl-N-methyl D-phenylalanyl-L-proline cyclohexyl ammonium salt are dissolved in 200 ml of diethyl ether and 120 ml of 1 N sulfuric acid. The diethyl ether phase is washed with with 2x50 ml of water, dried over anhydrous sodium sulfate and evaporated under reduced pressure at a temperature of at most 35 OC until a thick oil is obtained.

The residue (about 0.1 mole) is dissolved in 170 ml of anhydrous dimethyl formamide, cooled to a temperature of -150C and 11.2 ml (0.1 mole) of N-methyl-morpholine are added under stirring. Then 13.2 ml of i-butoxycarbonyl chloride are added dropwise within 3 to 5 minutes while the same temperature is maintained. after further 5-10 minute stirring the above dimethyl formamide suspension of the amino component is added to the mixed anhydride thus obtained, then 35 ml of triethyl amine are added to the reaction mixture under vigorous stirring at a temperature of -200C to -250C. The pH of the vapour space is controlled (the pH must be above 8) and in case of necessity further amount of triethyl amine is added.The reaction mixture is stirred for one hour at a temperature of -150C, for one hour at OOC, then it is diluted with 200 ml of benzene and 150 ml of water are added.

The phases are separated and the aqueous phase is extracted with 3x30 ml of benzene. The combined benzene solutions are washed with 2x50 ml of water, dried over anhydrous sodium sulfate and evaporated under reduced pressure at a temperature of at most 400C 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 bed as follows: 300 g (5-fold amount) of Kieselgel-60 adsorbent are suspended in ether and a filter bed is prepared with a diameter: height ratio of 3 to 1. The contaminating upper spot is eluted with a 95:5 mixture of ether and tetrahydrofuran, while the desired product is eluted uith pure tetrahydrofuran.The substance content of the fractions is controlled by thin-layer chromatography by using a 480:20:6:11 mixture of ethyl acetate/pyridine/acetic acid/water as eluent. The Rf value of the desired product is 0.71-0.75. The combined fractions are evaporated under vacuum at a temperature of at most 35 OC until a thick oil is obtained and the product is used at once or stored in dry-ice. Yield: 58.8 g (85 %).

Rf = 0.71 - 0.75 (ethyl acetate/pyridine/acetic acid/ater = 480 : 20 : 6 : 11.

= = +13.50 (c = 1, tetrahydrofuran).

Step 2 N-benzylOxyzarbonyl-N-methyl-D-phenylalanyl-L-prolyl-NG- benzyloxycarbonyl-L-arginine aldehyde 34.14 g (0.05 mole) of N-benzyloxycarbonyl-N methyl-D-phenylalanyl-L-prolyl-NG-benzyloxycarbonyl-L- arginine lactam (Example 5, Step 1) are dissolved in 150 ml of dry tetrahydrofuran cooled to a temperature of OOC. The solution is cooled to -200C and 0.0375 mole of lithium aluminium hydride dissolved in tetrahydrofuran are added under stirring within 5 to 10 minutes.The progress of reduction is followed by thin-layer chromatography by using a 240:20:6:11 mixture of ethyl acetate/pyridine/acetic acid and water. (The Rf values of the lactam used as starting material and the aldehyde are 0.8 and 0.5, respectively.) In case of necessity, further amount of lithium aluminium hydride is added, and at the end of reaction the mixture is poured in portions under constant stirring into 320 ml of 1 N sulfuric acid previously cooled to a temperature of 2 to 4 OC, The final pH should be between 2 and 3, this pH is adjusted by adding further amount of sulfuric acid if necessary. If a precipitate is formed, it is filtered off, the solution is diluted with water until it begins to become turbid, then it is extracted with 3xl00 ml of n-hexane.

The aqueous tetrahydrofuran phase is extracted with 250 ml of dichloromethane twice or three times and the combined dichloromethane solutions are washed with 2x50 ml of water, 2x50 ml of cold 5 % sodium hydrogen carbonate solution, then with- 2x50 ml of water. Thereafter the solution is dried over anhydrous sodium sulfate and evaporated in vacuo at a temperature of at most 400C.

The evaporation residue is dissolved in a 7:3 mixture of dichloromethane and acetone (120 ml) and made flow without suction through a filtering bed prepared from 108 g of Kieselgel-60 adsorbent with the aid of 200 ml of 7:3 mixture of dichloromethane and acetone, then the filtering bed is washed with 600 ml of the same eluent mixture. The process may last not longer than 20 minutes.

The combined filtrates are evaporated to 50-60 ml under reduced pressure at a temperature of at most 40 C, then this operation is repeated after adding 100 ml of benzene to the residue. The product is precipitated from the solution of about 50-60 ml by adding 100 ml of cyclohexane.

The precipitate thus obtained is washed with 2x30 ml of cyclohexane and dried over paraffin chips in a vacuum desiccator. Yield: 18 g (53 %).

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

[&alpha;]20D = +14.10 Cc = 1, tetrahydrofuran).

Total impurity content of the product: max. 2-3 %.

Claims (7)

1. An improved process for the preparation of tripeptide aldehydes of formula I R-D-Phe-Pro-Arg-H.H2S'04 I wherein R represents hydrogen or methyl, Phe stands for phenylalanine group, Pro is L-proline group and Arg stands for L-arginine group in pharmaceutically pure form, by condensing an NGprotected-L-arginine lactam of formula IV, H-Arg(Z)-lactam .HC1 IV wherein Arg is the same as defined hereinabove and Z stands for benzyloxycarbonyl, with a dipeptide of formula V, ZR(D-Phe)-Pro-OH V wherein R, Z, Phe and Pro are the same as defined hereinabove, through a mixed anhydride, reducing 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 defined hereinabove, with lithium aluminium hydride, and hydrogenolysing the thus-obtained protected tripeptide aldehyde of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II wherein R, Z, Phe and Arg are the same as defined hereinabove, in the presence of sulfuric acid, w h i c h comprises al) liberating the NG-protected L-arginine lactam in situ in the course of condensation of NG- protected-L-arginine lactam hydrochloride of formula IV, H-Arg(Z)-lactam. HCl IV wherein Z is the same as defined hereinabove, and dipeptide of formula V, ZR(D-Phe)-Pro-OH V wherein R, Z, Phe and Pro are the same as defined hereinabove, into a mixed anhydride in the reaction mixture by subsequently adding a base to the mixture of the said reaction components, then after reducing 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 hereinabove, decomposing the complex thus formed by adding the reaction mixture to an excess of 1 N sulfuric acid at a temperature of O to 5 OC, then carrying out the hydrogenolysis of the protected tripeptide of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, at a temperature below 100C in the presence of 1 to 2 molar equivalents of sulfuric acid or a2) 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 hereinabove, decomposing the complex thus formed by adding the reaction mixture to an excess of 1 N sulfuric acid at a temperature of O to 5 OC, then carrying out the hydrogenolysis of the thus-obtained protected tripep;;tide of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, at a temperature below 100C in the presence of 1 to 2 molar equivalents of sulfuric acid, or a3) carrying out the hydrogenolysis of the protected tripeptide of formula II, ZR-(D-Phe)-Pro-Arg(Z)-H II wherein R, Z, Phe, Pro and Arg are the same as defined hereinabove, at a temperature below 100C in the presence of 1 to 2 molar equivalents of sulfuric acid.

2. A process as claimed in claim 1, which c o m p r i s e s liberating the NG-protected-L-arginine lactam from the lactam hydrochloride of formula IV with the aid of triethyl amine.

3. A process as claimed in claim 1, w h i c h c o m p r i s e s carrying out the hydrogenolysis at a temperature of 6 to 8 OC.

4. A process as claimed in any of claims 1 to 3, w h i c h c o m p r i s e s carrying out the hydrogenolysis by bubbling hydrogen gas through the system.

5. A process as claimed in any of claims 1 to 3, w h i c h c o m p r i s e s carrying out the hydrogenolysis in ethanol or in tetrahydrofuran.

6. A process of preparing a tripeptide aldehyde of the general formula I as defined in claim 1, substantially as hereinbefore described in any one of Examples 1 to 5.

7. A tripeptide aldehyde of the genral formula I as defined in claim 1, produced by a process as claimed in any one of claims 1 to 6.