CS202097B2 - Method of preparing analogues of sematostatine - Google Patents

Description

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

Y-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Ehe-L-Thr-L-Ser-L- Cys-OH wherein Y represents D-Val or D-Ala and pharmaceutically acceptable acid addition salts thereof. The invention also relates to intermediate compounds prepared in the synthesis of these tetradecapeptides.

Somatostatin (also known as somatotropin release inhibitory factor) is a tetradecapeptide of formula | ---------- η

L-Ala-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-L-Trp-L-lys-L-Thr-L-Phe-L-Thr-L-Ser- L-Cys-OH

This tetradecapeptide was isolated from sheep hyphotalan extracts and was found to be effective in inhibiting growth hormone secretion, also known as samatotropin. See P. Brazeau, W. Vale, R. Burgus, N. Ling, M. Butcher, J. River and R. Ouillemin, Science, 179. 77 (1973).

In addition, a compound usefully referred to as D-Trp®-somatostatin was recently published in Brown et al., Endocrinology, 98, No. 2, 336-343 (1976).

The structure of the biologically active tetradecapeptides of the formula and their non-toxic acid addition salts differs from the structure of somatostatin and their respective salts by the presence of the D-tryptophan residue at position 8 instead of the L-tryptophan residue, and the D-valine or D-alanine residue at position 1 instead of L- alanine residue. Suitably, the tetradecapeptides of the formula I are referred to as D-Val ', D-Trp®-somatostatin and D-Ala', D-Trp'-somatostatin. The invention therefore relates to a process for the preparation of compounds of the formula I

HY-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- cys-OH (I) and their pharmaceutically acceptable non-toxic acid addition salts, and intermediates of formula II

RY-Gly-L-Cys (R1) -L-Lys _(R2) -L-Asn-L-Phe-L-Phe-D-Trp _(R5) -L-Lys _(R2) -L-Thr (R 3) -L-Phe-L-Thr (R ₃ ) -L-Ser (R 4) -L-Cys (R 1) -X (II) wherein

Y is D-Val or D-Ala,

R is hydrogen or an alpha-amino protecting group,

R 1 is hydrogen or a thio protecting group,

R ₂ represents hydrogen or a protecting group of the .epsilon.-amino, each of R

R 3a represents hydrogen or a hydroxy protecting group,

R 5 is hydrogen or formyl and X is hydroxy or a resin of the formula

wherein the resin is polystyrene with the proviso that when

X is hydroxy, each of the symbols

R 1, R ₂ , R ₂ , R 4, R 4 and R 8 represent hydrogen and when X represents a residue of the resin formula

-O-CH 2 -Q 2 each of the symbols

R 1, R 2, R 3 and R 4 are different from hydrogen.

Novel tetradecapeptides of the general formula I

HY-Gly-L-Cy-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- Cys-OH (I) wherein

Y is D-Val or D-Ala according to the invention is prepared by reacting the corresponding straight-chain tetradecapeptide of formula III

Y-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- Cys-OH (III) wherein

Y represents D-Val or D-Ala, acting with an oxidizing agent. This reaction converts two sulfhydryl groups into a disulfide bridge.

Suitable pharmaceutically acceptable non-toxic acid addition salts are addition salts with organic and inorganic acids, for example those derived from hydrochloric, sulfuric, sulfonic acids, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic acids. , nitric, benzoic, ascorbic, p-toluene sulfonic, benzenesulfonic, naphthalenesulfonic and propionic. Preferred acid addition salts are acetates. The acid addition salts are prepared by conventional methods.

As examples of intermediate compounds of formula II

RY-Gly-L-Cys (R) -L-Lys _(R2) -L-Asn-L-Phe-L-Phe-D-Trp (R5) -L-Lys _(R2) -L-Thr ( R 3) -L-Phe-L-Thr (R 3) -L-Ser (R ₄ ) -L-Cys (R 1) -X _(II) wherein various symbols are as defined above, compounds of the formulas may be mentioned

RD-Val-Gly-L-Cys (R) -L-Lys (R ₂ ) -L-Asn-L-Phe-L-Phe-D-Trp (R 5) -L-Lys (R ₂ ) -L -Thr (R 3) -L-Ehe-L-Thr (R 3) -L-Ser (R ₄ ) -L-Cys (R 1) -X a

RD-Ala-Gly-L-Cys (R1) -L-Lys _(R2) -L-Asn-L-Phe-L-Phe-D-Trp (R5) -I, Lys _(R2) -L -Thr (R 3) -L-Phe-L-Thr (R 3) -L-Ser (R 4) -L-Cys (R 1) -X.

Preferred intermediate compounds are those of formula III

Y-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- Cys-OH (III) wherein Y is as defined above.

Other preferred intermediate compounds are those of the following formulas:

HD-Val-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phr-L-Thr-L-Ser- L-Cys-OH

HD-Ala-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser- L-Cys-OH

N- (BOC) -D-Val-Gly-L- (PMB) Cys-L- (CBzOC) -Lys-L-Asn-L-Phe-L-Phe-D-Trp-L- (CBzOG) -Lys -L- (Bzl) Thr

-L-Phe-L- (Bzl.) Thr-L- (Bzl) Ser-L- (PMB) Cys-O-CH ₂ -4 7) Resins

N- (BCC) -D-Ala-Gly-L- (PMB) Cys-L- (CBzOC) -Lys-L-Asn-L-Phe-L-Phe-D-Trp-L- (CBzOC) Lys- L- (Bzl) Thr-L-Phe-L- (Bzl) Thr-L- (Bzl) Ser-L- (PMB) Cys-0 — CHJ Resins

The above formulas defining intermediate compounds also include compounds containing amino, hydroxy, and thio (sulfhydryl) protecting groups. Protecting groups must have properties meeting two aspects. On the one hand, protecting groups must prevent the reactive moiety present in a molecule from undergoing a reaction during exposure of the molecule to conditions that would otherwise result in the disappearance of the reactive moiety. However, the protecting group must, on the other hand, be of a nature such that it can be readily cleaved while regenerating the original reactive group under conditions that would not adversely affect other parts of the molecule. Suitable groups for this purpose, i.e. to protect amino, hydroxy and thio groups, are well known to those skilled in the art. The disclosures of these protecting groups and their uses have been discussed in full. One of them is the work of Protective Groups in Organic Chemistry, red. JFW McOmie, Ed., Plenum Press, New York, 1973.

In the above formulas defining the intermediate compounds, the R cell is an alpha-amino hydrogen or an alpha-amino protecting group. The amino protecting groups are well known to all those skilled in the art of peptide chemistry, many of which are listed in McOmie, supra, in Chapter 2 by J. W. Barton. Illustrative examples of such protecting groups include benzyloxycarbonyl-, p-chlorobenzyloxycarbonyl-, β-bromobenzyloxycarbonyl-, o-chlorobenzyloxycarbonyl-, 2,6-dichlorobenzyloxycarbonyl-, 2,4-dichlorobenzyloxycarbonyl-, o-bromobenzyloxycarbonyl-, o-bromobenzyloxy -, p-nitrobenzyloxycarbonyl-, tert-butyloxycarbonyl- (BOC), t-amyloxycarbonyl-, 2- (p-biphenylyl) isopropyloxycarbonyl (BpOC), adamantyloxycarbonyl-, isopropyloxycarbonyl-, cyclopentyloxycarbonyl-, cyclohexyloxycarbonyl-, cyclohexyl- trityl) and p-toluenesulfonyl. A preferred alpha-amino protecting group for R is t-butyloxycarbonyl.

R 1 represents a cell of a hydrogen atom or a sulfhydryl group of cysteine or a protecting group of a sulfhydryl substituent. Many of these protecting groups are described in McOmie, cited in Chapter 7 above, by R.G. Hickey, V.R. Rao and W.G. Rhodes. Illustrative examples of these protecting groups include p-methoxybenzyl, benzyl, β-tolyl, benzhydryl, acetamidomethyl, trityl, β-nitrobenzyl, tert-butyl, isobutyloxymethyl and any of a large number of trityl derivatives. <Further examples can be found, for example, in the encyclopedia Houben-Weyl, Methodes der Organischen Chemie, Synthes von Peptiden, Vol. 15/1 and 15/2, (1974), Stuttgart, Germany. A preferred sulfhydryl protecting group R 1 is p-methoxybenzyl.

R2 represents a hydrogen cell at the epsilon-amino group of a lysine residue or a suitable epsilon-amino protecting group. Illustrative examples may be the groups listed above as suitable for alpha-amino protection. Typical groups suitable for this purpose include benzyloxycarbonyl-, tert-butyloxycarbonyl-, tert-amyloxycarbonyl-, cyclopentyloxycarbonyl-, adamantyloxycarbonyl-, β-methoxybenzyloxycarbonyl-, p-chlorobenzyloxycarbonyl-, β-bromobenzobenzyloxy, β-bromobenzobenzyl, β-bromobenzobenzyloxycarbonyl- 6-dichlorobenzyloxycarbonyl-, 2,4-dichlorobenzyloxycarbonyl-, 6-bromobenzyloxycarbonyl-, p-nitrobenzyloxycarbonyl-, isopropyloxycarbonyl-, cyclohexyloxycarbonyl-, cycloheptyloxycarbonyl- and p-toluenesulfonyl.

As will be apparent from the description below, the process for preparing the tetradecapeptides of formula I comprises periodically cleaving the alpha-amino protecting group from the terminal amino acid present in the peptide chain. The only limitation with respect to the identity of the epsilon-amino protecting group of the lysine residue is therefore that the protecting group must be such that it does not cleave under the conditions used to selectively remove the .alpha.-amino group. Appropriate choice of alpha-amino and epsilon-amino protecting groups is readily apparent to those of ordinary skill in peptide chemistry, but the relative ease with which each protecting group can be cleaved is to be considered. For example, groups such as 2- (p-biphenylyl) isopropyloxycarbonyl (BpOC) and trityl are very labile and can be cleaved in the presence of a weak acid. A moderately strong acid such as hydrochloric acid, trifluoroacetic acid or boron trifluoride in acetic acid is required to cleave other groups such as tert-butyloxycarbonyl-, tert-amyloxycarbonyl-, adamantyloxycarbonyl- and p-methoxybenzyloxycarbonyl groups. Even stronger acids are required to cleave other protecting groups, such as the cleavage of benzyloxycarbonyl, halobenzyloxycarbonyl, β-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl and isopropyloxycarbonyl groups requiring strongly acidic conditions, i.e. the use of hydrogen bromide, trifluoroacetate or hydrogen fluoride. Of course, using stronger acids also cleaves all the more labile groups. Thus, when choosing amino protecting groups, it is necessary to ensure that the alpha-amino group is more labile than the epsilon-amino protecting group and the cleavage conditions must be selective so that only the alpha-amino protecting group is cleaved. A preferred combination of protecting groups satisfying these conditions is a combination of o-chlorobenzyloxycarbonyl- or cyclopentyloxycarbonyl of <<2 and tert-butyloxycarbonyl, as the alpha-amino protecting group used for each amino acid added to the polypeptide chain.

The groups R3 and R4 represent hydroxyl hydrogens or protective groups of the alcoholic hydroxy groups of threonine and lilac. Many such protecting groups are described in the above-cited McMie's work, Chapter 3, by C. B. Reese. Typical examples of such protecting groups include C1-C4 alkyl groups such as methyl, ethyl and tert-butyl, benzyl, substituted benzyl such as β-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl and o-chlorobenzyl. C1-C3 alkanoyl such as formyl, acetyl and propionyl, triphenylmethyl (trityl). When R 3 and R 4 are protecting groups, both groups are preferably benzyl.

R 5 is either hydrogen or formyl and defines a tryptophan moiety. The formyl group serves as a protecting group. The use of this protecting group is not obligatory and therefore R5 may be either hydrogen (a group not protected by nitrogen) or formyl (a group protected by nitrogen).

Group X defines the nature of the carboxyl terminal group of the tetradecapeptide chain. It may be a hydroxy group, in which case it is a free carboxy group. In addition, X may be a solid resin support to which the terminal carboxyl group of the peptide is bound during its synthesis. The remainder of the solid resin can be represented by the formula

It applies that, when X represents hydroxyl, each of R, R ,, R2 ^{»R3> R4 and R5} are hydrogen. When X represents a solid resin support, each of R 1, F 1, R 2, R 3 and R 4 is each a protecting group.

The following abbreviations are used throughout the description, most of which are commonly introduced in the art.

Ala - alanine Asn - asparagine Cys - cysteine Gly - glycine Lys - lysine Phe - phenylalanine Ser - serine Thr - threonine Trp - tryptophan

Val - valine

DCC - Ν, Ν'-dicyclohexylcarbodiimide

DMF-N, N-dimethylformamide

BOC - tert.butyloxycarbonyl

PMB - p-methoxybenzyl

CBzOC - o-chlorobenzyloxycarbonyl

CPOC - cyclopentyloxycarbonyl

Bzl - benzyl

Forformyl

BpOC-2- (p-biphenyl) isopropyloxycarbonyl.

While the choice of particular protecting groups to be used in the preparation of compounds of Formula I is within the skill of one of ordinary skill in peptide synthesis, it will be understood that the sequence of the reaction to be carried out affects the choice of particular protecting groups. In other words, the protecting group chosen must be stable both to the reactants used and to the conditions of the subsequent reaction steps. For example, as already noted to some extent, the particular protecting group used must remain intact under the conditions used to cleave the alpha-amino protecting group of the terminal amino acid residue of the peptide fragment in preparation for condensation of the next amino acid fragment to the peptide chain. It is also important to select as a protecting group a group that remains intact during the building of the peptide chain and which can be easily cleaved after completion of the synthesis of the desired tetradecapeptide product. However, all these factors are within the skill of the art.

As can be seen from the above discussion, the tetradecapeptides of formula I can be prepared by solid phase synthesis. This synthesis consists in the gradual build-up of the peptide chain, starting at the terminal carbon end of the peptide. In most cases, the cysteine is first coupled to the resin via its carboxyl function, by reaction of cysteine and protected amino and sulfhydryl protected with a chloromethylated or hydroxymethylated resin. The preparation of a hydroxymethyl resin is described in Bodanszky et al., Chem. Indian. (London), 38, 1597-98 (1966). Chloromethylated resin is commercially available (Lab Systems, Inc., San Mateo, California, USA).

Alternatively, the cysteine may bind to the resin by first activating its carboxyl functional group in a conventional manner. For example, cysteine can be reacted with a resin in the presence of a carboxy oxy activating compound such as N, N'-dicyclohexylcarbodiimide (DCC). _;

Upon binding of the free carboxy group of the cysteine to the resin support, the peptide chain begins to build gradually, by progressively condensing the individual amino acids to the N-terminal portion of the peptide chain. Thus, it is necessary at each stage to cleave the alpha-amino protecting group from the amino acid in the terminal portion of the peptide fragment, and then join the following amino acid residue to the free and reactive N-terminus of the terminal amino acid. The cleavage of the alpha-amino protecting group may be carried out in the presence of an acid such as hydrobromic, hydrochloric, trifluoroacetic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, and acetic acid to give the corresponding acid addition salt of the product.

Another method of cleaving the amino protecting group is by treatment with boron trifluoride. For example, by treatment with diethylether boron trifluoride in glacial acetic acid, the amino-protected peptide fragment is converted to a complex with boron trifluoride, which is then converted to the free peptide fragment by treatment with a base such as aqueous potassium bicarbonate. Any of these methods may be used provided that it is achieved by cleavage of the N-terminal alpha-amino protecting group without disrupting any other protecting groups present in the peptide chain. In view of this requirement, it is preferable to cleave the N-terminal protecting group using trifluoroacetic acid. Typically, the cleavage is carried out at a temperature of about 0 ° C to about room temperature.

After cleavage of the protecting group at the N-terminal position, the product obtained is usually in the form of an acid addition salt which has been used to cleave the protecting group. This product can then be converted to the free amino compound by treatment with a weak base, typically a tertiary amine such as pyridine or triethylamine.

Now the peptide chain is ready for reaction, with the following amino acid. The reaction with the amino acid can be carried out by any of several known techniques. To attach the following amino acid to the N-terminus of the peptide chain, an amino acid having a free carboxy group but having a suitably protected alpha-amino group and all other optionally reactive groups present is used. The amino acid needs to be activated to react with the N-terminus of the peptide chain. One method of activation that can be used in the synthesis is to convert the amino acid to a mixed anhydride. Here, the free carboxylic function of the amino acid is activated by reaction with another acid, typically with an acid chloride derivative in the form of an acid chloride. Examples of such acid chlorides that can be used to form mixed anhydrides include ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate and pivaloyl chloride.

Another way of activating the carboxyl function of an amino acid for coupling with a peptide chain is by converting the amino acid into a reactive ester derivative. Examples of such reactive esters are 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, p-nitrophenyl ester, 1-hydroxybenzotriazole ester and N-hydroxysuccinimide ester. Another method of linking the C-terminal portion of an amino acid to a peptide fragment is a method in which the condensation is performed in the presence of at least an equimolar amount of N, N'-dicyclohexylcarbodiimide (DCC). The latter process is preferably used in the preparation of the tetradecapeptide of formula II wherein X is a group of formula

O-CH.

resin

After preparing the peptide chain with the desired amino acid sequence, the resulting peptide can be removed from the resin support. This is done by treating the tetradecapeptide with the reactive groups protected by hydrogen fluoride. The action of hydrogen fluoride cleaves the peptide from the resin, but in addition cleaves all other protecting groups present on the reactive groups of the peptide chain, as well as the protecting group of the alpha-amino group of the terminal amino acid. When the peptide is cleaved from the resin while hydrogen fluoride is deprotected, the reaction is preferably carried out in the presence of anisole. The presence of anisole has been found to inhibit the potential alkylation of certain amino acid residues present in the peptide chain. In addition, the cleavage is preferably carried out in the presence of ethyl mercaptan. Ethyl mercaptan serves to protect the indole ring of the triethanophan residue and furthermore facilitates the conversion of protected cysteine residues into the thiol form. Also, when R1 is a formyl group, the presence of ethyl mercaptan facilitates the cleavage of the formyl group by hydrogen fluoride. ,

After cleavage as described above, a straight chain peptide containing 14 amino acid residues is obtained. In order to obtain the final product of formula (I), it is necessary to oxidize the straight chain tetradecapeptide under conditions such that the sulfhydryl groups present in the molecule, one at each cysteine residue, are oxidized to form a disulfide bridge. The oxidation can be carried out by treating the dilute linear tetradecapeptide solution with one of a number of oxidizing agents, such as iodine and potassium ferricyanide. Air may also be used as the oxidizing agent, the pH of the mixture typically being maintained from about 2.5 to about 9.0, preferably from about 6.2 to about 7.2. When air is used as the oxidizing agent, the concentration of the peptide solution is usually less than about 0.4 mg peptide / ml solution and usually about 50 µg / ml.

The compounds of formula I can be administered to warm-blooded animals, including humans, and are particularly useful for relaxing smooth muscles. In particular, the gastrointestinal tract can be brought to a state of relaxation by parenteral administration of small amounts of these compounds and, preferably, D-Val ', D-Trp®-somatostatin. This effect, which results in reduced bowel motility, is particularly desirable in hypotonic gastrointestinal radiography. In addition, the compounds of the invention are useful in the treatment of colonic spasms, polyrospasm and other spasmatic conditions of the gastrointestinal tract, urinary tract spasms and gallbladder spasms.

To achieve smooth muscle relaxation, the compounds of the invention are generally administered at a dosage of from about 0.1 µg to about 3 µg per kg body weight of the recipient, and preferably from about 0.3 to about 1.5 µg per kg body weight. Parenteral administration, either intramuscular, subcutaneous or intravenous, is used. Preferably, the compounds of the invention are administered intravenously or intramuscularly.

Liquid unit dosage forms for parenteral administration are usually prepared by admixing the compound with a pharmaceutical carrier, for example isotonic saline, isotonic glyoin, lactose, mannitol, dilute acetic acid, bacteriostatic water, for example water containing about 1% benzyl alcohol and phosphate buffer solutions and suitable combinations any standard carriers. The carrier is usually present in a weight ratio of about 25: 1 to about 1000: 1 relative to the active compound.

Depending on the carrier and concentration used, the compound may either be suspended or dissolved in a suitable sterile vehicle, preferably water. To prepare solutions, the compound and the carrier may be dissolved in the vehicle of choice, filtered and filled with vials or injections, which are then sealed. Advantageously, adjuvants such as local anesthetics, preservatives or buffers may be dissolved in the vehicle. To enhance stability, the compound in association with the carrier can be dissolved in water, the aqueous solution placed in a vial, and then lyophilized. The dry lyophilized solid is then sealed in a vial and a vial of vehicle is added to the package to reconstitute the formulation before use. Parenteral suspensions may be prepared in substantially the same manner except that the compound is suspended in the carrier instead of being dissolved therein.

The compounds of formula I are also effective, although not always to the same extent, as growth hormone release inhibitors. This inhibitory effect is beneficial in those cases where it is desirable to treat excessive somatotropin secretion. Excessive secretion of somatotropin may be associated with adverse disorders such as juvenile diabetes and acromegaly. These compounds also have other physiological effects, such as inhibition of gastric acid secretion, useful in the treatment of ulcers, inhibition of exocrine pancreatic secretion, potentially useful in the treatment of pancreatitis, and inhibition of insulin and glucagon secretion.

The compounds can be administered by various routes, such as orally, sublingually, subcutaneously, intramuscularly, and intravenously. The dosage range for sublingual or oral administration is preferably about 1 mg to about 100 mg / kg body weight. The dosage for intravenous, subcutaneous or intramuscular administration in these indications is usually from 1 µg to about 1 mg / kg body weight, and preferably from about 50 µg to about 100jug / kg body weight. It is understood that the dosage range is largely dependent on the particular disorder being treated and its severity.

The compounds of formula I may be administered orally or sublingually in association with a pharmaceutical carrier, for example in the form of tablets or capsules. Conventional inert diluents and carriers such as magnesium carbonate or lactose in conjunction with conventional disintegrating agents such as corn starch and alginic acid and glidants such as magnesium stearate are used as pharmaceutical carriers. Typically, the amount of carrier or diluent is in the range of about 5 to about 95 based on the final formulation and preferably about 50 to about 85% based on the final formulation. Appropriate flavor enhancers may also be added to the final formulation. When the compounds of formula I are to be administered parenterally, suitable carriers are, for example, those described above in connection with the use of these compounds to achieve smooth muscle relaxation. The following examples illustrate the preparation of compounds of formula (I) and intermediates. The examples are illustrative only and do not limit the scope of the invention in any way.

He did

N-tert-butyloxycarbonyl-L-cysteinyl (S-p-methoxybenzyl) methylated polystyrene resin

To 1000 ml of Ν, Ν-dimethylformamide (DMF) containing the cesium salt of N-tert-butyloxycarbonyl- (Sp-methoxybenzyl) cysteine (prepared from 17.5 g of free acid) was added 100 g of chloromethylated polystyrene resin (Lab Systems, Inc.). , 0.75 mmol (Cl / gram). The mixture was stirred at room temperature for 5 days. The resin was filtered off and washed three times with a mixture of 85% DMF and 15% water and DMF and then two more times with DMF. A solution of cesium acetate (16 g, 83.4 mmol) was added to the resin suspended in 1000 ml of DMF. The mixture was stirred at room temperature for 9 days. The resin was then filtered off and washed three times with a mixture of 85% DMF and 15% water and DMF three times. The resin was then washed with chloroform and suspended four times in chloroform in the separator and the liquid was drained to remove dust. The resin was filtered off, washed with 95% ethanol and then washed three times alternately with benzene and 95% ethanol three times. The resin was dried under vacuum at 30 ° C to give 115.3 g of the title product. Amino acid analysis shows that per gram of resin there is 0.254 mmol of cysteine. Cysteine is determined as cysteic acid in the hydrolysis product, using a 1: 1 mixture of dioxane and concentrated hydrochloric acid, to which a small amount of dimethylsulfoxide has been added.

Example 2

N-tert-butyloxycarbonyl-D-valyl-glycyl-L- (S- p-methoxybenzyl) cysteinyl-L- (N -o-chlorobenzyloxycarbonyl ^d) lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D -tryptofyl-L- (N -o-chlorobenzyloxycarbonyl ^R) lysyl-L- (O-benzyl) threonyl-L-phenylalanyl-L- (O-benzyl) threonyl-L- (O-benzyl) seryl-L- (Sp (methoxybenzyl) cysteinylmethylated polystyrene resin

The product of Example 1 (5.0 g) was placed in a reaction vessel of the Beckman 990 Automatic Peptide Synthesizer and twelve of the remaining thirteen amino acids were added to the product. The resulting protected tridecapeptide bound to the resin is divided into two equal portions and a terminal residue is attached to the product contained in one of these portions. The amino acids used and their sequence are as follows:

1. N-tert-Butyloxycarbonyl- (O-benzyl) -L-serine;

2. N-tert-butyloxycarbonyl- (O-benzyl) -L-threonine;

3. N-tert-butyloxycarbonyl-L-phenylalanine;

4. N-tert-butyloxycarbonyl- (O-benzyl) -L-threonine

5. N * - tert-butyloxycarbonyl-N-o-chlorobenzyloxycarbonyl-L-lysine;

6. N-tert-Butyloxycarbonyl-D-tryptophan

7. N-tert-butyloxy-L-phenylalanine;

8. N-tert-butyloxycarbonyl-L-phenylalanine;

9. N-tert-butyloxycarbonyl-L-asparagine p-nitrophenyl ester;

10. N-tert-Butyloxycarbonyl-N-o-chlorobenzyloxycarbonyl-L-lysine;

11. N-tert-Butyloxycarbonyl- (S-p-methoxybenzyl-L-cysteine)

12. N-tert-Butyloxycarbonylglycine a

13. N-tert-Butyloxycarbonyl-D-valine.

The sequence of operations to cleave the neutralizing protective group and the amino acid attachment for each of the amino acids introduced into the peptide is as follows:

1. Wash three times with chloroform (10 ml / g resin each) for 3 minutes.

2. cleavage of the BOC group by treatment with 29% trifluoroacetic acid,% chloroform and 6% triethylsilane at a rate of 20 ml / g for 20 minutes, repeating the procedure twice,

3. Wash twice with chloroform (10 ml / g resin each time for 3 minutes).

4. one wash with methylene chloride (10 ml / g resin, for 3 minutes),

5. Wash three times with a mixture of 90% tert-butyl alcohol and 10% target. amyl alcohol (always 10 ml / g resin, always for 3 minutes),

6. wash three times with methylene chloride (10 ml / g resin each) for 3 minutes,

7. neutralization of 3% triethylamine in methylene chloride (10 ml / g resin) for 3 minutes - repeated three times,

8. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

9. Wash with a mixture of 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

10. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

11. Add 1.0 mmol / g of protected amino acid resin and 1.0 mmol / g of N, N'-dicyclohexylcarbodiimide resin (DCC) in 10 ml / g methylene chloride resin and mix for 120 minutes;

12. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

13. Wash with a mixture of 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

14. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

15. neutralization by treatment with 10 ml / g of 3% triethylamine resin in methylene chloride for 3 minutes - repeated three times,

16. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

17. Washing with a mixture of 90% tert-butyl alcohol and 10% target. amyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

18. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

19. wash 10 ml / g resin with dimethylformamide for 3 minutes - repeat three times,

20. Add 1.0 mmol / g of protected amino acid resin and 1.0 mmol / g of Ν, Ν'-dicyclohexylcarbodiimide resin (DCC) in 10 ml / g resin 1: 1 mixture DMP and methylene chloride for 120 minutes,

21. wash 10 ml / g of dimethylformamide for 3 minutes - repeat three times,

22. Wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

23. Wash with a mixture of 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeat three times,

24. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

25. neutralization by treatment with 3 ml / g of 3% triethylamine resin in methylene chloride for 3 minutes - repeated three times,

26. wash with methylene chloride (10 ml / g resin) for 3 minutes - repeat three times,

27. washing with a mixture of 90% t-butyl alcohol and 10% t-butyl alcohol (10 ml / g resin) for 3 minutes - repeated three times, and

28. Wash with methylene chloride (10 mL / g resin) for 3 minutes - repeat three times.

The above sequence of operations is used to attach each of the amino acids except glycine and asparagine. The attachment of glycine is carried out only using steps 1 to 18. The asparagine residue is introduced via the reactive p-nitrophenyl ester. In doing so, stage 11 is replaced by the following three stages:

a) washing of 10 ml / g resin with dimethylformamide for 3 minutes - repeated three times,

(b) addition of 1.0 mmol / g of N-tert-butyloxycarbonyl-L-asparagine p-nitrophenyl ester resin in 10 ml / g of a 1: 3 mixture of dimethylformamide and methylene chloride and stirring for 720 minutes and "

c) washing 10 ml / g resin with dimethylformamide for 3 minutes - repeated three times.

Also, step 20 was modified by adding p-nitrophenyl ester of N-tert-butoxycarbonyl-L-asparagine in a 3: 1 mixture of dimethylformamide and methylene chloride and stirring for 720 minutes.

The resulting resin bound peptide was dried under vacuum. The product is hydrolyzed at reflux in a mixture of concentrated hydrochloric acid and dioxane for 72 hours. Amino acid analysis of the resulting product gives the following results (lysine is used as standard) Asn, 1.00; 2Thr, 2.18; Ser, 0.95; Gly, 1.00; Val, 0.99; 3Phe, 3.45; 2Lys, 2.00.

Example 3

D-valyl-glycyl-L-cysteinyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyll-L-lysyl-L-threonyl-L-thyonyl-L-threonyl-L-seryl-L -gysWin

To a mixture of 7.2 mL of anisole and 7.2 mL of ethyl mercaptan was added 3.914 g (at a substitution level of 0.155 mmol / g) of the protected terapeptide on the resin of Example 2. The mixture was cooled in liquid nitrogen and 80 mL of liquid hydrogen fluoride was distilled. The resulting mixture was allowed to warm to 0 ° C and stirred for 2 hours. The hydrogen fluoride is then distilled off. Ether was added to the remaining mixture and the resulting mixture was cooled to 0 ° C. The resulting solid was filtered and washed with ether. The product was dried and the deprotected tetradecapeptide was extracted from the resin mixture using 1M acetic acid and 50% acetic acid. The acetic acid solution is then lyophilized to dryness in the dark. The resulting pale yellow solid was suspended in a mixture of 10 mL degassed 0.2 M acetic acid and 4 mL glacial acetic acid. Heat the slurry gently with 6 mL of 50% acetic acid until a clear yellow solution is obtained and this solution is applied to a Sephadex G-25F column. Chromatography is performed under the following conditions:

solvent: degassed 0.2 M acetic acid column dimensions: 75 x 1,500 mm temperature: 26 ° C flow rate: 1,670 ml / h fraction volume: 25.05 ml ·

Absorbance at 280 nm of each fraction is plotted against Fraction Number. A curve with a large wide maximum and an adjacent arm (inflexion) is obtained. UV-spectroscopy shows that the major part of the maximum is the product. Fractions 207 to 233 are combined (i.e. eluate from 5 160 to 5837 ml, maximum corresponds to a total flow of 5 515 ml eluate).

The pooled fractions do not contain a product corresponding to the curve arm. UV-spectroscopy shows that the combined fractions contained 470 mg of the product (yield 46.4%). Ellman titration of the aliquot showed that the sulfhydryl group content was 95% of theory.

Example 4

Oxidation to D-Vall, D-Trp®-somatostatin

The solution of reduced D-Val 1, D-Trp 4 -somatostatin (677 ml) from Example 3 was diluted to 50 µg / ml with distilled water. Concentrated ammonium hydroxide was added to adjust the pH of the mixture to 6.7. The solution was stirred at 4 ° C in the dark for 64 hours and then determined by Ellman titration that oxidation was complete.

The mixture was concentrated in vacuo to a volume of 45 mL and 45 mL of glacial acetic acid was added. The mixture was desalted on a Sephadex G-25P column. Chromatography is performed under the following conditions:

solvent: degassed 50% acetic acid column size: 50 x 2150 mm temperature: 26 ° C flow rate: 148 'ml / h fraction volume: 17.3 ml

The absorbance at 280 nm of each fraction is plotted against the fraction order. The obtained curve contains two large maxima. The first peak consists of aggregated product forms and the second peak is the monomer product. The substance corresponding to the second maximum was collected (fractions 116-155 (2000-2685 ml)). UV spectroscopy showed that the sample contained 279 mg of the product (yield 59.4%). The solution is lyophilized in the dark to dryness.

The resulting white solid was separated into two approximately equal portions and each portion was re-chromatographed. The first portion was dissolved in 25 mL of degassed 50% acetic acid and absorbed on a Sephadex G-25F column. Chromatography is performed under the following conditions:

solvent: degassed 50 # acetic acid column size: 50 x 2, 50 mm temperature: 26 ° C flow rate; 148 ml / h fraction volume: 17.3 ml.

The absorbance at 280 nm of each fraction is plotted against the fraction order. The fraction obtained is plotted against the fraction order. The obtained curve contains two large maxima. Fractions containing the second peak were collected, i.e. fractions 119-125 were pooled (eluate from 2,128 to 2,256 mL). UV spectroscopy shows that the sample contained 65.3 mg of product. The solution is lyophilized to dryness in the dark to give the desired product.

The second part is re-chromatographed in the same way as the first part and similar results are obtained. The products obtained in the two chromatographic procedures were combined and, according to UV-spectroscopy, the total yield was 126 mg (45.2 # yield of purified product). The combined product was dissolved in 15 mL of degassed 0.2 M acetic acid and applied to a Sephadex G-25F column. Chromatography is performed under the following conditions:

solvent: degassed 0.2 M acetic acid, column dimensions: 50 χ 1,500 mm temperature: 26 ° C flow rate: 466 ml / h fraction volume: 16.3 ml.

Absorbance at 280 nm of each fraction is plotted against fraction order. The curve obtained contains one large maximum. UV-spectroscopy shows that the major part of the maximum is a product of very good purity. Fractions 160 to 80 were combined (from a flow rate of 2692 to a flow rate of 2934 ml, maximum corresponding to a flow rate of 2685 ml) and the combined fractions were lyophilized to dryness in the dark, UV-spectroscopy showed the presence of 90.4 mg of product theory).

Optical rotation = -56.1 ° (in 1 # acetic acid).

Amino Acid Analysis:

Val 1.0, Gly 0.97, 2Cys ,, 62, 2Lys 2.00, Asn 1.01, 3Phe 2.87, Trp 1.02, 2Thr 1.83,

Ser 0.81.

The above results are expressed as ratios relative to the unit amount of lysine (Lys / 2 = 1.0). All values are averages of two 21-hour hydrolyses performed without aseptors. Tryptophan is determined by UV-spectroscopy (as a ratio to Lys / 2); the amount of lilac is not corrected for losses due to hydrolysis.

The product obtained contains less impurities. If desired, the product can be further purified by preparative high pressure liquid chromatography (HPLC).

An alternative method of oxidizing reduced D-Val ^, D-Trp®-somatostatin to D-Val ^,

D-Trp ⁸ -somatostatin is treatment with potassium ferricyanide. The oxidation is carried out in an aqueous solution, the pH of which is adjusted to 6.7 as described above. An aqueous solution of potassium ferricyanide is added to the mixture until a final concentration of approximately 3.3 times the amount of reduced D-Vall, D-Trp ⁸ -somatostatin is reached. The solution was stirred in the dark at room temperature for about 2 hours. Completion of the oxidation was verified by Ellman titration.

Example 5

N-tert-butyloxycarbonyl-D-alanyl-glycyl-L- (Sp-methoxybenzyl) cysteinyl-L- (N -o-chlorobenzyloxycarbonyl ^e) lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L - N - (4-O-chlorobenzyloxycarbonyl) lysyl-L- (O-benzyl) threonyl-L-phenylalanyl-L- (O-benzyl) threonyl-L- (O-benzyl) seryl-L- (Sp-methoxybenzyl) cysteinyl- methylated polystyrene resin

This compound was prepared using the second portion of the tridecapeptide prepared according to Example 2, and N-tert-butyloxycarbonyl-D-alanine was added to this fragment instead of N-tert-butyloxycarbonyl-D-valine.

Amino acid analysis of the resulting product after 21 hours of hydrolysis at reflux in a 1: 1 mixture of concentrated hydrochloric acid and dioxane gives the following results (lysine is used as standard):

Asn 1.16, 2Thr 2.14, Ser 1.04, Gly 1.09, Ala 1.17, 3Phe 2.88, 2Lys 2.00.

Example 1 a d 6

D-alanyl-glycyl-L-eysteinyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenylalanyl-D-tryptophenyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L- seryl-L-cysteine

The title compound was prepared as described in Example 3 using 3.722 g (at a substitution level of 0.156 mmol / g) of the product of Example 5. Purification of the product was performed by Sephadex G-25F column chromatography. Chromatography is performed under the following conditions:

solvent: degassed 0.2 M acetic acid column dimensions: 75 χ 1,500 mm temperature: 26 ° C flow rate: 1,658 ml / h fraction volume: 24.87 ml.

The absorbance at 280 nm of each fraction is plotted against the fraction number. A curve with a large wide maximum and an adjacent arm (inflexion) is obtained. UV-spectroscopy shows that the major part of the maximum is the product. Fractions 206 to 230 (i.e., from a flow of 5,098 to a flow of 5,720 mL of solvent) were pooled.

The pooled fractions do not contain a product corresponding to the arm, curve. UV-spectroscopy shows a theoretical amount of 403.9 mg (41.9% yield) of product in the sample. Ellman titration of the aliquot showed that the content of free sulfhydryl groups was 93% of theory.

Example 1 a d 7

Oxidation to D-Ala ', D-Trp®-somatostatin ·

The reduced D-Ala ', D-Trp ⁸ -somatostatin of Example 6 was treated as described in Example 4. The solution of Example 6 (622 ml, theoretical product content 403.9 mg) was diluted with distilled water to a concentration of 50 µg / ml. . Concentrated ammonium hydroxide was added to adjust the pH of the mixture to 6.7. The solution was stirred at room temperature in the dark for 41 hours.

The mixture was concentrated to about 40 ml in vacuo and diluted with 40 ml glacial acetic acid. The mixture is absorbed on a Sephadex G-25F column. Chromatography is performed under the following conditions:

solvent: degassed 50% acetic acid column dimensions: 50 x 2150 mm temperature: 26 ° C flow rate: 151 ml / h fraction volume: 17.61 ml.

The absorbance at 280 nm of each fraction is plotted against the fractionation order. The obtained curve contains two large maxima. The first maximum consists of aggregated product forms and the second maximum is a monomeric product. The substance corresponding to the second maximum was collected (fractions 113-155 (from 1971 flow to 2,728 mL of eluate)). The solution was lyophilized to dryness in the dark.

The resulting white solid was separated into two approximately equal portions and each portion was re-chromatographed. The first portion was dissolved in 22 mL of degassed 50% acid and applied to a Sephadex G-25F column. Chromatography is performed under the following conditions:

solvent: degassed 50% acetic acid column dimensions: 50 x 2150 mm temperature: 26 ° C flow rate: 153 m / h fraction volume: 17.85 ml.

The absorbance at 280 nm of each fraction is plotted against the fraction order. The obtained curve contains two large maxima. Fractions containing the second maximum fraction are collected, i.e., fractions 121-127 (flow volume of solvent 2,142-2,267 ml) are combined. UV-spectroscopy of the sample indicated that the product content was 56.7 mg. The solution was lyophilized to dryness in the dark. The desired product is obtained.

The second part is re-chromatographed in the same way as the first part and similar results are obtained. The products obtained in the two chromatography procedures were combined and, according to UV-spectroscopy, the overall yield was 126.3 mg (31.3% yield based on reduced form). The combined product was dissolved in 21 mL of degassed 0.2 M acetic acid and applied to a Sephadex G-25F column. Chromatography is performed under the following conditions:

solvent: degassed 0.2 M acetic acid column dimensions: 50 x 1500 mm. temperature: 26 ° C flow rate: 449 ml / h fraction volume: 15.71 ml.

Absorbance at 280 nm of each fraction is plotted against fraction order. The curve obtained contains one large maximum. UV-spectroscopy shows that the major part of the maximum is the product. Fractions 169-188 (eluent 2,640-2,953 ml, maximum 7070 ml) were combined and lyophilized to dryness in the dark. UV-spectroscopy showed the presence of 85.2 mg of the product (yield 67.5%).

Optical Rotation = -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 1.9a,

Ser 0.88.

The above results are expressed as ratios relative to the unit amount of ly15 sine (Lys / 2 = 1.0). All values are averages of two 21-hour hydrolyses performed without acceptors. Tryptophan is determined by UV-spectroscopy (as a ratio to Lys / 2), the amount of serine is not corrected for hydrolysis losses.

D-Val ', D-Trp®-somatostatin has been tested in dogs as an agent for the in vivo inhibition of gastric acid secretion. Six dogs with a chronic fistula and Heidenhain sac are induced gastric acid secretion by infusion of the C-terminal tetrapeptide gastrin at a dosage of 0.5 µg / kg.h. Each dog also serves to control itself. After one hour of steady-state hydrochloric acid secretion, dogs are infused for 1 hour with D-Trp®-somatostatin at a dose of 0.15 µg / kg.h. Sampling of gastric acid is continued for a further 1.5 hours at a sampling interval of 15 minutes. Samples with an automatic titrator titrated to pH 7. The maximal inhibitory effect of D-Val-D-Trp ⁸ -somatostatinu was extrapolated against the dose-response curve of somatostatin, and the relative efficacy of analog to that of somatostatin are expressed as% activity. D-Val ', D-Trp ⁸ -somatostatin inhibits steady-state gastric acid secretion induced by C-terminal tetrapeptide gastrin by 48.22 ± 6.45% (standard mean error of measurement). This effect is equivalent to that of 0.175 µg / kg h of somatostatin. The relative activity of this substance towards somatostatin is therefore 116%. A purer sample of D-Val ', D-Trp ⁸ -somatostatin, administered at doses of 0.200, 0.166 and 0.138 µg / kg.h, inhibits the steady-state secretion induced by the C-terminal tetrapeptide gastrin of 77.63, 71.57 and 67 , 8 #. The activity relative to somatostatin is 302 to 325%.

D-Ala ', D-Trp®-somatostatin tested at a dose of 0.20 µg / kg.h under the same conditions inhibits steady-state secretion induced by C-terminal tetrapeptide gastrin by 73.61 t 3.66% (standard mean error). This effect is equivalent to 0.550 µg / kg.h of somatostatin. The activity of this substance with respect to somatostatin is 275 #.

D-Val ', D-Trp®-somatostatin and D-Ala), D-Trp- ^8- somatostatin have also been tested for their effect on conscious dog bowel motility. Three dogs with intralumenal catheters in the antrum, douden and pylor are used as test animals. Pressure changes in the lumen of the intestine are recorded in a Visicorder using voltage meters and miniature light beam galvanometers. After the steady-state has been established, the test compound is infused intravenously over 10 minutes. The test compound initially raises and then decreases the intralumenal pressure in the pylorus, while the pressure in the duodenum and anthra remains reduced during the test. The lowest effective dose required to increase pyloric pressure and reduce duodenal and anthraous pressure is about 0.05 Aig / kg.10 min for D-Val ', D-Trp ⁸ -somatostatin and about 0.1 µg / kg.10 min for D-Ala ', D-Trp ⁸ -somatostatin. In comparison, the same value for somatostatin alone is 0.125 to 0.25 Aig / kg. 10 min.

D-Val, D-Trp ⁸ -somatostain and D-Ala 4, D-Trp®-somatostatin were also tested for their efficacy for growth hormone release. The procedure was carried out using normal male Sprague-Dowley rats weighing 100-120 g (Laboratory Supply Company, Indianapolis, Indiana). The assay is a modification of the method of P. Brazeau, W. Vale and R. Guilleman, Endocrinologists, 94: 184 (1974). A total of five groups of eight rats were used in the test to test each compound. Sodium pentobarbital is intraperitoneally administered intraperitoneally to stimulate growth hormone secretion. One group serves as a control and receives only a salt solution. Animals from the two groups are s.c. administered somatostatin subcutaneously to animals from one group at 2 / tg / animal and animals from the other group at 50 / tg / animal. from one group at a dose of 10 µg / animal and animals from the other group at a dose of 0.4 µg / animal. Serum growth hormone concentrations are measured 20 minutes after the simultaneous administration of sodium pentobarbital and the test compound. The degree of inhibition of serum growth hormone concentration relative to the control group and the relative potency of the test compounds relative to somatostatin alone are determined.

At a dose of 0.4 µg / animal and 1.0 µg / animal, D-Val ', D-Trp®-somatostatin inhibits the increase in growth hormone secretion by 14 and 42% compared to the control group. Somatostatin does not

202097 1.6 at a dose of 2 µg / rat no effect on increasing growth hormone secretion, whereas at a dose of 50 µg / rat it inhibits the increase in growth hormone secretion by 56% compared to the control group.

At a dose of 0.4 µg / rat and 10 µg / rat D-Ala 1, D-Trp ⁸ -somatostatin inhibits the increase in growth hormone secretion by 54 and 91 µl, respectively, compared to the control group. Somatostatin at a dose of 2 µg / rat and 50 µg / rat inhibited the increase in growth hormone secretion by 40 and 87% compared to the control group.

D-Val ', D-Trp ⁸ -somatostatin and D-Ala', D-Trp ⁸ -somatostatin have also been tested for their in vivo potency to inhibit glucagon and insulin secretion after stimulation with L-Alanine. Ordinary non-blooded dogs of both sexes are left overnight without food. Blood samples are taken and then intravenous infusion of saline containing somatostatin or test compound is started. In addition, L-alanine is administered intravenously over a period of 15 minutes. The saline solution containing somatostatin or the test compound is infused for 15 minutes after the end of the L-alanine infusion. Infusion of L-alanine causes a sudden increase in serum glucagon and insulin levels, which returns to control concentrations after the end of the β-alanine infusion. This procedure revealed that the minimum dose of D-Val ', D-Trp ⁸ -somatostatin for inhibiting glucagon secretion is 0.04 to 0.11 µg / kg / min and for inhibiting insulin secretion is below 0.004 µg / kg / min. min. The minimum dose of D-Ala ', D-Trp ⁸ -somatostatin inhibiting both glucagon and insulin secretion is less than 0.03 zig / kg / min.

Claims (5)

SUBJECT OF THE INVENTION A process for the preparation of somatostatin analogs of general formula I Y-Gly-L-Cy-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- cys-OH (I), wherein Y is D-Val or D-Ala, and pharmaceutically acceptable non-toxic acid addition salts thereof, characterized in that the corresponding straight-chain tetradecapeptide of formula III Y-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- Cys-OH (III), wherein Y is as defined above, acts with an oxidizing agent. 2. The process of item 1 for preparing a compound of formula HD-Val-Gly-L-Cy-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser- L-cys-OH and pharmaceutically acceptable non-toxic acid addition salts thereof, characterized in that the corresponding straight-chain tetradecapeptide of the formula HD-Val-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phr-L-Thr-L-Ser- L-Cys-OH acts with an oxidizing agent. 3. A process according to claim 2, wherein the oxidizing agent is air. 4. The process of item 1 for the preparation of a compound of formula HD-Ala-Gly-L-cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L- Phe-L-Thr-L-Ser-L-Cys-OH and pharmaceutically acceptable non-toxic acid addition salts thereof, characterized in that the corresponding straight-chain tetradecapeptide of the formula HD-Ala-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser- 1 H-Cys-OH acts with an oxidizing agent. 5. A process according to claim 3, characterized in that air is used as the oxidizing agent.