Process for preparing somatostatin analogs and intermediates thereto.

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(72) Inventor:

1980 06 15

1981 01 26 © Dependence:

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360 675

- 2 No. 366075

The invention relates to a process for the preparation of new tetradecapeptides of the formula

Y-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-LI__

I,

Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH wherein Y is D-Val or D-Ala, and their pharmaceutically acceptable acid addition salts. Somatostatin, also known as the somatotropin releasing inhibitory factor, is a tetradecapeptide of the formula

L-Ala-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-LTrp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- Cys-OH.

This tetradecapeptide was isolated from sheep hypothalamus extracts and its effect is that of inhibiting secretion of growth hormone (GH), also known as somatotropin, see P. Brazeau, W. Vale, R. Burgus, N. Ling, M. Butcher, 3. Rivier and R. Guillemin, Science, Vol. 179, p. 77 (1973),

In addition, over the as D-Trp ^8thSomatostatin has been reported by Brown et al., Endocrinology, Vol. 93, No. 2, pp. 336-343 (1976).

The biologically active tetradecapeptides of formula (I) above differ in structure from that of somatostatin by the presence of a D-tryptophan residue at position 8 in place of an L-tryptophan residue and a D-valine or D-alanine residue at position 1 Place of an L-alanine residue. For the sake of simplicity, the tetradecapeptides of formula (I) will be referred to as D-Val¹, D-Trp ^8th-Somatostatin and D-Ala¹, D-Trp^8th-Somatostatin called.

The process according to the invention for the preparation of the novel tetradecapeptides of the formula (I) is characterized in that the corresponding straight-chain tetradecapeptide of the formula

Y-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp, (ΠΙ)

L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH, wherein Y has the meaning given above, is reacted with an oxidizing agent. Through this reaction, the two sulfhydryl groups are converted into a disulfide bridge.

Pharmaceutically acceptable non-toxic acid addition salts are salts with organic or inorganic acids such as hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic and propionic acid. The preferred acid addition salts are those made with acetic acid. All of these salts can be obtained by conventional methods.

The practice of the method according to the invention can be effected by treating a dilute solution of the linear tetradecapeptide with an oxidizing agent, eg 3od or potassium ferricyanide. Air can also be used as an oxidizing agent. The ρ value of the mixture is generally between about 2.5 and 9.0 and preferably between 6.2 and 7.2. When air is used as the oxidizing agent, the concentration of the peptide solution is generally not higher than about 0.4 mg peptide / ml solution and is preferably about 50 pg / ml.

The compounds of formula (I) can be administered to warm blooded mammals, including humans, and are particularly well suited for smooth muscle relaxation. In particular, the gastrointestinal tract may be obtained by parenteral administration of small amounts of these compounds, and preferably D-Val¹, D-Trp^8th-Somatostatin be relaxed. This effect,

3,606,675, which leads to a reduction in intestinal motility, is particularly desirable in hypotensive gastrointestinal radiography. In addition, these compounds are useful in the treatment of spastic colon, pyolorospasm and other spastic conditions of the gastrointestinal tract as well as ureteral and biliary colic.

To achieve smooth muscle relaxation, these compounds are usually administered at a dose of about 0.1 to 3 pg / ug body weight, and preferably about 0.3 to 1.5 pg / kg body weight of the recipient. Administration is parenteral and may be intramuscular, subcutaneous or intravenous. Preferably, the compounds are administered intravenously or intramuscularly.

The straight-chain tetradecapeptides of the formula (III) used as starting material in the process according to the invention can be prepared by solid-phase synthesis. This synthesis consists of a stepwise construction of the peptide chain beginning at the C-terminal end of the peptide chain. First, cysteine ​​is linked to the resin by reacting amino-protected, S-protected cysteine ​​with a chloromethylated resin or hydroxymethyl resin with its carboxyl function. The preparation of a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London), Vol. 38, pp. 1597-98 (1966). The chloromethylated resin is commercially available (Lab Systems, Inc., San Mateo, California, V.St.A.).

To link the C-terminal cysteine ​​with the resin, the protected cysteine ​​is first converted to its cesium salt. This salt is then processed according to the method of BF Gisin, Helv. Chim. Acta, Vol. 56, p. 1476 (1973). However, the cysteine ​​can also be linked to the resin by activation of a carboxyl function by known procedures. For example, the cysteine ​​can be reacted with the resin in the presence of a carboxyl group activating compound such as N, N'-dicyclohexylcarbodiimide (DCC).

After linking the cysteine ​​with free carboxyl group to the resin support, the remainder of the construction of the peptide consists in staging each amino acid to the N-terminal part of the peptide chain. This addition of an amino acid requires cleavage of the α-amino protecting group from the amino acid representing the N-terminal portion of the peptide fragment, followed by attachment of the next amino acid ester to the free and reactive N-terminal amino acid. The cleavage of the α-amino protecting group is carried out in the presence of an acid such as hydrobromic acid, hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid or acetic acid, to form the corresponding acid addition salt, Another known method for cleaving the amino protecting group uses boron trifluoride. For example, by boron trifluoride diethyl etherate in glacial acetic acid, the amino-protected peptide fragment in a BF₃Complex which can then be converted to the deblocked peptide fragment by treatment with a base such as aqueous potassium bicaronate. Jpde of these methods can be used as long as care is taken to work so that cleavage of the N-terminal α-amino protecting group does not affect the other protecting groups present on the peptide chain. For this purpose it is preferred to effect the cleavage of the N-terminal protecting group by means of trifluoroacetic acid. In general, the cleavage is carried out at a temperature of about 0 "C to room temperature.

The product obtained after cleavage at the N-terminus is usually in the form of an acid addition salt with the acid used to cleave the protecting group. The product can then be converted into the free terminal amino compound by reaction with a weak base, for example a tertiary amine, such as pyridine or triethylamine.

The peptide chain is then in the state in which it can be reacted with the next amino acid. This can be done by any of the various known ways of working. For attachment of the next amino acid to the N-terminal peptide chain, an amino acid is used which has a free carboxyl group but is protected at the α-amino function as well as at any other active sites present. The amino acid is subjected to conditions that activate the carboxyl function for the attachment reaction. One such activation measure that can be used in the synthesis is to convert the amino acid to a mixed anhydride. The free carboxyl function of the amino acid is activated by reaction with another acid, for example a carboxylic acid, for example in the form of its acid chloride, active 4 No. 3,606,675.

Another method of activating the carboxyl function of the amino acid for attachment is that of converting the amino acid into an activated ester derivative. Examples of such active esters are a 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, p-nitrophenyl ester and an ester formed with 1-hydroxybenzotriazole or N-hydroxysuccinimide. Another method for linking the C-terminal amino acid to the peptide fragment is to carry out the addition reaction in the presence of at least an equimolar amount of N, N'-dicyclohexylcarbodiimide (DCC).

Then, when the desired amino acid sequence is present, the peptide can be removed from the resin carrier. For this purpose, the protected resin carrier tetradecapeptide is treated with hydrogen fluoride. This removes the peptide from the resin. In addition, however, all remaining protecting groups on the reactive sites of the peptide chain as well as the α-amino protecting group are cleaved off at the N-terminal amino acid. When hydrogen fluoride is used to cleave the peptide from the resin and to remove the protecting groups, it is preferred to carry out the reaction in the presence of anisole. It has been found that the presence of anisole inhibits the possible alkylation of certain amino acid residues present in the peptide chain. In addition, it is preferred to carry out the cleavage in the presence of ethylmercaptan.

The product obtained after the cleavage reaction is a straight chain peptide of formula (III) having 14 amino acid residues.

The invention is further illustrated by the following examples.

Example 1: Preparation of the straight-chain tetradecapeptide starting material

A) Nt-butyloxycarbonyl-Sp-methoxy-benzyl-L-cysteinyl-methylated polystyrene resin

To 1000 ml of Ν, Ν-dimethylformamide (DMF) containing the cesium salt of Nt-butyloxycarbonyl- (Sp-methoxybenzyl) -cysteine ​​(prepared from 17.5 g of the free acid) is added 100 g of chloromethylated polystyrene resin (Lab Systems, Inc , 0.75 mmol Cl / g). The mixture is stirred for 5 days at room temperature. Then the resin is filtered off and washed three times alternately with a mixture of 85% DMF and 15% water and with DMF and then twice with DMF. A suspension of the resin in 1000 ml of DMF is treated with a solution of 16 g (83.4 mmol) of cesium acetate. The mixture is stirred for 9 days at room temperature. Thereafter, the resin is filtered off and washed alternately three times each with a mixture of 85% DMF and 15% water and with DMF. The resin is washed with CHCl 3 and then suspended four times in a separatory funnel in CH 2 Cla with the liquid removed each time to remove fines. The resin is filtered off, washed with 95% ethanol and then alternately three times each with benzene and 95% ethanol. Drying the resin in vacuo at 30 ° C gives 115.3 g of the title product. Amino acid analysis yields 0.254 mmol Cys / g resin. The cysteine ​​is determined to be cysteic acid after acid hydrolysis, which is carried out using a 1: 1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethylsulfoxide has been added. washed with 95% ethanol and then alternately three times with benzene and 95% ethanol. Drying the resin in vacuo at 30 ° C gives 115.3 g of the title product. Amino acid analysis yields 0.254 mmol Cys / g resin. The cysteine ​​is determined to be cysteic acid after acid hydrolysis, which is carried out using a 1: 1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethylsulfoxide has been added. washed with 95% ethanol and then alternately three times with benzene and 95% ethanol. Drying the resin in vacuo at 30 ° C gives 115.3 g of the title product. Amino acid analysis yields 0.254 mmol Cys / g resin. The cysteine ​​is determined to be cysteic acid after acid hydrolysis, which is carried out using a 1: 1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethylsulfoxide has been added.

B) Nt-butyloxycarbonyl-D-valyl-glyc-L- (Sp-methoxybenzyl) -cysteinyl-L- (Ν^ε -o-chlorbenzyloxycarbonyU-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L- (N^e-o-chlorobenzyloxycarbonyl) lysyl-L (Q-benzyl) -threonyl-L-phenylalanyl-L- (0-benzyl) -threonyl-L- (O-benzyl) -seryl-L- (Sp-methoxybenzyl) - cysteinylmethylated polystyrene resin

5.0 g of the product obtained according to section A) are introduced into the reaction vessel of an automatic peptide synthesizer, Beckman 990, and twelve of the remaining thirteen amino acids are added using the automatic synthesizer. The formed protected tridecapeptide resin is divided into two equal proportions, and one of these fractions introduces the final residue. The amino acids used and the order of their use are as follows: (1) Nt-butyloxycarbonyl- (O-benzyl) -L-serine; (2) Nt-butyloxycarbonyl- (O-benzyl) -L-threonine; (3) Nt-butyloxycarbonyl-L-phenylalanine; (4) Nt-butyloxyo-carbonyl- (O-benzyl) -L-threo-5 No. 366075 nin; (5) N "-t-butyloxycarbonyl-N^ε -o-chlorobenzyloxycarbonyl-L-lysine; (6) N "-t-butyloxycarbonyl-D-tryptophan; (7) Nt-butyloxycarbonyl-L-phenylalanine; (8) Nt-butyloxycarbonyl-L-phenylalanine;

(9) N-t'-butyloxycarbonyl-L-asparagine p-nitrophenyl ester; (10) N "-t-butyloxycarbonyl-N^ε -o-chlorobenzyloxycarbonyl-L-lysine; (11) Nt-butyloxycarbonyl- (Sp-methoxybenzyl) -L-cysteine; (12) Nt-butyloxycarbonylglycine; and (13) Nt-butyloxycarbonyl-D-valine. The order of deprotection, neutralization, attachment and rejoining for the introduction of each individual amino acid into the peptide is as follows: (1) Three washes of 3 min each with chloroform (10 ml / g resin); (2) removal of the BOC group by two treatments of 20 minutes each with 10 mg / g resin of a mixture of 29% trifluoroacetic acid, 48% chloroform, 6% triethylsilane and 17% methylene chloride; (3) two washes of 3 minutes each with chloroform (10 ml / g resin); (4) a 3 min wash with methylene chloride (10 ml / g resin); (5) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); (6) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (7) Neutralization by three treatments of 3 minutes each with 3% triethylamine in methylene chloride (10 ml / g resin); (8) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (9) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); (10) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (11) Addition of 1.0 mmol / g resin of the protected amino acid and 1.0 mmol / g of resin N, Ν'-dicyclohexylcarbodiimide (DCC) in 10 ml / g resin of methylene chloride, followed by mixing for 120 minutes; (12) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (13) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); (14) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (15) neutralization by three treatments of 3 minutes each with 3% triethylamine in methylene chloride (10 ml / g resin); (16) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (17) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); (18) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (19) three washes of 3 minutes each with DMF (10 ml / g resin); (20) Addition of 1.0 mmol / g resin of protected amino acid and 1.0 mmol / g resin N, Ν'-dicyclohexylcarbodiimide (DCC) in 10 ml / g resin of a 1: 1 mixture of DMF and methylene chloride, followed by mixing during 120 min; (21) three washes of 3 minutes each with DMF (10 ml / g resin); (22) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (23) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); (24) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (25) neutralization by three treatments of 3 minutes each with 3% triethylamine in methylene chloride (10 ml / g resin); (26) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (27) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); and (28) three washes of 3 minutes each with methylene chloride (10 ml / g resin). (26) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (27) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); and (28) three washes of 3 minutes each with methylene chloride (10 ml / g resin). (26) three washes of 3 minutes each with methylene chloride (10 ml / g resin); (27) three washes of 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin); and (28) three washes of 3 minutes each with methylene chloride (10 ml / g resin).

The above treatment sequence is used to align each individual amino acid except the glycine and asparagine residues. The alignment of the glycine is carried out using only steps 1 to 18. The asparagine residue is introduced via its active p-nitrophenyl ester. To this end, the above step (11) is turned to the following three-step sequence: (a) three washes of 3 minutes each with DMF (10 ml / g resin); (b) adding 1.0 mmol / g resin of the p-nitrophenyl ester of Nt-butyloxycarbonyl-L-asparagine in 10 ml / g resin of a 1: 3 mixture of DMF and methylene chloride, followed by mixing for 720 min; and (c) three washes of 3 minutes each with DMF (10 ml / g resin). Also, the above step (20) is achieved by using the p-nitrophenyl ester of Nt-butyloxycarbonyl-L-asparagine in a 3:

The finished peptide resin is dried in vacuo. The product is hydrolyzed by refluxing in a 1: 1 mixture of concentrated hydrochloric acid and dioxane for 72 hours. Amino acid analysis of the resulting product using lysine as standard gives the following values: Asn, 1.00; 2Thr, 2,18; Ser, 0.95; Gly, 1.00; Val, 0.99; 3Phe, 3.45; 2Lys, 2.00.

Nr.360675

- 6 C) D-Valyl-glycyl-L-cysteinyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl- L-seryl-L-cysteine

To a mixture of 7.2 ml of anisole and 7.2 ml of ethylmercaptan is added 3.914 g (at a substitution value of 0.155 mmol / g) of the protected tetradecapeptide resin of Example 2. The mixture is cooled in liquid nitrogen and treated by distillation with 80 ml of liquid hydrogen fluoride. The resulting mixture is allowed to warm to 0 ° C and stirred for 2 h. The hydrogen fluoride is removed by distillation. The remaining mixture is mixed with ether and cooled to 0 ° C. The resulting solid is filtered off and washed with ether. The product is dried and the deprotected tetradecapeptide is extracted from the resin mixture using 1m acetic acid and 50% acetic acid. The acetic acid solution is immediately freeze-dried in the dark. The pale yellowish solid thus obtained is suspended in a mixture of 10 ml of degassed 0.2M acetic acid and 4 ml of glacial acetic acid. The suspension is gently warmed with 6 ml of 50% acetic acid until a clear yellow solution is formed, which is applied to a column of Sephadex G-25F. The details of the chromatography are as follows: solvent degassed 0.2M acetic acid; Column size 7.5 χ 150 cm; Temperature 26 ° C; Flow rate 1670 ml / h; Fraction volume 25.05 ml. Column size 7.5 χ 150 cm; Temperature 26 ° C; Flow rate 1670 ml / h; Fraction volume 25.05 ml. Column size 7.5 χ 150 cm; Temperature 26 ° C; Flow rate 1670 ml / h; Fraction volume 25.05 ml.

The absorbance of each fraction at 280 mp is plotted against the fraction number, resulting in a wide broad peak range followed by a shoulder. Most of the peak range corresponds to the product due to UV spectroscopy. The fractions to be pooled and their effluent volumes are as follows: Fractions 207 to 233 (5160 to 5837 ml, peak = 5515 ml).

This factions federation does not cover the shoulder at the back. UV spectroscopy shows the presence of 470 mg of product (yield - 46.4%). An Ellman titration of a sample shows a free sulfhydryl content of 95% of theory.

Preparation of the new tetradecapeptide by the process according to the invention

D) oxidation to D-Val¹, D-Trp^8thsomatostatin

677 ml of the solution of reduced D-Val obtained according to Section C)¹, D-Trp^8th-Somatostats are diluted with distilled water to a concentration of 50 pg / ml. The p_H~ Value of the mixture is adjusted to 6.7 with concentrated ammonium hydroxide. The solution is stirred for 64 h in the dark at 4 ° C, after which an Ellman titration indicates that the oxidation is complete.

The mixture is concentrated in vacuo to a volume of 45 ml and treated with 45 ml of glacial acetic acid. Thereafter, the mixture is desalted on a Sephadex G-25 F column. The details of the chromatography are as follows: solvent, degassed 50% acetic acid, column size 5.0 × 215 cm, temperature 26 ° C., flow rate 148 ml / h, fraction volume 17.3 ml.

The absorbance of each fraction at 280 mp is plotted against the fraction number, yielding two large peaks. The first peak corresponds to the aggregated forms of the product and the second peak corresponds to monomeric product. The material corresponding to the second peak is pooled (fractions 116 to 155, 2000 to 2685 ml). UV spectroscopy indicates that the sample contains 279 mg of product (Yield = 59.4%). The solution is freeze-dried in the dark.

The resulting white solid is rechromatographed in approximately two equal parts. The first part is dissolved in 25 ml of degassed 50% acetic acid and adsorbed on a column of Sephadex G-25F. Chromatography details: solvent, degassed 50% acetic acid; Column size 5.0 χ 215 cm; Temperature 26 ° C; Flow rate 148 ml / h; Fraction volume 17.3 ml.

The order of absorption of each fraction at 280 mp against the fraction number gives two large peaks. A standard cut of the second tip is made. Fractions 119 to 125 (effluent volumes 2128 to 2256 ml) are pooled. UV spectroscopy shows the

I

Presence of 65.3 mg product in this sample. The desired product is achieved by freeze-drying the solution in the dark.

The second part, like the first one, is rechromatographed to give comparable results. The two good products are combined and come together

No.360675 according to UV spectroscopy 126 mg (45.2% purified product). The combined product is dissolved in 15 ml degassed 0.2M acetic acid and applied to a column of Sephadex G-25F. Chromatography details: solvent degassed 0.2 M acetic acid; Column size 5.0^x 150 cm; Temperature 26 ° C; Flow rate 466 ml / h; Fraction volume 16.3 ml.

By applying the absorbance of each fraction at 280 mp to the fraction number, a large peak range is obtained. UV spectroscopy shows that most of the peak range is excellent product. Fractions 160 to 180 (2592 to 2934 ml, peak = 2685 ml) are combined and freeze-dried in the dark. UV spectroscopy gives the presence of 90.4 mg of product (71.7% yield).

Optical rotation [a] "= -56.1 ° (1% acetic acid).

Amino acid analysis: Val, 1.0; Gly, 0.97; 2Cys, 1.62; 2Lys, 2.00; Asn, 1.01; 3Phe, 2,87; Trp, 1.02; 2Thrs, 1.83; Ser, 0.81.

The above results are expressed as ratios of Lys / 2 = 1.0. All values ​​are averages of two 21 hour hydrolyses without rinse. Tryptophan is determined by UV spectroscopy (in relation to Lys / 2). Serine is not corrected for losses during hydrolysis.

The above product contains small amounts of impurities. If desired, the product can be further purified by preparative high pressure liquid chromatography (HPLC).

Another possible method for the oxidation of the reduced D-Val¹'D-Trp' somatostatin to D-Val¹, D-Trp^8th -Somatostatin is in the treatment with potassium ferricyanide. The oxidation is carried out in an aqueous solution adjusted to p ^ 6.7 as described above. To the mixture is added an aqueous solution of potassium ferricyanide to a final concentration approximately 3.3 times that of the reduced D-Val¹, D-Trp^8thSomatostatin corresponds. The solution is stirred for about 2 h in the dark at room temperature. The completeness of the oxidation is determined by an Ellman titration.

Example 2: Preparation of the straight-chain tetradecapeptide starting material

A) Nt-butyloxycarbonyl-D-alanyl-glycyl-1-L- (Sp-methoxybenzyl) cy- styrene-L- (Ν^ε -o-chlorobenzyloxycarbonyl) lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L- (N^£-ü-chloro-benzyloxycarbonyl) lysyl-L- (0-benzyl) threonyl-L-phenylalanyl-L- (O-benzyl) -threonyl-L- (0-benzyl) -seryl-L- (Sp-methoxybenzyl) cysteinyl-methylated polystyrene resin

This compound is prepared using the second part of Example 1, section

B) and Nt-butyloxycarbonyl-D-alanine in place of Nt-butyloxycarbonyl-D-valine.

Amino acid analysis of the product obtained after 21 hours of hydrolysis in a 1: 1 mixture of concentrated hydrochloric acid and dioxane at reflux showed the following values, with lysine being used as standard: Asn, 1.16; 2Thr, 2,14; Ser, 1.04; Gly, 1.09; Ala, 1:17; 3Phe, 2,88; 2Lys, 2.00.

B) D-Alanyl-glycyl-L-cysteinyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-iysyl-L-threonyl-L-phenylalanyl-L-threonyl-L- seryl-L-cysteine

The title compound is prepared according to the procedure described in Example 1, Section C) using 3.772 g (at a substitution value of 0.156 mmol / g) of the product obtained according to Example 2, Section A). The product is purified by chromatography on a column of Sephadex G-25 F. Chromatography conditions: solvent degassed 0.2 M acetic acid; Column size 7.5^x 150 cm; Temperature 26 ° C; Flow rate 1658 ml / h; Fraction volume 24.87 ml.

By applying the absorbance of each fraction at 280 mp to the fraction number, a large broad peak range with a subsequent shoulder is obtained. Due to UV spectroscopy, the majority of the maximum value range corresponds to the product.

The fractions to be pooled and their effluent volumes are as follows: Fractions 206 to 230 (509B to 5720 ml).

This faction unification does not include the subsequent shoulder. UV spectroscopy shows that the sample contains 403.9 mg of product (yield = 41.9%). An Ellman titration of a sample gives a free sulfhydryl content of 93% of theory.

- 8 No. 366075

Preparation of the new tetradecapeptide by the process according to the invention

C) Oxidation to D-Ala^l'D-Trp'-somatostatin

The obtained according to Example 2, Section B) reduced D-Ala ¹ 'D-Trp'-somatostatin is treated as described in Example 1, Section D). 622 ml of the solution (theory 403.9 mg) are diluted with distilled water to a concentration of 50 pg / ml and then adjusted to a Pjj of 6.7 with concentrated ammonium hydroxide. The mixture is stirred for 41 h at room temperature in the dark. The mixture is concentrated in vacuo to about 40 ml and then diluted with 40 ml of glacial acetic acid. The mixture is then adsorbed on a column of Sephadex G-25F. Chromatography conditions: solvent degassed 50% acetic acid; Column size 5.0 x 215 cm; Temperature 26 ° C; Flow rate 151 ml / h; Fraction volume 17.61 ml.

Orders of absorption of this fraction at 280 mp against the fraction number yield two large broad peaks. The first peak range corresponds to aggregated forms of the product and the second range corresponds to good monomeric product. The product represented by the second peak range (fractions 113 to 155, 1971 to 2728 ml) is combined and freeze-dried in the dark. The resulting solid is rechromatographed in approximately two equal portions. The first portion is dissolved in 22 ml degassed 50% acetic acid and the solution is applied to a column of Sephadex G-25F. Chromatography conditions: solvent degassed 50% acetic acid; Column size 5.0 x 215 cm; Temperature 26 ° C; Flow rate 153 ml / h; Fraction volume 17.85 ml.

By ordering the absorbance of each fraction at 280 mp against the fraction number, two large peak ranges are obtained. A standard cut of the second peak range is made by combining fractions 121 to 127 (effluent volumes from 2142 to 2267 ml). ÜV spectroscopy indicates the presence of 56.7 mg of product in this sample. The desired product is obtained by freeze drying and solution in the dark.

The second portion is rechromatographed in the same manner as the first. The products are combined (126.3 mg by UV spectroscopy - 31.3% yield from the reduced form). The product is dissolved in 21 ml degassed 0.2M acetic acid and applied to a column of Sephadex G-25F. Conditions of chromatography; Solvent degassed 0.2 M acetic acid; Column size 5.0 χ 150 cm; Temperature 26 ° C; Flow rate 449 ml / h; Fraction volume 15.71 ml.

By applying the absorbance of each fraction at 280 mp against the fraction numbers, a large peak range is obtained. UV spectroscopy shows that most of this range corresponds to the product. Fractions 169 to 188 (2640 to 2953 ml, peak = 2705 ml) are combined and freeze-dried in the dark. UV spectroscopy gives the presence of 85.2 mg product (67.5%).

Optical rotation [α] = -54.9 ° (1% acetic acid).

Amino acid analysis; Ala, 1.05; Gly, 1.0; 2Cys, 1.58; 2Lys, 2.0; Asn, 1,10; 3Phe, 2,92; Trp, 1.02; 2Thr, 1,98; Ser, 0.88.

The above results are expressed as ratios to Lys / 2 = 1.0. All values ​​are averages of two 21 hour hydrolyses without rinse. Tryptophan is determined by UV spectroscopy (in relation to Lys / 2). Serine is not corrected for losses during hydrolysis.