CH641153A5 - Thymosin alpha-1 fragments and their preparation - Google Patents

Abstract

Thymosin alpha 1 fragments of the general formula R<1>-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (I> in which R<1> represents hydrogen, H-Glu-Lys-Lys- or H-Asp-Leu-Lys-Glu-Lys-Lys-, and their pharmaceutically tolerated salts, and a process for preparing them, which process consists in eliminating the protective groups from a protected peptide of the general formula R<2>-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu-(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (II) in which R<2> represents Boc, Boc-Glu(OBz1)-Lys(Z)-Lys(Z)- or Boc-Asp(OBz1)-Leu-Lys(Z)-Glu(OBz1)-Lys(Z)-Lys(Z), Bzl represents benzyl, and Z represents benzyloxycarbonyl, and, if desired, converting the resulting compound into a pharmaceutically tolerated salt. These compounds have an immunopotentiating effect.

Description

The present invention therefore relates to thymosin a ^ fragments of the general formula

R ^ Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (I)

wherein

R1 represents hydrogen, H-Glu-Lys-Lys- or H-Asp-Leu-Lys-Glu-Lys-Lys-,

and their pharmaceutically acceptable salts, and a process for their preparation, which is characterized in that the protective groups of a protected peptide of the general formula

R2-Glu (OBzl) -V al-V al-Glu (OBzl) -Glu (OBzl) -Ala - Glu (OBzl) -Asn-OBzl (II)

wherein

R2 Boc, Boc-Glu (OBzl) -Lys (Z) -Lys (Z) - or

Boc-Asp (OBzl) -Leu-Lys (Z) -Glu (OBzl) -Lys (Z) -Lys (Z) -, •

Bzl Benzyl and

Z represent benzyloxycarbonyl,

is split off and, if desired, the compound obtained is converted into a pharmaceutically acceptable salt.

The general formula I summarizes the compounds H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu - Asn-OH,

A-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH and H-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH together.

The protective groups can easily be removed from a protected peptide of the formula II by methods known per se, for example by treatment with anhydrous acid such as hydrogen fluoride, preferably in the presence of anisole.

The protected peptides of the formula II can be prepared by methods known per se. The following strategy was used in the present case:

First, H-Glu (OBzl) -OH was coupled to Boc-Ala-OSu to form the dipeptide fragment Boc-Ala-Glu (OBzl) -OH, which was then combined with HCl. H-Asn-OBzl using the DCC / HOSu method from Wünsch and Drees, Chem. Ber. 99, 110 (1966). The hydrochloride salt of the asparagine benzene ester was prepared from Boc-Asn-OBzl, which in turn was synthesized from commercially available Boc-Asn-OH and benzyl bromide using the cesium salt method. The Boc protecting group was removed by treatment with 4N HCl in dry tetrahydrofuran for 30 minutes.

The reaction of H-Glu (OBzl) -OH with Boc-Glu (OBzl) - OSu provided Boc-Glu (OBzl) -Glu (OBzl) -OH in the form of a colorless, clear oil, which was then used for the synthesis of the protected pentapeptide Boc-Glu (OBzl) -Glu (OBzl) -Ala - Glu (OBzl) -Asn-OBzl was used, the reaction in the presence of DCC / HOSu with HCl. H-Ala-Glu (OBzl) - Asn-OBzl, which was obtained from Boc-Ala-Glu (OBzl) -Asn-OBzl by treatment with 4N HC1 / THF. The protected pentapeptide was obtained in pure, crystalline form in good yield.

To produce the protected octapeptide Boc-Glu- (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn - OBzl, the protected tripeptide Boc-Glu- (OBzI) was first -VaI-Val-OH produced. Boc-Val-OSu was converted with free valine to Boc-Val-Val-OH, which after de-blocking with 4N HCl in THF and subsequent reaction with Boc-Glu (OBzl) -OSu the desired tripeptide in crystalline form as the cyclohexylamine salt Boc -Glu (OBzl) -Val - Val-OH. CHA delivered. The cyclohexylamine salt was converted to the free acid and then by DCC in the presence of HOSu with HCl. H-Glu (OBzl) -Glu (OBzl) -Ala-Glu- (OBzl) -Asn-OBzl, which consists of Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu- (OBzl) -Asn-OBzl Treatment with HCl in THF was coupled. The protected octapeptide Boc-Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn - OBzl was obtained in a purified form as an amorphous solid and by hydrogenolysis and subsequent treatment converted into the free octapeptide Glu-Val-Val-Glu-Glu-Ala-Glu-Asn with trifluoroacetic acid in the usual way. It was purified by ion exchange column chromatography and provided a uniform material by thin layer chromatography and paper electrophoresis.

For the production of the protected undecapeptide Boc - Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) - Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl a tripeptide was first synthesized from Boc-Lys (Z) -OSu and H-Lys (Z) -OH. The dipeptide Boc-Lys (Z) -Lys (Z) -OH obtained was treated with 4N

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HCl treated in THF and the salt obtained HCl. H-Lys- (Z) -Lys (Z) -OH was then reacted with Boc-Glu (OBzl) -OSu. The tripeptide Boc-Glu (OBzl) -Lys (Z) -Lys- (Z) -OH thus obtained was analyzed with DCC and the like. HOSu activated according to Weygand et al, Z. Naturforsch. 21b, 426 (1966) and the solution of the activated tripeptide ester Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OSu formed in combination with the trifluoroacetate of H-Glu (OBzl) -V al- V al-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) - Asn-OBzl obtained from the corresponding protected octapeptide by treatment with TFA for 30 minutes. After adding a small amount of a base, the desired protected undecapeptide Boc-Glu (OBzl) -Lys (Z) - Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala -Glu- (OBzl) -Asn-OBzl obtained. Cleavage of the protective groups from the protected undecapeptide with anhydrous hydrofluoric acid gave the free undecapeptide Glu-Lys-Lys-Glu-Val - Val-Glu-Glu-Ala-Glu-Asn, which was uniform in paper electrophoresis after ion exchange column chromatography.

The protected tetradecapeptide Boc-Leu-OSu was synthesized according to a similar scheme and was coupled to H-Lys- (Z) -OH and yielded Boc-Leu-Lys (Z) -OH. After removal of the Na-Boc group with 4N HCl in THF and reaction with Boc-Asp (OBzl) -OSu, the protected tripeptide Boc-Asp (OBzl) -Leu-Lys (Z) -OH was obtained in crystalline form. It was converted into the activated ester Boc-Asp (OBzl) - Leu-Lys (Z) -OSu and with the trifluoroacetate of H-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) - Val-Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, obtained by treating the corresponding protected undecapeptide with TFA, condensed. The desired product, Boc-Asp- (OBzl) -Leu-Lys (Z) -Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val - Val-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, was obtained in good yield and was uniform in thin-layer chromatography. The free tetradecapeptide Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn was obtained from the protected compound by treatment with anhydrous hydrofluoric acid and purification on an ion exchange column.

The new octa-, undeca- and tetradecapeptides and their pharmaceutically acceptable salts can be administered parenterally to warm-blooded mammals, i.e. intravenously, subcutaneously or intramuscularly. These compounds have a strong immunopotentiating effect at a daily dose of approximately 1-100 mg per kg body weight when administered intravenously. Of course, the dose required depends on the particular condition in which the individual to be treated is, the degree of the disease or the duration of the treatment. A suitable dosage unit for pharmaceutical use is 1 mg of the above-mentioned peptides. Immediately before administration, the lyophilized preparation can be reconstituted by adding sterile water or saline.

The present invention also includes the pharmaceutically acceptable salts of the above peptides. Suitable salts are the sodium and potassium salts or salts with a strong organic base such as guanidine. In addition, the preparations can contain the counterions to the cations mentioned, such as chloride, bromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate or ascorbate.

Of the abbreviations used in the text mean: Boc = tert-butyloxycarbonyl; Bzl = benzyl; DCC = di-cyclohexylcarbodiimide; DMF = dimethylformamide; THF = tetrahydrofuran; HOSu = N-hydroxysuccinimide; Triton B = 40% methanolic solution of trimethylbenzylammonium hydroxide; NMM = N-methylmorpholine; CHA = cyclohexylamine; DCHA = dicyclohexylamine; Z = benzyloxycarbonyl; DMSO = dimethyl sulfoxide: TFA = trifluoro

acetic acid; DC = thin layer chromatography; Et3N = triethylamine; HOBT = 1-hydroxybenzotriazole.

The following examples describe the synthesis of the peptides according to the invention in detail.

example 1

a) 11.0 g of Boc-Asn-OH were dissolved in 200 ml of methanol and 20 ml of water. The solution was adjusted to a pH of 7.0 with a 20% strength aqueous solution of cesium carbonate (approx. 55 ml). The mixture was evaporated to dryness, the residue was concentrated twice at 45 ° C. from 120 ml DMF and the white residue thus obtained was stirred with 8.9 g benzyl bromide in 120 ml DMF for 6 hours 15. After concentration to dryness and treatment with a large amount of water, the product solidified. It was filtered off, dissolved in ethyl acetate, washed with water, dried over sodium sulfate, brought to dryness and recrystallized from ethyl acetate / petroleum ether. From-20 loot: 13.8 g (90.3%) Boc-Asn-OBzl, mp 120-122 ° C, [a] D26 = -17.29 ° (c = 1, DMF).

13.7 g Boc-Asn-OBzl were dissolved in 80 ml THF and treated with 500 ml 4N HCl in THF. The mixture was allowed to stand for 45 minutes during which time a precipitate began to form. Treatment with 1000 ml of ether provided a white solid material which was filtered off, washed with ether and dried in vacuo over sodium hydroxide. Yield: 10.3 g (94%) HCl. H-Asn-OBzl, mp 122-126 ° C, [a] D25 = + 6.82 °.

30 b) 7.0 g of finely ground H-Glu (OBzl) -OH and 8.8 g of Boc-Ala-OSu were stirred for 48 hours in 250 ml of DMF and 6 ml of NMM. Additional NMM was added to keep the reaction slightly basic. Then the solvent was removed and the residue was partitioned between 300 ml of ethyl 35 acetate and 500 ml of water containing 2 ml of 10% sulfuric acid. The organic phase was washed three times with water, dried over sodium sulfate and brought to dryness. The residue was taken up in a small amount of ether and treated with a large 40 volume of petroleum ether. A white amorphous precipitate formed, which was uniform by thin layer chromatography. Yield: 11.0 g (91.5%) Boc-Ala-Glu- (OBzl) -OH, mp 84-88 ° C, [a] D25 = 8.08 ° (c = 1, DMF).

10.4 g Boc-Ala-Glu (OBzl) -OH, 6.56 g HCl. H-Asn-OBzl 45 and 5.9 g HOSu were dissolved in 250 ml DMF at 0 ° C. 5.7 g of DCC and 3.5 ml of triethylamine were added. The mixture was then stirred at 0 ° C for 2 hours and at 25 ° C for 40 hours with the further addition of triethylamine to keep the reaction slightly basic. The insoluble by-products were filtered off while the filtrate was evaporated to dryness. The oily residue obtained solidified on treatment with water. The crude product was taken up in chloroform, washed three times with water, dried over sodium sulfate and concentrated. A form-55 the precipitate was filtered off and the filtrate was treated with petroleum ether. A crystalline product was obtained. Yield: 8.0 g (51.4%) Boc-Ala-Glu (OBzl) -Asn - OBzl, mp 102-105 ° C, [a] D25 = 12.5 ° (c = 1, DMF) .

c) 4.74 g of finely ground H-Glu (OBzl) -OH and 0.7 g of 60 Boc-Glu (OBzl) -OSu were stirred in DMF in the presence of 3.6 ml of MMM for 36 hours. The solution obtained was concentrated and the viscous residue was treated with water. The precipitate was taken up in ethyl acetate, washed with 5% acetic acid and three times with water, dried over 65 sodium sulfate and concentrated. 14.03 g of a clear oil were obtained, which was left to stand under petroleum ether. Finally, 10.2 g (90%) of oily Boc-Glu (OBzl) -Glu (OBzl) -OH were obtained, [a] D25 = -7.59 ° (c = 1,

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DMF). The product was uniform by thin layer chromatography.

d) 28.2 g of Boc-Ala-Glu (OBzl) -Asn-OBzl were treated with 1.1 liters of 4N HCl in THF for 1 hour. An oil was obtained by concentration, which was taken up twice more with THF and concentrated again. When treated with a large amount of ether, the oil changed to solid HCl .H - Ala-Glu (OBzl) -Asn-OBzl, which was mixed with 25.6 g Boc-Glu- (OBzl) -Glu (OBzl) -OH, 10.6 g of HOSu and 10.9 g of DCC in 540 ml of DMF were stirred at 0 ° C. for one hour and then at 25 ° C. for 48 hours. Triethylamine was added to keep the reaction slightly basic. The insoluble by-products were filtered off while the filtrate was concentrated to dryness. The crude product was taken up in chloroform, washed three times with water, dried over sodium sulfate and concentrated. When treated with petroleum ether, the product solidified. Recrystallization from isopropanol gave 28.9 g (59.8%) of Boc-Glu (OBzl) -Glu- (OBzD-Ala-Glu (OBzl) -Asn-OBzl, mp 169-175 ° C, [a] D25 =

- 11.78 ° (c = 1, DMF).

3.9 g of Boc-Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl was treated with 15 ml of 4N HCl in THF for 30 minutes. Then 210 ml of ether was added. The precipitate was filtered off, washed with water and recrystallized from ethanol and ether. Yield: 2.58 g (75.1%) HCl. H - Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, mp 148 to 151 ° C, [a] D25 = -3.65 ° (c = 1, DMF).

e) 12.6 g of Boc-Val-OSu and 4.68 g of valine were reacted with one another in 250 ml of DMF in the presence of 2 ml of triethylamine for 96 hours. Additional triethylamine was added to keep the reaction slightly basic. The insoluble material was filtered off while the filtrate was concentrated at 45 ° C. The residue was partitioned between ether and dilute sulfuric acid (approx. 1%) and the organic phase was washed three times with water,

dried over sodium sulfate and concentrated. The product obtained was recrystallized from ether and petroleum ether. Yield: 12.2 g (96.4%) Boc-Val-Val-OH, mp 155-158 ° C, [a] D25 = + 1.10 ° (c = 1, DMF).

40.5 g of Boc-Val-Val-OH were treated with 1.8 liters of 4N HCl in THF for one hour. Concentration and treatment with ether provided 34.5 g HCl. H-Val-Val-OH in the form of a white amorphous powder which was reacted with 55.6 g of Boc-Glu- (OBzl) -OSu in 1 liter of DMF for 24 hours in the presence of 54 ml of triethylamine. The mixture was freed from insoluble material and evaporated to dryness. The oily residue was taken up in 1.5 l of ethyl acetate, the solution was washed twice with 5% acetic acid and three times with water, the organic phase was dried over sodium sulfate and evaporated to dryness. A colorless, clear oil was obtained which did not crystallize. It was dissolved in 3.2 1 ether and adjusted to pH 7.5 with 17 ml CHA. The solid residue obtained was filtered off and recrystallized from methanol and ether. Yield: 58.9 g (72.7%) Boc-GIu (OBzI) -VaI-VaI-OH. CHA, mp 158-160 ° C, [a] D25 = 33.41 ° (c = 1, MeOH).

To a suspension of 1.69 g Boc-Glu (OBzl) -Val-Val - OH. CHA in 40 ml of water and 40 ml of ethyl acetate were added to 4 ml of 1 M sulfuric acid in a shaking funnel. After shaking vigorously, the product dissolved and the organic phase was washed several times with water, dried over sodium sulfate and concentrated to an oil. The free tripeptide thus obtained was treated with 2.58 g of HCl. H-Glu- (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl condensed in 15 ml DMF in the presence of 0.012 g HOSu, 0.3 ml NMM and 0.63 g DCC, the reaction mixture Was held at 0 ° C for 1 hour and at 25 ° C for 60 hours. Additional NMM was added to keep the reaction slightly basic. Insoluble by-products were filtered off while the filtrate was concentrated at 45 ° C. The oily residue obtained solidified when water was added. The crude product was dissolved in 50 ml DMF and reprecipitated with 300 ml metha-5 noi. Yield: 2.25 g (58.7%) Boc-Glu- (OBzl) -V al-V al-Glu (OBzl) -Glu- (OBzl) -Ala-Glu (OBzl) -Asn - OBzl, F 277-280 ° C, [a] D25 = -12.43 ° (c = 1, DMF).

0.72 g of this product were hydrogenated over 0.5 g of a 5% Pd / BaS04 catalyst for 3 hours at 3.4 atmospheres in a mixture of 40 ml DMF, 30 ml methanol and 2 ml water. The mixture was filtered and the filtrate was evaporated to dryness. It was then treated with 5 ml of TFA for 30 minutes and the residue obtained after concentration was treated with ether several times. The resulting white residue was taken up in 20 ml of water and, after lyophilization, gave 0.47 g of a crude product, which was equilibrated with ammonium acetate buffer on a column (3 × 32 cm) of a strongly basic polystyrene resin (Bio-Rad AG1-X 2) of pH 8.1 (2% acetic acid-20, which was adjusted to pH 8.1 with ammonia) was added. The column was successively filled with 200 ml of 0.025 M ammonium acetate (pH 5.5), 0.025 M HOAc, 0.05 M HOAc, 0.1 M HOAc, 0.25 M HOAc, 0.5 M HOAc, 0.75 M each HOAc and 1 M HOAc eluted. Fractions of 25 12 ml each were collected and each fraction was examined by thin layer chromatography. The fractions containing the desired product (fractions 225-229) were combined and, after two lyophilizations, gave 0.223 g (48.1%) of pure Glu-Val-Val-Glu-Glu-Ala-Glu-Asn, 30 which was thin-layer chromatographically paper electrophoresis table was uniform.

Example 2

35 15 g Boc-Lys (Z) -OH were stirred with 5.8 g HOSu and 8.66 g DCC in 200 ml THF for 3 hours. An insoluble by-product was filtered off and the filtrate was concentrated to dryness. The remaining syrup (24.2 g) was treated with 150 ml of isopropanol and 150 ml of petroleum ether 40 and gave 21 g of Boc-Lys (Z) -OSu in an oily form. It was reacted with 10.6 g of H-Lys (Z) -OH in 250 ml of DMF in the presence of 5.5 ml of triethylamine for 72 hours. Additional triethylamine was added during the reaction to keep the reaction mixture slightly basic. The mixture 45 was then filtered and the filtrate was concentrated at 45 ° C. The oily residue obtained was treated with one liter of 5% acetic acid. The precipitate formed was extracted with ethyl acetate and the organic phase was washed with water, dried over sodium sulfate and concentrated to an oil which was crystallized from 300 ml of ethyl acetate and 10 ml of DCHA. Recrystallization from methanol and ether gave 22.7 g (72.5%) Boc-Lys (Z) -Lys- (Z) -OH. DCHA, mp 160-162 ° C, [a] D25 = -2.21 ° (c = 1, MeOH).

10 g Boc-Lys (Z) -Lys (Z) -OH. DCHA were partitioned between 55 one liter of ethyl acetate and one liter of 0.1 N H2S04. The organic phase was washed three times with water, dried over sodium sulfate and evaporated to dryness. The free acid obtained, Boc-Lys (Z) -Lys (Z) -OH, was treated with freshly prepared 4N HCl 6o in THF for 30 minutes. The solvent and excess acid were removed by concentration at 30 ° C, while the residue was taken up twice with THF and concentrated again. The residue obtained, HCl. H-Lys (Z) - Lys (Z) -OH, solidified on treatment with ether, was filtered off and washed several times with ether. The product obtained, 6.7 g, was dissolved in 70 ml of DMF, cooled in an ice bath, mixed with 1.63 ml of triethylamine and 5.54 g of Boc-Glu (OBzl) -OSu. The mixture became

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Stirred at 0 ° C for 1 hour and at 25 ° C for 24 hours. Additional triethylamine was added to maintain a pH of 7.5. Finally, it was acidified with a few ml of acetic acid (pH 3.5) and the solvent was evaporated off. The residue obtained was taken up in ethyl acetate, washed three times with water, dried over sodium sulfate and evaporated to dryness. After treatment with ether and recrystallization from ethyl acetate, 7.26 g (69.5%) of Boc-Glu (OBzl) -Lys (Z) -Lys-Z) -OH,

Mp 153-155 ° C, [a] D25 = -2.71 ° (c = 1, THF).

1.7 g Boc-Glu (OBzl) -VaI-Val-Glu (OBzl) -Glu (OBzl) -Ala - Glu (OBzl) -Asn-OBzl was treated with 24 ml TFA for 30 minutes. After removing the excess acid by evaporation at 30 ° C, the residue was treated with ether. The powder obtained was washed carefully with ether and petroleum ether, dried in vacuo over sodium hydroxide and gave 1.71 g of the trifluoroacetate of the octapeptide. Then the activated ester was Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OSu in situ by reacting 0.995 g of Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -OH with Generated 0.16 g HOSu and 0.274 g DCC in 15 ml DMF for 3 hours at 0 ° C. 1.71 g of the octapeptide salt CFjCOOH were added to this solution. H-Glu (OBzl) -Val-Val-Glu (OBzl) -Glu- (OBzl) -Ala-Glu (OBzl) -Asn-OBzl added together with 0.2 ml triethylamine. After adding a few more drops of triethylamine and 15 ml of DMF, the mixture was stirred for 3 days at 25 ° C., acidified with acetic acid and then treated with water. The white precipitate was filtered off, washed with water, methanol and ether, so that 2.25 g of a crude product with a melting point of 310-313 ° C. were obtained, which was dissolved in DMF and reprecipitated with methanol. Yield: 1.75 g (68.3%) Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzI) -Val-Val-Glu (OBzl) -Glu (OBzl) -Ala --Glu (OBzl) -Asn-OBzl, mp 314-316 ° C, [a] D25 = 13.68 ° (c = 1, DMSO). The material was uniform by thin layer chromatography.

0.5 g Boc-Ghi (OBzl) -Lys (Z) -Lys (Z) -Glu- (OBzl) -Val-Val - Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn -OBzl were in

2 ml of TFA dissolved and stirred for 15 minutes with 15 ml of hydrofluoric acid at 0 ° C. After evaporating excess acid at 0 ° C, the residue was dissolved in 5% aqueous acetic acid, the solution washed three times with ether, concentrated and lyophilized. 0.34 g of crude Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn was obtained, which was purified by chromatography on an ion exchange column. Yield: 0.13 g (42.1%), [a] D25 = -85.65 ° (c = 1, H20).

Example 3

4.0 g of Boc-Leu-OSu and 3.42 g of H-Lys (Z) -OH were condensed in 75 ml of DMF in the presence of 1.7 ml of triethylamine for 48 hours. The pH was kept at 7.5 by periodic addition of triethylamine. Insoluble material was filtered off and the filtrate was evaporated to dryness. The residue was dissolved in 200 ml of ether, the solution was treated with 3 ml of DCHA, the crystalline precipitate was washed with ether and recrystallized from methanol and ether. Yield: 5.7 g (69.5%) Boc-Leu-Lys (Z) -

-OH. DCHA, mp 140-142 ° C, [a] D25 = -7.20 ° (c = 1, MeOH).

2.97 g Boc-Leu-Lys (Z) -OH. DCHA was converted to the free acid (partitioned between ethyl acetate and 0.1 N 5 H2S04) and the colorless oil obtained (2.2 g) was treated with 4N HCl in 40 ml THF for 30 minutes. Excess acid and solvent were removed by heating to 30 ° C and the residue was treated with ether. The remaining oil was dissolved in ether, concentrated a-io and concentrated twice more after the addition of ether. The residue was finally stirred overnight with 1.85 g of Boc-Asp (OBzl) -OSu in the presence of 1.85 ml of triethylamine. The mixture was evaporated to dryness and gave an oily residue which was taken up in ethyl 15 acetate, washed three times with water, dried over sodium sulfate and evaporated to dryness. The crude product thus obtained was crystallized from ethyl acetate and petroleum ether and gave 1.52 g (49.6%) of pure Boc-Asp (OBzl) -Leu-Lys (Z) -OH, mp 109-111 ° C., [a ] D25 = -16.4 ° 20 (c = 1, DMF).

1.2 g Boc-Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val-Val - Glu (OBzl) -Glu (OBzl) -Ala-Glu (OBzl) -Asn- OBzl were treated with 35 ml TFA for 30 minutes. Excess acid was quickly stripped off and the rest treated with ether several times to give 1.2 g of the undecapeptide TFA salt in the form of a white powder. The salt was dissolved in a mixture of 5 ml DMF and 2 ml DMSO and with in situ by stirring 0.381 g Boc-Asp (OBzl) -Leu-Lys (Z) -OH for 3 hours at 0 ° C 30 with 0.126 g HOSu and 0.124 g DCC in 3 ml DMF-produced Boc-Asp (OBzl) -Leu-Lys (Z) -OSu. The mixture was stirred at 0 ° C for 2 hours and then at 25 ° C for 3 days, during which time triethylamine was added periodically to maintain a slightly basic environment. 35 The resulting gelatinous substance was treated with 5% acetic acid and the resulting white precipitate was filtered off, washed with water, methanol and ether and gave 1.28 g of crude Boc-Asp (OBzl) -Leu-Lys- (Z) - Glu (OBzl) -Lys (Z) -Lys (Z) -Glu (OBzl) -Val-Val-Glu (OBzl) -40 -Glu (OBzl) -Ala-Glu (OBzl) -Asn-OBzl, F. 325 -326 ° C. Precipitation from DMF / DMSO (10 ml / 5 ml) and methanol (230 ml) gave 1.22 g (80.2%) of pure material, mp 326-327 ° C, Md25 = -15.71 ° ( c = 1, DMF / DMSO).

1.128 g of this product was mixed with 7 ml of anisole for 45 and treated with 25 ml of anhydrous hydrofluoric acid at 0 ° C. for 15 minutes. Excess acid was removed at 0 ° C and the remaining residue was partitioned between water and ether. The aqueous phase was washed twice with ether, concentrated to half the original volume and lyophilized. 0.69 g of raw material was obtained which was chromatographed in the manner described above for the octapeptide. The material eluted in fractions 101-120 was combined, lyophilized and gave 0.25 g of a product which was still slightly contaminated. It was therefore re-chromatographed on the same column and gave 0.155 g (22%) of pure Asp-Leu-Lys-Glu-Lys-Lys-Glu - V al-V al-Glu-Glu-Ala-Glu-Asn, [ a] D25 = -86.27 ° (c = 1, 0.1 N HCl). The material was uniform on paper electrophoresis.

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Claims (5)

641153 2nd PATENT CLAIMS 1. Thymosin o ^ fragments of the general formula R ^ Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (I) wherein R1 represents hydrogen, H-Glu-Lys-Lys- or H-Asp-Leu-Lys-Glu-Lys-Lys-, and their pharmaceutically acceptable salts. 2. A compound according to claim 1, of the formula H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu - Ala-Glu-Asn-OH. 3. A compound according to claim 1, the formula H-Glu-Lys-Lys-Glu-V al-V al-Glu-Glu-Ala-Glu-Asn-OH. 4. A compound according to claim 1, of the formula H-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH. 5. A process for the preparation of the thymosin (^ fragments according to claim 1 and their pharmaceutically acceptable salts, characterized in that the protective groups of a protected peptide of the general formula R2-Glu (OBzl) -V al-V al-Glu (OBzl) -Glu (OBzl) -Ala - Glu (OBzl) -Asn-OBzl. (II) wherein R2 Boc, Boc-Glu (OBzl) -Lys (Z) -Lys (Z) - or Boc-Asp (OBzl) -Leu-Lys (Z) -Glu (OBzl) -Lys (Z) -Lys (Z), Bzl Benzyl and Z represent benzyloxycarbonyl, is split off and, if desired, the compound obtained is converted into a pharmaceutically acceptable salt. Thymosin a1 is a heat-stable, strongly acidic polypeptide consisting of 28 amino acid units of the following sequence: 5 10 H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu- 15 20 -Ue-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-25 -V al-V al-Glu-Glu-Ala-Glu-Asn-OH. (I) This peptide, which is a potent immunopotentiating agent, was derived from the thymosin fraction 5 by Goldstein et al. isolated by a combination of ion exchange chromatography and gel filtration [Proc. Nati. Acad. Be. USA 74: 725-729 (1977)]. It has now surprisingly been found that the carboxyl-terminal octa-, undeca- and tetra-decapeptides of thymosin aj also have an immunopotentiating activity, i.e. influence the regulation, differentiation and function of T cells.