CH633524A5 - Somatostatin analogs and their preparation - Google Patents

Description

The present invention relates to peptide derivatives of somatostatin, to their methods of preparation, to pharmaceutical compositions containing them, as well as to products used in their preparation.

We know that the factor inhibiting the release of somatotropin (SRIF), of cyclic type, known under the name of somatostatin, has, according to Brazeau et al., "Science", 179,77 (1973), the following structure:

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

S S (A)

all amino acids being of the natural configuration or L.

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Several methods of synthesis of somatostatin have been described in the literature, in particular the solid phase method of Rivier, "J. Am. Chem. Soc. ", 96.2986 (1974), and the solution methods of Sarantakis et al.," Biochemical Biophysical Research Communica-10 tions ", 54.234 (1973), and Immer et al.," Helv. Chim. Acta ”, 57, 730 (1974). There is much research in progress on peptides, the aim of which is to improve the pharmacological activity of somatostatin by modifying its structure synthetically.

The reduced form (open chain form) of somatostatin is (RS) is the linear tetradecapeptide of the formula:

SH SH (B)

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

20 The reduced form (B) was prepared by total synthesis [see Rivier et al., “C. R. Acad. Sci. Ser. p. Sei. Natur. " (Paris), 276, 2737 (1973), and Sarantakis and McKinley, "Biochem. and Biophys. Res. Communications ", 54, 234 (1973)] and this form can be converted to somatostatin (A) by oxidation, so that a bridging bond is formed between the two sulfhydryls of the two amino acid fragments of cysteinyl in the tetradecapeptide.

Various polypeptides which can be considered to be structural modifications of somatostatin have been synthetically prepared, and these preparations have been described in the chemical literature. Such polypeptides have certain structural characteristics in common with somatostatin and differ from it in that specific functional groups or amino acid fragments, originally present in the somatostatin molecule, are either missing or replaced by d other functional groups or amino acid moieties. The present invention relates to new synthetic biologically active polypeptides which can be considered as a structural modification of somatostatin. The polypeptides of the invention differ from somatostatin in the following reports:

io a) the Ala1-Gly1 segment is present, missing or replaced by Gly-Gly, Gly-Gly-Gly, Ala-D-Ala, acetyl, or benzoyl;

b) is replaced by Arg or His;

c) the remainder Asn5 is replaced by His, Glu, or Asp, and d) the remainder Trp8 is either present or replaced by D-Trp or 6-45 F-D-Trp.

All amino acid fragments and optically active amino acids are of the L configuration unless otherwise indicated.

Consequently, the present invention provides therapeutically active chemical compounds corresponding to formula I:

50

X-Cys-Xi-X2-Phe-Phe-X3-Lys-Thr-Phe-Thr-Ser-X4-OH (I)

I I

SA SA

in which A represents hydrogen or the two symbols A 55 signify a direct bond between the sulfur atoms; X represents hydrogen, Ala-Gly, Gly-Gly, Ala-D-Ala, Gly-Gly-Gly, acetyl or benzoyl; Xi represents His or Arg; X2 represents His, Glu or Asp, X3 represents Trp, D-Trp or 6-F-D-Trp, and X4 represents Cys or D-Cys, as well as the pharmacologically acceptable salts of the above compounds.

In addition, the invention contemplates the linear form of the compounds of formula I, that is to say the non-cyclic reduced compounds of formula la below, which contain two free sulfhydryl groups, as well as the addition salts. pharmaceutically acceptable acids of these compounds.

SH SH

I I

X-Cys-Xj-X2-Phe-Phe-X3-Lys-Thr-Phe-Thr-Ser-X4-OH (la)

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The symbols identifying the amino acids and the amino acid fragments in the polypeptides described in this case are the symbols which have been adopted by the IUPAC-IUB Committee on Biochemical Nomenclature Recommendation (1971), and have been described in "Archives of Biochemistry and Biophysics" , 150, 1-8 (1972). The symbol 6-F-D-Trp denotes D-tryptophan, where the 6 position is substituted by fluorine.

The tangible materials obtained according to the invention have the inherent physical properties of being white to light-colored solids, which are essentially insoluble in chloroform, benzene, etc., but which show solubility in water and in aqueous acid solutions, for example hydrochloric acid or acetic acid solutions. The compositions according to the invention do not show clearly distinguishable melting points and they can be purified, for example by chromatographic means. Hydrolysis of the compositions according to the invention, for example in 4N methanesulfonic acid, allows the determination of their amino acid content, which is compatible with the structures as defined above.

The tangible materials obtained according to the invention exhibit pharmaceutical activity, in particular the characteristic of being able in practice to inhibit the release of one or more of the hormones formed by somatotropin, glucagon, insulin, which is demonstrated by conventional pharmacological test methods. By controlling the dosage, the action selectivity allows the inhibition of the release of somatotropin and glucagon, without influencing the endogenous insulin secretion rates.

In addition, tangible materials obtained according to the invention can be used in admixture with insulin for the treatment of warm-blooded animals suffering from diabetes mellitus.

The invention also encompasses, according to a more particular aspect, the chemical compounds of formula I, in which X is hydrogen, X3 is preferably D-Trp and X4 is preferably Cys.

According to a more particular aspect, the invention encompasses:

a) a compound of formula I, in which X represents hydrogen, Ala-Gly, Ala-D-Ala, Gly-Gly or Gly-Gly-Gly; Xt is His; X2 is His; X3 is Trp or D-Trp, and X4 is Cys.

b) a compound of formula I, in which X represents hydrogen, Ala-Gly, Ala-D-Ala, Gly-Gly or Gly-Gly-Gly; Xj is Arg; X2 is His; X3 is Trp or D-Trp, and X4 is Cys or D-Cys.

c) a compound of formula I, in which X represents hydrogen, Xj is His, X2 is His, X3 is Trp or D-Trp, and X4 is Cys or D-Cys.

d) a compound of formula I, in which X represents hydrogen, Ala-Gly, Gly-Gly-Gly, Ala-D-Ala, acetyl or benzoyl;

Xj is Arg or His;

X2 is Glu or Asp;

X3 is Trp or D-Trp, or 6-F-D-Trp, and

X4 is Cys or D-Cys;

X! and X2 are preferably both His, or else Xj represents Arg and X2 represents Glu. Very particularly preferred compounds are:

H-Cys-Arg-His-Phe-Phe-D-Trp-Lys

I

S S

I

Thr-Phe-Thr-Ser- Cys-OH,

H-Cys-His-His-Phe-Phe-T rp-Lys-

Thr-Phe-Thr-Ser-Cys-OH,

H-Cys-His-His-Phe-Phe-D-Trp-Lys I

S S

I

Thr-Phe-Thr-Ser-Cys-OH,

H-Cys-His-His-Phe-Phe-D-T rp-Ly s-

I

S S

I

Thr-Phe-Thr-Ser-D-Cys-OH H-Cys-Arg-Glu-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys

I I

S S

and H-Cys-His-Glu-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys

I I

S S

as well as their reduced forms (open chain).

The invention also contemplates protected intermediates for the compounds of formula I, in particular compounds corresponding to the formula:

R1-Cys-B1-B2-Phe-Phe-X3-Lys-Thr-Phe-Thr-Ser-X4-OR

! I I I I I

Sa R6 R7 R8 R9 Sß (II)

in which R is a carboxyl protecting group (for example an alkyl, an aralkoyl) or a polystyrene resin support, linked to the intervention of a methylene group, that is to say that in this case , R represents - CH2 - polystyrene resin support; R1 represents an α-amino protecting group or an Ala-Gly, Gly-Gly, Ala-D-Ala or Gly-Gly-Gly group protected in α-amino, or alternatively acetyl or benzoyl;

R2 R3

I I

Bj represents His or Arg, where R3 is a protective group for the side chain nitrogen atoms of arginine and R2 is hydrogen or a protective group for the imidazole nitrogen atom of histidine;

B2 represents His as defined above, Glu or Asp, where R4 and Rs

I I I

R2 R4 Rs are respectively protective groups for the side-chain carboxy groups of glutamic acid and aspartic acid; R6 is a protecting group for the side chain amino group of lysine; R7, R8 and R9 are each protective groups for the hydroxyl groups of threonine and serine; X3 represents Trp, D-Trp or 6-F-D-Trp; X4 represents Cys or D-Cys, and a and ß each represent a protecting group for sulfhydryl or hydrogen, or a and ß represent a direct bond between the sulfur atoms.

Examples of α-amino protecting groups for R1 or as part of R1 are: 1) protecting groups of the acyl type, illustrated by formyl, trifluoroacetyl, phtalyl, p-toluenesulfonyl (tosyl), nitrophenylsulfenyl, etc. ; 2) protective groups of the aromatic urethane type, illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyls, in particular p-chlorobenzyloxycarbonyl and p-nitrobenzyloxycarbonyl; 3) protective groups of the aliphatic urethane type, illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycar-bonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, amyloxy-carbonyl; 4) protective groups of the cycloalkyl urethane type, illustrated by cyclopentyloxycarbonyl, adamantylcarbonyl, cyclohexyloxycarbonyl; 5) protective groups of the thio-urethane type, such as phenylthiocarbonyl; 6) alkyl-type protecting groups, as illustrated by triphenylmethyl (trityl); 7) trialkylsilane groups, for example trimethyl-silane. The preferred α-amino protecting group is tert-butyloxycarbonyl.

Examples of R3 are nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl, with preference for tosyl.

Protection via the nitro or tosyle group takes place on one or other of the nitrogen atoms N ™ or Nral of arginine, while the

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protecting groups of the oxycarbonyl type protect the N8 atom and one or the other of the nitrogen atoms Nra or NMl.

Examples of R2 are tosyl, benzyloxycarbonyl, adamanty-loxycarbonyl and tert-butyloxycarbonyl, preferably the tosyl group.

The lysine side chain amino group is protected by Rs which can be exemplified by tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl, nitrobenzyloxycarbonyl, etc., the 2-chlorobenzyloxycarbonyl group being preferred.

Examples of protective groups R7, R8 and R9 for the hydroxyl group of threonine and serine are acetyl, benzoyl, tertiary butyl and benzyl, with preference for the latter.

Examples of R4 and R5 are benzyl and t-butyl.

Examples of the protective groups a and ß for the sulfhydryl group of the amino acid fragment of cysteine are benzyl; substituted benzyles in which the substituent consists of at least one of the methyl, methoxy, nitro or halo radicals (for example 3,4-dimethylbenzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, etc.), trityl , benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyrannyl, acetamidomethyl, benzoyl, s-sulfonic salt, with a preference for the p-methoxybenzyl group.

The compounds of formula I can be prepared by removing all the protective groups and the polystyrene resin support when there is one, starting from a compound of formula II as defined above, then with, if desire, oxidation or reduction of the product to give the cyclic or open chain form and, if desired, isolation in the form of the free base or in the form of a salt acceptable in pharmacology.

The separation of the protective groups can be carried out by methods known in practice for the protective groups in question. The protecting groups are preferably removed during a single phase, for example using hydrofluoric acid in the presence of anisole.

The polystyrene resin support can be separated at the same time as the removal of the protective groups, for example using hydrofluoric acid in the presence of anisole, or it can be split by transesterification to give a protected intermediate on the carboxyl of formula II, in which R is an ester function. By way of example, a methanolysis gives a methyl ester intermediate of formula II, in which R represents methyl. The hydrolysis of these esters gives the required C-terminal carboxyl function.

By appropriate choice of protecting groups, the protecting groups a and ß can be selectively separated from the compounds of formula II to give the free disulfhydryl derivatives of formula II, in which a and ß represent hydrogen. For example, when a and ß are a trityl or acetamido radical, they can be selectively separated by the action of a mercuric salt or a silver salt to obtain the corresponding disulfhydryl derivative in the form of its corresponding mercuric or silver salt. The latter salt can be subjected to the action of hydrogen sulfide to obtain the corresponding free disulfhydryl derivative of formula II.

The free disulfhydryl derivatives of formulas I and II can each be cyclized by oxidation using an oxidizing agent, for example iodine, oxygen (such as air), 1,2-diiodoethane or sodium ferricyanide or potassium, to obtain the corresponding compound of formula I, in which the two groups A form a direct bond, and of formula II, in which a and β form a direct bond.

The final polypeptide products and their necessary intermediates can be prepared by the well known solid phase method, as described, for example, by Merrifield, "J. Am. Chem. Soc. ”, 85, 2149 (1963), or by a classic synthesis. The present invention therefore therefore also provides a process for the preparation of a compound of formula II, in which a and ß are protective groups and R represents a polystyrene resin support, this process comprising the sequential combination of suitably protected amino acids and / or activated required under solid phase synthesis conditions to give a support of 5-chloro-thylated or hydroxymethylated resin. Alternatively, the compounds of formula II, in which a and ß are protecting groups and R is a carboxyl protecting group, can be prepared by combining the required suitably protected and / or activated amino acids or amino acid groups of this kind by known methods for the formation of peptide bonds to obtain the desired sequence of amino acids. The protecting groups a and ß can then be separated selectively and the product can be oxidized to form the cyclic disulfide of formula II, in which a and ß form a direct bond.

Methods of activating amino acids prior to combination, and the combination methods themselves are well known in the art and reference may be made in particular to the manual of Schroeder and Lubke mentioned above.

In the choice of a particular side chain protecting group to be used in the synthesis of the peptides according to the invention, the following rules should be followed: a) the side chain protecting group must be stable with respect to the reagent and under the reaction conditions chosen for the separation of the α-amino protecting group at each stage of the synthesis; b) the protective group must retain its protective properties (i.e. not separate under the conditions of combination), and c) the side chain protective group must be separable at the end of the synthesis comprising the desired sequence of amino acids under reaction conditions which will not alter the peptide chain.

In the conventional method as applied to the compounds according to the present invention, the desired peptide is constructed by condensation of amino acids or groups of amino acids which are protected as necessary. Condensation reactions can be carried out using methods generally for the formation of amide bonds in peptide and penicillin chemistry. To promote easy condensation of the amino acids, it is preferable to use a condensing agent. Examples of condensing agents are carbodiimides, for example N, N'-dicyclohexylcarbodiimide (DCQ, N, N '-. 40 diisopropylcarbodiimide. Alternatively, condensation can be achieved by activating one of the groups or both. Examples of the activated form of the terminal carboxyl are acid chloride, acid anhydride, acid azide and activated ester. It will be apparent to those skilled in the art that the proposed process for carrying out the condensation reactions must be compatible with the protective groups existing on the amino acids.

In the construction of the molecule by the conventional method, it is preferable to condense a protected peptide of the formula:

50 R1-Cys-B1-B2-Phe-OH

I

Sa (Via)

with a protected peptide of formula: s, H-Phe-X3-Lys-Thr-Phe-Thr-Ser-X4-OR

II III

R6 R7 R8 R9 Sß

wherein R is a carboxyl protecting group (preferably benzyl), R1, R6, R7, R8, R9, Blt B2, X3 and X4 are as defined above, and a and ß are protecting groups, preferably p-methoxybenzyle, in order to obtain a compound of formula II which is stripped of its protection and cyclized.

In the solid phase process, as applied to the compounds according to the present invention, the cysteine protected in a-amino and in 65 sulfhydryl is first attached to a chloro-methylated polystyrene resin, with then separation of the protective group d a-amino using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or HCl in dioxane. The

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removal of protection is carried out at a temperature between approximately 0 ° C and room temperature. Other conventional reagents and separation conditions for the removal of α-amino protecting groups can be used as described by Schroeder and Lubke, "The Peptides", 1, 72-75 (Academic Press, 1965). After removal of the α-amino protecting group, the following desired protected amino acids are individually combined with the sequence supported on the resin, and this in series. Alternatively, small peptide fragments can be prepared, for example by the solution process, and introduced into the solid phase reaction in the desired order. Each protected amino acid or each protected amino acid sequence is introduced into the solid phase reaction in an excess of about four times. The combination is carried out in dimethylformamide, methylene chloride or a mixture of these two solvents. The success of each combination reaction at each stage of the synthesis is determined by the ninhydrin reaction, as described by E. Kaiser et al., "Analyt. Biochem. ”, 34, 595 (1970). When an incomplete combination has occurred, the reaction is repeated before the α-amino protecting group is separated for the introduction of the next amino acid or the next sequence of amino acids.

Preferred combination reagents are 1-hydroxybenzotriazole and diisopropylcarbodiimide, but other useful reagents are known to those skilled in the art.

After the desired amino acid sequence has been synthesized, the polypeptide is separated from the resin support by treatment, for example with hydrofluoric acid and anisole, to obtain the linear polypeptide completely stripped of its protections. Cyclic disulfide can be produced by air oxidation or, for example, by oxidation with K3Fe (CN) 6.

The non-toxic addition salts of the linear and cyclic polypeptides are produced by methods well known in practice starting from hydrochloric, hydrobromic, sulfuric, phos-phoric, polyphosphoric, maleic, acetic, citric acids,

benzoic, succinic, malonic, ascorbic, etc., the acetic acid salt being preferred.

The protecting groups used throughout the solid phase synthesis are well known in the art. The protective groups of α-amino, used with each amino acid introduced into the sequence of the final polypeptide, are: 1) protective groups of the acyl type, illustrated by formyl, trifluoroacetyl, phtalyl, p-toluenesulfonyl (tosyl), nitrophenylsulfenyl, etc. . 2) protective groups of the aromatic urethane type, illustrated by benzyloxycarbonyl and substituted benzyloxycarbonyls, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; 3) protective groups of the aliphatic urethane type, illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycar-bonyl, allyloxycarbonyl; 4) protective groups of the cycloalkyl urethane type, illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; 5) protective groups of the thio-urethane type, such as phenylthiocarbonyl; 6) alkyl-type protecting groups, as illustrated by triphenylmethyl (trityl); 7) trialkylsilane groups, for example trimethylsilane. The preferred α-amino protecting group is tert-butyloxycarbonyl.

The histidine imidazole nitrogen atom, designated by Nim, is protected by a group which may be tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl, preferably the tosyl group.

Protection for the lysine side chain amino group can be provided by a group which may be tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl, nitrobenzyloxycarbonyl, etc., preferably the 2-chlorobenzyloxycarbonyl group.

The nitrogen atoms of the arginine side chain, designated by Ns, are protected by a group which may be nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl or tert-butyloxycarbonyl, preferably tosyl. Protection via the nitro or tosyle group is done on one or other of the nitrogen atoms N <° or N ™ 1, while protective groups of the oxycarbonyl type protect the atom N8 and one or the other nitrogen atoms N® or Noi.

5 Protection for the hydroxyl group and threonine and serine can be done by acetyl, benzoyl, tertiary butyl, benzyl. The benzyl group is preferred for this purpose.

The protecting group for the sulfhydryl group of the amino acid fragment of cysteinyl is a group chosen from class 10 comprising benzyl and substituted benzyles, in which the substituent consists of at least one of the following radicals: methyl, methoxy, nitro, or halo (e.g. 3,4-dimethylbenzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, etc.), trityl, benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, is benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, methyl, tetrahydropyranyl, s-sulfonic salt, etc., with p-methoxybenzyl being preferred.

The compositions according to the present invention, like somatostatin itself, can exist either in the open chain monomeric form (called reduced form), or in the cyclic monomeric form (called oxidized form). Each of these forms can be produced using a process essentially identical to that used to obtain the corresponding form of somatostatin itself. These methods are known to specialists in this field. The reduced form is represented here by the structure in which A is hydrogen; in this way, there are free thiol substituents on the two amino acid fragments of Cys. The oxidized form is represented here by the same structure when the two groups A represent a direct bond, that is to say that there is a single bond between the sulfur atoms carried by the two amino acid fragments of Cys, so that a monomer ring is formed.

In addition, the compounds can exist in the so-called reduced polymer form (see for example United States patent No. 3926937), this form being obtainable by the process described in practice for obtaining a reduced polymer somatostatin. This polymer form can be illustrated by the formula:

- (H>

M

S

I

X-L-Cys-Xi-X2-L-Phe-L-Phe-X3-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L- X4-OH

(II)

- (H>

M

NOT

in which X, X1 and X2 have the definition given for the formula I, M is 0 or 1, and N is an integer from 2 to 100 50 inclusive. In the structure in parentheses, when X is different from H, the sulfur-sulfur bonds are formed at random between Cys3-Cys3, Cys3-Cys14, and Cys14-Cys14 (where X denotes H, Cys1 instead of Cys3, etc.) . The structure is cyclic when M is equal to 1, that is to say when the compound does not contain any free SH group and there is a bond between the sulfur atoms carried by the Cys terminal fragments. For the purposes of the present invention, the reduced polymer forms are, from a qualitative point of view, entirely equivalent to the compounds claimed more particularly.

The pharmacological activity of the peptides according to the present invention characterizes the compounds as being useful in the treatment of acromegaly and diabetes in the same way as in the case of somatostatin itself. The administration of the peptides can be done by the usual conventional routes for somatostatin and related polypeptides, as directed by a physician, and in an amount dictated by the degree of dysfunction determined by the physician. The compounds can be administered alone or in combination with conventional carriers and adjuvants, acceptable in pharmacies, in the form of unit doses.

633,524

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As previously indicated, the compositions according to the present invention are also advantageous in admixture with insulin for the treatment of warm-blooded animals suffering from diabetes mellitus. Reference may be made, for example, to U.S. Patent No. 3,912,807, which provides for the use of an effective amount of a composition comprising somatostatin in admixture with insulin for the treatment of warm-blooded animals with diabetes mellitus.

In therapeutic use as agents for the treatment of acromegaly, juvenile diabetes and diabetes mellitus, treatment is initiated with small doses which are less than the optimum dose of the compound. Then the dose is increased in small amounts until the optimal effect is achieved under the circumstances involved. In general, the compounds of the invention are administered at a dosage rate which will generally give effective results without causing harmful or unpleasant side effects. The doses can however be modified according to the requirements relating to the patient and according to the compound which is used. For convenience, the total daily dose can be subdivided and administered in portions over the day, as desired.

Accordingly, the invention further encompasses pharmaceutical compositions comprising a compound of formula I or a pharmacologically acceptable salt thereof, in association with a pharmacologically acceptable carrier or vehicle. These pharmaceutical compositions are preferably in the form of unit doses.

The following examples will illustrate the present invention even more completely without limiting the scope thereof.

Example 1:

tert-Butyloxycarbonyl-Sp-Methoxybenzyl-L-Cysteinyl-Nim-Tosyl-L-Histidyl-Nim-Tosyl-L-Histidyl-L-Phénylalanyl-L-Phénylalanyl-D-Tryptophyl-N £ -2-Chlorobenzyloxycarbonyl-L-Lysyl -0-Benzyl-L-Thréonyl-L-Phénylalanyl-O-Benzyl-L-Thréonyl-O-Benzyl-L-Séryl-Sp-Méthoxybenzyl-L-Cystéinyl-Hydroxyméthylpolystyène, ester

A chloromethylated polystyrene resin (Lab Systems, Inc.), crosslinked at 1% with divinylbenzene, is esterified with BOC-Cys- (SMBzl) OH according to Gisin, "Helv. Chim. Acta ”, 56.1976 (1973). The polystyrene resin ester is treated according to program A for the incorporation of BOC-Ser (Bzl) OH, BOC-Thr (Bzl) OH, BOC-Phe-OH, BOC-Thr (Bzl) OH, BOC- Lys (Clz) OH, BOC-D-Trp-OH, BOC-Phe-OH, BOC-Phe-OH, BOC-His (Tos) OH, BOC-His (Tos) OH, and BOC-Cys (SMBzl) OH , in order to obtain the peptidore-sine mentioned in the heading.

Program A:

1.wash with CH2C12 x 3;

2. treatment with TFA-CH2C12-EDT (1/1/5%, v / v) for 5 min;

3. treatment as following 2 for 25 min;

4. washing with CH2C12 x 3;

5. washing with DMF;

6. treatment with 12% TEA in DMF, twice, for 3 min;

7. washing with DMF;

8. washing three times with CH2C12;

9. treatment with 4 equivalents of the corresponding amino acid derivative in CH2Cl2-DMF and stirring for 5 min;

10. addition in two portions of 5 equivalents of DIC dissolved in CH2Cl2 over a period of 30 min, reaction time of 6 h;

11. washing with DMF x 3;

12. washing with CH2C12 x 3;

13. Ninhydrin test reaction, according to Kaiser et al., "Annal. Biochem. ", 34,595 (1970).

In the case of an incomplete reaction, the operating phases 9 to 13 are repeated above.

Example 2:

Cyclic disulfide (1-12) of L-Cysteinyl-L-Histidyl-L-Histidyl-L-

Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-

Phenylalanyl-L-Threonyl-L-Sêryl-L-Cysteine

The peptidoresin of Example 1 (9 g) is mixed with 18 mg of anisole and treated with liquid HF (180 ml) for 45 min in an ice bath. The excess HF is removed in vacuo as quickly as possible (about 1 h) and the remainder is extracted with 2M glacial acetic acid, then filtered. The filtrate is washed with ether and the aqueous layer is oxidized with K3Fe (CN) 6 at a pH of 7. The excess oxidant is separated by an ion-exchange resin Bio-Rad AG-3, then the peptide is absorbed on a Bio-Rex 70 ion exchange resin and eluted with a pyridine buffer of pH 7 to obtain, after lyophilization, 1.5 g of material which is applied to a column of Sephadex G15 ( 2.5 x 160 cm) and which is eluted with 15% aqueous acetic acid. The fractions (5.2 ml each) of tubes 88-90 are combined and lyophilized to give the compound mentioned in the heading, 174 mg.

Rf (BWA, 4/1/1) 0.45 Rf (BWAP, 30/24/6/20) 0.64

Amino acid analysis:

Thr (2): 1.87 Ser (l): 0.89 Cys (2): 1.16 Phe (3): 3 Lys (l): 1.05 His (2): 1.76 Trp: not determined .

Example 3:

The in vivo pharmacological activity of the dodecapeptide prepared according to Example 2 was established by the following methods with the results indicated.

Suppression of growth hormone

A subcutaneous injection of peptide solubilized or suspended in physiological saline solution is made to male Charles River CD rats, which are not fasted. Corresponding rats which have undergone a subcutaneous injection of a control saline solution serve as control animals so that each experimental rat is accompanied by a control rat. The rats are kept in separate cages and, 20 min before the end of the test period, they receive an injection into the peritoneum of Nembutal at a dose of 50 mg / kg. Blood samples are obtained by a cardiac puncture and the plasma is separated for radioimmunological determination of the growth hormone (GH) concentration in ng / ml. Periods of 2 and 4 h after injection are provided to verify the duration of the activity of the peptide in suppressing peripheral GH levels in circulation. Comparisons between the GH values obtained in the control rats and in the experimental rats at each of the times indicated are estimated by Student's test method t and the statistical significance p at the rate of 0.05 or at a lower rate is used as an activity index.

Compound (dose)

2h

4h

Witness

Example 2 (1 mg / kg)

118 ± 20 32 ± 6 p = <0.01

115 ± 23 62 ± 13 p = <0.01

Suppression of growth hormone, glucagon and insulin Male albino rats are divided into three groups (9 rats per group) and injected into the peritoneum of Nembutal at a rate of 50 mg / kg. 15 min after the injection of Nembutal, the following are injected subcutaneously according to the group: a) the compound tested, normally at a rate of 10-2000 ng / kg; b) SRIF at 200 µg / kg, or c) physiological saline. 10 min later, 0.5 ml of arginine (300 mg / ml, pH 7.2) is injected into the heart. The rats are decapitated 5 min after receiving the arginine and their blood is collected in Trasylol-EDTA. Appropriate small amounts are then determined for growth hormone, glucagon and insulin. An active compound is a compound that

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significantly alters the plasma level of any of these hormones compared to control animals treated with saline. Comparisons between control and experimental values are estimated statistically by analysis of variance, and statistical significance p at the rate of 0.05 or less is used as an activity index.

Compound (dose | ig / kg)

GH (ng / ml)

Insulin (| iU / ml)

Glucagon (Pg / ml)

Witness

Example 2 (100)

163 ± 29 20 ± 8 p <<0.01

278 ± 32 202 + 49 p => 0.05

69 ± 13 20 ± 8 p = <0.01

Example 4:

tert-Butyloxycarbonyl-Sp-Methoxybenzyl-L-Cysteinyl-NS "-Tosyl-L-Arginyl-N'm-Tosyl-L-Histidyl-L-Phénylalanyl-L-Phénylalanyl-D-Tryptophyl-N £ -2-Chlorobenzyloxycarbonyl- L-Lysyl-0-Benzyl-L-Threonyl-L-Phenylalanyl-O-Benzyl-L-Threonyl-O-Benzyl-L-Séryl-Sp-Métoxyoxy-benzyl-L-Cystéinyl-Hydroxymétylpolystyène, ester

A chloromethylated polystyrene resin (Lab Systems, Inc.), crosslinked at 1% with divinylbenzene, is esterified with Boc-Cys (SMBzl) -OH according to Gisin, "Helv. Chim. Acta ”, 56.1976 (1973). The polystyrene resin ester is treated according to program A below for the incorporation of Boc-Ser (Bzl) -OH, Boc-Thr (Bzl) -OH, Boc-Phe-OH, Boc-Thr (Bzl ) -OH, Boc-Lys (ClZ) -OH, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-His (Tos) -OH, Boc-Arg (Tos) -OH and Boc-Cys (SMBzl) -OH, in order to obtain the peptidoresin cited in the heading.

Program A:

1. washing three times with CH2 Cl2;

2. treated with TFA-CH2C12-EDT (1/1/5%, v / v) for 5 min;

3. treat as follows 2 for 25 min;

4. washing with CH2C12 x 3;

5. washing with DMF;

6. treated with 12% TEA in DMF, twice for 3 min;

7. washing with DMF;

8. washing with CH2C12 x 3;

9. treating with 4 equivalents of the corresponding amino acid derivative in CH2Cl2-DMF and stirring for 5 min;

10. 5 equivalents of DIC dissolved in CH2Cl2 are added in two portions over a period of 30 min, reaction time of 6 h;

11. washing with DMF x 3;

12. washing with CH2C12 x 3;

13. a test is carried out by the reaction with ninhydrin according to Kaiser et al. "Annal. Biochem. ”, 34, 595 (1970).

In the case of an incomplete reaction, the operating phases 9 to 13 are repeated above.

Examples:

Cyclic disulfide (1-12) of L-Cysteinyl-L-Arginyl-L-Histidyl-L-

Phenylalaryl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-

Phenylalanyl-L-Threonyl-L-Sêryl-L-Cystèine

The peptidoresin of the previous example (8.5 g) is mixed with 16 ml of anisole and treated with liquid HF (100 ml) for 45 min. The excess HF is removed in vacuo as quickly as possible and the remainder is taken up in 25% aqueous acetic acid. The polymer support is separated by filtration and the filtrate is washed with ether. The aqueous layer is poured into 3.51 of water, the pH is adjusted with NH4.OH to 7, and the sulfhydryl compound is then oxidized with K3Fe (CN) 6. The pH is adjusted to 5 with glacial acetic acid and the inorganic oxidant is separated with Bio-Rad AG 3. The peptide material is absorbed on Bio-Rex 70 and eluted with a pyridine buffer of pH 7 to obtain the crude compound, 2 g. This material is passed through a column of SephadexG-15 (2.5 x 160 cm) and eluted with 15% aqueous AcOH. The material of fractions 47 to 93 (5.2 ml for each fraction) (886 mg) is applied to a column of CM-Sephadex G-25 and eluted with a gradient of progressive NH4OAc (NH40Ac 0.1 to 0.3 molar) to obtain the compound mentioned in the heading. This material is applied to a Sephadex LH 20 column (2.5 x 92 cm) and eluted with 10% aqueous AcOH. The compound cited in the heading emerges in the pure state in fractions 55 to 63 (4.1 ml each). Production: 159 mg.

Rf (BWA, 4/1/1): 0.46 Rf (BWAP, 30/24/6/20): 0.65

Amino acid analysis:

Thr (2): 1.94 Ser (1): 0.91 Cys (2): 1.79 Phe (3): 3 His (1): 1.02 Lys (1): 0.85 Arg (1) : 0.95 Trp: not determined.

Example 6:

The in vivo pharmacological activity of the compound prepared according to Example 5 was determined by the method according to the results indicated.

Suppression of growth hormone, glucagon and insulin Male albino rats are divided into two groups (9 rats per group) and injected into the peritoneum of Nembutal at a rate of 50 mg / kg. 15 min after the injection of Nembutal, the test compound is injected subcutaneously into one of the groups and a physiological saline solution into the other group. 10 min later, 0.5 ml of arginine (300 mg / ml, pH 7.2) is injected into the hearts of the rats. They are decapitated 5 min after receiving the arginine and the blood is collected in Trasylol-EDTA. Appropriate small amounts are subject to determination for growth hormone (GH), glucagon (GLUN) and insulin (INS). An active compound is one that significantly changes the plasma level of any of these hormones compared to rats treated with saline. Comparisons between control and experimental values are estimated statistically by analysis of variance, and statistical significance p is used at the rate of 0.05 or less as an activity index.

Results

Exped

Dose

GH

INS

GLUN

rience

(fig / kg)

(ng / ml)

(ONE / ml)

(Pg / ml)

1

257 + 49

177 ± 19

61 ± 9

100

61 + 5

52 + 16 *

0 + 0 *

2

• - •

131 ± 43

262 ± 26

42 +6

10

71 ± 21 *

238 ± 72

4 + 3 *

* p <0.01

Example 7:

tert-Butyloxycarbonyl-Sp-Methoxybenzyl-L-Cysteinyl-im-Tosyl-L-Histidyl-im-Tosyl-L-Histidyl-L-Phënylalanyl-L-Phênylalanyl-D-Tryptophyl-e-2-Chlorobentyloxycarbonyl-L-Lysyl- O-Benzyl-L-Thro-nyl-L-Phenylalanyl-O-Benzyl-L-Threonyl-O-Bentyl-L-Sêryl-Sp-M-thoxybenzyl-D-Cystenyl-Hydroxymethylpolystyrene, ester

A chloromethylated polystyrene resin (Lab Systems, Inc.), crosslinked at 1% with divinylbenzene, is esterified with Boc-D-Cys (SMBzl) OH according to Gisin, "Helv. Chim. Acta ”, 56.1976 (1973). The polystyrene resin ester is treated according to program A for the incorporation of Boc-Ser (Bzl) OH, Boc-Thr (Bzl) OH, Boc-Phe-OH, Boc-Thr (Bzl) OH, Boc- Lys (ClZ) OH, Boc-D-Trp-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-His (Tos) OH, Boc-His (Tos) OH and Boc-Cys (SMBzl) OH, in order to obtain the peptidoresin cited in the heading.

5

10

15

20

25

30

35

40

45

50

55

60

65

633,524

8

Example 8:

Cyclic disulfide (1-12) of L-Cysteinyl-L-Histidyl-Histidyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl- D-Cysteine, diacetate salt

The peptidoresin of the previous example (10 g) is mixed with 20 ml of anisole and treated with liquid HF (150 ml) in an ice bath for 45 min and excluding air. The excess of liquid HF is separated under vacuum and the rest is taken up in 50% aqueous AcOH. The mixture is filtered and the filtrate is diluted with water to 3.51. The pH is adjusted to 7 with dilute NaOH and the disulfhydryl peptide is oxidized with K3F (CN) 6 to obtain the cyclic disulfide. The pH of the mixture is adjusted to 5 with glacial AcOH and treated with Bio-Rad AG 3. The peptide material is absorbed on Bio-Rex 70 (H + form) and eluted with a pyridine buffer (Py-H20-AcOH , 30/66/4, v / v). The fractions containing the peptide material are combined and lyophilized to give 1.6 g of crude material. The latter is applied to a Sephadex LH-20 column (2.5 x 90 cm) and eluted with 10% aqueous AcOH (fractions of 5.8 ml each). The material which emerges in fractions 97-101 is combined and lyophilized to obtain the dodecapeptide mentioned in the heading, 171 mg.

Thin layer chromatography, glass plates previously coated with Avicel,

Rf (BWA, 4/1/1, v / v): 0.32 Rf (BWAP, 30/24/6/20, v / v): 0.56

Amino acid analysis:

Thr (2): 1.85 Ser (1): 0.84 Cys (2): 1.29 Phe (3): 3 Lys (1): 1.05 His (2): 1.41 Trp (1) : 0.75. The in vivo pharmacological activity of the dodecapeptide prepared according to Example 8 was established by the methods detailed at the end of Example 3. The results obtained are as follows.

Suppression of growth hormone

Compound (dose)

2h

4h

Witness

Example 8 (1 mg / kg) ** <0.001

127 ± 22 20 ± 3 **

59 + 20 107 ± 34

Suppression of growth hormone, glucagon and insulin

Compound

Insulin

Glucagon

(dose, ng / kg)

GH (ng / ml)

(nU / ml)

(Pg / ml)

Witness

279 ± 53

323 ± 30

42 + 6

Example 8 (100)

64 ± 20 *

165 ± 21 *

5 + 2 *

Witness

201 ± 35

397 ± 36

117 + 11

Example 8 (100)

110 ± 22+

318 ± 54

70 ± 7 *

* p <0.01, + p <0.05

From the above, it can be seen that, at low doses, the peptide of Example 8 is specific for the suppression of glucagon and growth hormone, without influencing the secretion of insulin.

Example 9:

tert-Butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-Ns "-tosyl-L-arginyl-y-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-trypto-phyl-Nt-2-chlorobenzyloxycarbonyl-L -Lysyl-0-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-Sp-methoxyben-zyl-L-cysteine-Hydroxymethylpolystyrene, ester

A chloromethylated polystyrene resin is esterified with Boc-Cys (SMBzl) -OH, according to the method of Gisin, "Helv. Chim. Acta ”, 56.1976 (1973). The resin / amino acid ester is then treated according to program A as developed below, Boc-Ser (Bzl) -OH being used as protected amino acid in phase 9

of this program. This program A is then repeated in order to incorporate in succession the following amino acids in the peptidoresin:

Boc-Thr (Bzl) -OH

Boc-Phe-OH

Boc-Thr (Bzl) -OH

Boc-Lys (ClZ) -OH

Boc-D-Trp-OH

Boc-Phe-OH

Boc-Phe-OH

Boc-Glu (OBzl) -OH

Boc-Arg (Tos) -OH

Boc-Cys (SMBzl) -OH

Program A (for the treatment of resin ester):

1. washing with methylene chloride (CH2C12), three times;

2. treatment with trifluoroacetic acid / methylene chloride (1/1, v / v) containing 5% of 1,2-ethanedithiol, for 5 min;

3. repetition of phase 2 for 25 min;

4. washing with CH2Cl2, three times;

5. washing with dimethylformamide (DMF);

6. treatment with 12% triethylamine in DMF for 3 min;

7. washing with DMF;

8. washing with CH2C12, three times;

9. treatment with 4 equivalents of the appropriate protected amino acid in CH2C12-DMF and stirring for 5 min;

10. addition in two parts, over a period of 30 min, of

5 equivalents of diisopropylcarbodiimide dissolved in CH2Cl2, allowing the reaction to develop for 6 h;

11. washing with DMF, three times;

12. washing with CH2C12, three times;

13. test by reaction with ninhydrin according to the method of Kaiser et al., "Annal. Biochem. ", 34,595 (1970). In the case of an incomplete reaction, the preceding phases 9 to 13 are repeated.

Example 10:

Cyclic disulfide (1-12) of L-Cysteinyl-L-arginyl-L-glutamyl-L-

phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phê-

nylalanyl-L-threonyl-L-seryl-L-cysteine

The peptidoresin of Example 9 (8.5 g) is suspended in 16 ml of anisole and treated with anhydrous liquid hydrofluoric acid (HF) for 45 min in an ice bath, then the product is separated. excess HF under vacuum and the remainder is extracted with 10% aqueous acetic acid. The filtrate is washed with ether and the aqueous layer is poured into 51 of water, then the pH is brought to 7 with dilute ammonium hydroxide. The mixture is stirred in the open air for 48 h, then the pH is adjusted to 5 with glacial acetic acid, and the peptide is absorbed on Amberlite CG-50 (H + form). The peptide material is eluted with 50% aqueous acetic acid and lyophilized to give 850 mg of solid material.

The crude product is applied to a Sephadex LH-20 column (2.5 x 150 cm) and eluted with 10% acetic acid. Fractions 94-130 are combined and lyophilized to give 483 mg of material which is applied to a column of Sephadex G25 (1.5 x 115 cm) and which is eluted with 10% aqueous acetic acid. . Fractions 43-53 are pooled and lyophilized to obtain the above-mentioned peptide and lyophilized to obtain the above-mentioned peptide, 286 mg.

Thin layer chromatography, glass plates previously coated with Avicel,

Rf (BWA, 4/1/1, v / v): 0.40 Rf (BWAP, 30/24/6/20, v / v): 0.65

Amino acid analysis:

Thr (2): 1.92 Ser (1): 0.89 Glu (1): 1.03

Cys (2): 1.49 Phe (3): 3 Lys (1): 1.05 Trp (1): 0.81

Arg (1): 1.02.

5

10

15

20

25

30

35

40

45

50

55

60

65

9

633,524

Example 11:

tert-Butyloxycarbonyl-Sp-methoxybenzyl-L-cysteinyl-N'm-tosyl-L-histìdyl-y-benzyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryp-tophyl-Ne-2-chlorobenzyloxycarbonyl- L-lysyl-0-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-Benzyl-L-seryl-Sp-methoxyben- 5 zyl-L-cysteine Hydroxymethylpolystyrene, ester

A chloromethylated polystyrene resin is esterified with Boc-Cys (SMBzl) -OH, according to the method of Gisin, "Helv. Chim.

Acta ”, 56.1976 (1973). The amino acid resin ester is then treated according to program A (see example 9), Boc-Ser (Bzl) -OH 10 being used as protected amino acid in phase 9 of this program. This program A is repeated in order to successively incorporate the following amino acids into the peptidoresin:

Boc-Thr [Bzl) -OH Boc-Phe-OH Boc-Thr (Bzl) -OH Boc-Lys (ClZ) -OH Boc-D-Trp-OH Boc-Phe-OH Boc-Phe-OH Boc-Glu ( OBzl) -OH Boc-His (Tos) -OH Boc-Cys (SMBzl) -OH

Example 12:

Cyclic disulfide (1-12) of L-Cysteinyl-L-histidyl-L-glutamyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-phe-nylalanyl-L-threonyl- L-seryl-L-cysteine

The peptidoresin of Example 11 (8 g) is treated with anhydrous liquid HF as described in Example 10, and the linear disulfhydryl dodecapeptide is cyclized by oxidation with K3Fe (CN) 6 at pH 7.3 and with high dilution. The pH is brought to 5 with glacial acetic acid and the mixture is treated with ion-exchange resin Bio-Rad AG3-X4 (form Cl), then it is absorbed on Amberlite CG-50 (form H +). The peptide material is diluted with 30% aqueous acetic acid and lyophilized to give 500 mg of crude material. This is chromatographed on a column of Sephadex LH-20 to give the dodecapeptide mentioned in the heading.

Thin layer chromatography, glass plates previously coated with Avicel,

Rf (BWA, 4/1/1, v / v): 0.43 Rf (tert-AmOH /) y / W, 7/7/6, v / v): 0.74

Amino acid analysis:

Th (2): 1.92 Ser (1): 0.93 Glu (1): 0.94 Cys (2): 1.59 Phe (3): 3 Lys (l): 1.02 His (l) : 0.91 Trp (l): 0.81.

Example 13:

The biological activity of the peptides of Examples 10 and 12 was determined by the following method.

Male albino rats receive an injection of Nembutal into the peritoneum at a dose of 50 mg / kg. 15 min later, a subcutaneous injection of the test compound or physiological saline is administered. After 10 min, 0.5 ml of arginine (300 mg / ml, pH 7.2) is injected into the heart. 5 min after receiving the arginine, the rats are decapitated and the blood is collected in Trasylol-EDTA. A suitable small amount is tested for growth hormone, insulin and glucagon by radioimmunoassay. The results of this determination are as follows.

20

25

Compound

Dose

GH

Insulin

Glucagon

Number of animals

(Hg / kg)

(ng / kg)

(nU / ml)

(Pg / ml)

Witness

277 ± 69

301 ± 27

46 ± 8 *

10

Example 10

200

42 ± 6 *

115 ± 16 *

1.5 ± 1 *

10

Witness

-

216 ± 35

283 ± 28

55 ± 5

10

Example 10

40

28 ± 5 *

158 ± 57

12 ± 2 *

10

Witness

454 + 106

269 + 37

60 ± 10

10

Example 12

200

107 ± 29 *

205 ± 36

4 ± 2 *

10

Witness

233 + 35

343 ± 45

44 ± 4

10

Example 12

50

84 + 17 *

322 ± 40

10 ± 4 *

10

* p <0.01

The above results show that the peptides of Examples 10 and 12, representative of the other peptides according to the invention, are effective agents for reducing growth hormone and glucagon without strongly influencing the insulin levels at a dose of 40 mg / kg and 50 mg / kg respectively. The peptides of Examples 10 and 12 were also tested to determine the duration of their effect on the secretion of growth hormone in rats treated with Nembutal. The peptide of Example 10 showed inhibition 55

significant growth hormone for at least 4 h at a dose of 1000 mg / kg subcutaneously. The peptide of Example 12 did not show significant inhibition of growth hormone after 2 h and after 4 h.

The compounds described above can be administered to warm-blooded animals intravenously, subcutaneously, intramuscularly or orally, to control serum glucose in the treatment of diabetes. The dose required will vary with the particular condition being treated, the severity of the condition and the duration of treatment.

The active ingredient can be administered alone or in combination with pharmacy-acceptable vehicles or excipients. Appropriate pharmaceutical compositions will be apparent to those skilled in the art.

Example 14:

Classical synthesis of cyclic disulfide (1-12) of L-Cysteinyl-L-

Histidyl-L-Histidyl-L-Phênylalanyl-D-Tryptophyl-L-Lysyl-L-Thrëo-

nyl-L-Phenylalanyl-L-Threonyl-L-seryl-L-Cysteine a) N-Benzyloxycarbonyl-im-Benzyloxycarbonyl-L-Histidyl-L-Phëny-lalanyl, methyl ester (I)

A suspension of 42.3 g (0.1 mol) of Z-His (Z) -OH and 21.8 g (0.1 mol) of Phe-OMe-HCl (98%) in 1000 ml of CH2Cl2 is cooled to 0 ° C, then 22 g (0.12 mol) of DCC and 10.1 g (0.10 mol) of triethylamine are added. The mixture is allowed to reach room temperature overnight.

The separating dicyclohexylurea is removed by filtration and the filtrate is concentrated to give a gummy material. This material is dissolved in ethyl acetate, and the insoluble material is removed by filtration. EtOAc is washed with 5% citric acid, water, 5% ice-cold sodium bicarbonate solution and water. The organic layer is dried over sodium sulphate, concentrated to a third of its initial volume, then 500 ml of ether are added until the solution becomes cloudy, this solution then being left in a cold room overnight. A

50

633524

10

white solid which forms is separated and dried, 32 g, melting point 131-133 ° C; Thin layer chromatography: silica gel F-254, CH2Cl2: MeOH (9/1); Rf = 0.8.

b) Im-Benzyloxycarbonyl-L-Histidyl-L-Phenylalanyl, methyl ester, dihydrobromide salt (II)

30 g (0.5 mol) of Z-His (Z) -Phe-OMe (I) are dissolved in 300 ml of HBr-HOAc (32%) (the solution becomes cloudy after 2 min), and the mixture is left at room temperature for 30 min, then ether is added to cause precipitation. The soft solid material is filtered and dried overnight, 32 g of solid material,

melting point of 135-137 ° C; Thin layer chromatography: does not work.

c) N-Benzyloxycarbonyl-im-Benzyloxycarbonyl-L-Histidyl-im-Ben-zyloxycarbonyl-L-Histidyl-L-Phenylalanyl, methyl ester (III)

A suspension of 30.5 g (0.05 mol) of His (Z) -Phe-OMe-2HBr (II) and 21.7 g (0.05 mol) of Z-His (Z) -OH in 500 ml of CH2Cl2 is cooled to 0 ° C., then 11 g of DCC and 10.1 g (0.10 mol) of triethylamine are added. The mixture is allowed to reach room temperature overnight.

The dicyclohexylurea formed is separated by filtration and the filtrate is concentrated to a glassy material. The rest is dissolved in ethyl acetate and the EtOAc is washed with 10% citric acid, water, 5% ice-cold sodium bicarbonate solution and water . The EtOAc is dried over sodium sulfate, concentrated to about 1/3 of its volume, then ether is added to form a cloudy solution which is left in a cold room. A white solid separates, 29.5 g.

d) L-Histidyl-L-Histidyl-L-Phenylalanyl, methyl ester, tri-hydrochloride salt (IV) ^

I f

The mixture of 29 g of Z-His-His-Phe-OMe (IV), 10.8 ml of concentrated HCl, 180 ml of MeOH and two spatulas full of 10% Pd-C is hydrogenated for 5 h in using the hydrogenator Parr.

After separation by filtration, the methanol is concentrated to an oil and ether is added to form a solid. 18 g of raw material are used for the following reaction without further purification.

e) tert-Butyloxycarbonyl-S-p-Methoxybenzyl-L-Cysteinyl-L-Histi-dyl-L-Histidyl-L-Phenylalanyl, methyl ester (V)

Stirred 13 g of BOC-Cys (MBzl) -OH (0.038 mol), 5.4 g of hydroxybenzotriazole (0.040 mol) and 8.3 g of cyclohexylcarbodii-mide (0.040 mol) in 11 of methylene chloride cooled to ice for 30 min. 19.5 g of His-His-Phe-OMe-3HCl (0.0345 mol) and 10.5 g of N-methylmorpholine (0.096 mol) are added and the mixture is stirred overnight when it reaches room temperature . The suspension is filtered to separate the dicyclohexylurea and the filtrate is washed successively with water, 10% H 2 CO 2 and water,

then dried over Na2SO4. After filtration, the solvent is separated under vacuum, which leaves 21.0 g of crude product. This material is stirred in 100 ml of acetonitrile for 30 min at room temperature, then filtered. The precipitate is washed with acetonitrile, then with ether, and dried, producing 11 g; Thin layer chromatography: Rf = 0.12, silica gel 60, CHC13 / CH3 / OH-HAc,

9/1 / 0.5, v / v.

The filtrate from acetonitrile extraction contains the product and impurities and can be used for the extraction of other batches of raw material.

f) tert-Butyloxycarbonyl-S-p-Methoxybenzyl-L-Cystêinyl-L-Histi-dyl-L-Histidyl-L-Phênylalanine (VI)

11.0 g of BOC-Cys (MBzl) -His-His-Phe-OMe (0.0142 mol) are dissolved in 200 ml of methanol and 30 ml of IN potassium hydroxide are added. The solution is stirred at room temperature for 2 h, then the methanol is separated in vacuo. The rest is diluted with 200 ml of water, then filtered. The filtrate is adjusted to pH 5 with 10% citric acid and the gummy precipitate is left to stand while it crystallizes. The product is filtered, washed with water and dried under vacuum over P205; production of 7.3 g (67%); Thin layer chromatography: Rfde 0.52, silica gel 60, n-butanol ethyl acetate acetic acid H20.1 / 1/1/1, v / v; Rfde 0.43, silica gel 60, tert-amyl alcohol pyridine H20.7 / 6/7, v / v.

g) N-tert-Butyloxycarbonyl-O-Benzyl-L-Threonyl-L-Phenylalanine, methyl ester (VII)

A solution of Boc-L-Thr (Bzl) -OH (61.8 g, 0.2 mol) in THF is cooled to -15 ° C. 26.2 ml of isobutyl chloroformate (0 are added in portions) , 2 mol), keeping the temperature between - 15 and -10 ° C. After stirring at - 15 ° C for 15 min, a cold mixture of L-Phe-OMe HCl is added in portions (43.1 g, 0 , 2 mol) and N-methylmorpholine (22.4 mol) in DMF, while maintaining the temperature between -10 and -5 ° C. The mixture is stirred at 0 ° C for 2 h, then at room temperature overnight. The filtered reaction mixture is concentrated under vacuum and the rest is taken up in ethyl acetate. The ethyl acetate solution is washed successively with 5% KHS04, 5% KHC03 and a saline solution, then dried over Na2SO4. After concentration under vacuum, an oil is obtained which crystallizes on standing. The solid is recrystallized from isopropyl ether hexane, 74.9 g (80%); melting point 78-81 ° C; [a] ^ = +10.75 (c, 1.023, MeOH); Rf (CHC13): 0.35.

Analysis for C26H34N2Oa (470.55):

Calculated: C 66.36 H 7.28 N5.95%

Found: C 66.72 H 7.32 N5.85%

h) N-tert-Butyloxycarbonyl-O-Benzyl-L-Threonine-L-Phenylalanine (VIII)

Boc-Thr (Bzl) -Phe-OMe (23.5 g; 0.05 mol) is dissolved in a MeOH / dioxane mixture (100 ml, 1/1) and treated with N-NaOH (55 ml ) for 3 h until the starting material can no longer be detected by thin layer chromatography. Most of the solvent is evaporated in vacuo and the remainder is diluted with water, then acidified with 5% citric acid. The gum which separates is taken up in EtOAc, and washed with water (brine), then evaporated to dryness. The rest is crystallized from Et20-hexane, 19.8 g (87%), melting point 118-119 ° C; Rf (chloroform / methanol / acetic acid, 85/10/5): 0.80.

i) N-tert-Butyloxycarbonyl-O-Benzyl-L-Threonyl-O-Benzyl-L-Serine, methyl ester (IX)

N-tert-butyloxycarbonyl-O-benzyl-L-threonine (30.9 g, 0.1 mol) is dissolved in dry tetrahydrofuran (200 ml), cooled to -20 ° C and treated with N-methylmorpholine (11 ml), then with isobutyl chloroformate (13.4 ml). The cold reaction mixture is stirred for 5 min at -20 ° C., then treated with a solution of O-benzyl-L-serine, methyl ester, hydrochloride (25 g, approximately 0.1 mol) containing N-methylmorpholine (11 ml) in DME, and the mixture is allowed to reach room temperature overnight.

The solvent is removed in vacuo and the remainder is partitioned between water and ethyl acetate. The organic phase is washed with 5% citric acid, with water, with aqueous KHC03 and with water, then dried over MgSO4 and evaporated to dryness to obtain an oily residue which crystallizes from diethyl ether / hexane into a jelly-like material (29 g); Rf (CHCl3 / MeOH, 25/1); Rf (EtOAc / hexane, 1/1): 0.65.

j) N-tert-Butyloxycarbonyl-O-Benzyl-L-Threonyl-O-Benzyl-L-Serine

(X)

Boc-Thr (Bzl) -Ser (Bzl) -OMe (28.2 g, 0.0563 mol) is dissolved in methanol (about 50 ml) and treated with IN sodium hydroxide (75 ml) for 1.5 h at room temperature. The alkaline solution is neutralized to pH 7 with 10% citric acid and most of the methanol is separated in vacuo. The rest is diluted with a certain amount of water and acidified with 5% aqueous KHS04, then extracted with EtOAc. The organic phase is washed with water, then dried over Na2SO4 and

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evaporated to dryness to form an oil. The yield is quantitative. Rf (EtOAc / hexane, 1/1): 0.15 (long spot).

k) N-tert-Butyloxycarbonyl-O-Benzyl-L-Threonyl-O-Benzyl-L-Sé-ryl-S-p-Methoxybenzyl-L-Cysteine, benzyl ester (XI)

Boc-Thr (Bzl) -Ser (Bzl) -OH (24.3 g, 50 mmol) is dissolved in acetonitrile and dichloromethane (250 ml, 2/3) and mixed with H-Cys ( SMBzl) Bzl-TosOH (25 g, 50 mmol), then with triethylamine (6.8 ml) and N-hydroxybenzotriazole (6.8 g) and the mixture is cooled in an ice bath.

A solution of DCC (11 g, 53 mmol) in 50 ml of acetonitrile is added and the reaction mixture is stirred for 2 h under cold conditions, then for 2 d at room temperature. The separating DCU is removed by filtration and the filtrate is evaporated to dryness. The oily residue is partitioned between ethyl acetate and water, and the organic phase is washed with 10% citric acid, water, 5% KHC03 and brine, then dried over Na2SO4. The solvent is evaporated off and the oily residue is crystallized from Et 2 O / hexane to give a white solid, 24.5 g, melting point 87-90 ° C; Rf (chloroform / methanol, 25/1): 0.54; traces at 0.4 and 0.3 (heptane / EtOAc, 1/1): 0.64; traces at 0.25, positive at I2, Wîf = —14.7 (c, 0.98 DMF).

Elementary analysis for C44H52N3S09 (798.9):

Calculated: C 66.15 H 6.56 N 5.26%

Found: C 66.22 H6.95 N 5.29%

/) O-Benzyl-L- Threonyl-O-Benzyl-L-Séryl-S-p-Methoxybenzyl-L-Cysteine, benzyl ester, trifluoroacetate (XII)

Boc-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) -OBzl (8 g,

10 mmol) with anisole (100 mmol) and treated with TFA (100 ml) for 45 min. The solvent is evaporated in vacuo and the rest is dissolved in dry Et 2 O, then it is evaporated to dryness under high vacuum to obtain an oily compound, 7.2 g (90%); Rf (n-butanol / water / glacial acetic acid, 4/1/1): 0.9; Rf (heptane / EtOAc): 0.0-0.1 long spot, positive for I2 and ninhydrin.

m) N-tert-Butyloxycarbonyl-O-Benzyl-L- Threonyl-L-Phënylalanyl-O-Benzyl-L-Thréonyl-O-Benzyl-L-Séryl-Sp-Méthoxybenzyl-L-Cys-têine, benzy l ester ( XIII)

Boc-Thr (Bzl) -Phe-OH (prepared by the method of Example III) (9.2 g, 20 mmol) is dissolved in DMF (100 ml) and mixed with N-hydroxysuccinimide (3 , 4 g) and a solution of Thr (Bzl) Ser (Bzl) -Cys (SMBzl) OBzl, trifluoroacetate salt (20 mmol) in DMF (20 ml), neutralized with triethylamine to pH 7. The mixture is cooled in an ice bath and treated with DCC (4.5 g) for 2 hr cold, then for 2 d at room temperature. The DCU is filtered and the filtrate is evaporated to dryness. The rest is triturated with water to obtain a precipitate which is taken up in EtOAc, then washed with 5% citric acid, water, 5% Na2CO3 and water. , dried over Na2SO4 and evaporated to dryness. The rest is solidified from Et20 / hexane,

It can be crystallized from MeOH and is very soluble in CHCl3; 17.3 g (70%); melting point 105-107 C; Rf (CHCl3 / MeOH, 10/1): 0.90; [a] 2D5 = -3.6 (c: 1, DMF).

Analysis for C64H75N5S0i2H20 (1156.2):

Calculated: C 66.46 H 6.71 N6.05%

Found: C 66.68 H 6.59 N 6.20%

n) tert-Butyloxycarbonyl-D-Tryptophyl-Ne-Benzyloxycarbonyl-L-ly-syl, methyl ester (XIV)

Boc-D-Trp-OH (30.4 g, 0.1 mol), Lys (Z) -OMe HCl (33 g, 0.1 mol) and 13.6 ml of triethylamine are dissolved in 400 ml of DMF and cooled in an ice bath, then 22 g of dicyclohexylcarbodiimide are added and the mixture is left to reach room temperature overnight. The separating dicyclohexylurea is filtered and the filtrate is concentrated to a small volume, then an excess of water is added to form a gummy material. This material is taken up in ethyl acetate and washed with 10% aqueous citric acid, water, saturated NaHCO3 and water, then dried over Na2SO4 and evaporated to dryness. The rest is triturated with ether, then with hexane to give a crystalline solid, 28 g; Thin layer chromatography: silica gel G, hexane / EtOAc (1/1, v / v), Rf: 0.3.

o) tert-Butyloxycarbonyl-L-Phenylalanyl-D-Tryptophyl-N £ -Benzy-loxycarbonyl-L-lysine (XV)

A. Add Boc-Z> -Trp-Lys (Z) -OMe (22 g, 44 mmol) to a pre-cooled mixture of 220 ml of TFA and 25 ml of anisole, then stir for 30 min. an ice bath. The TFA is separated as quickly as possible at 27 ° C. on a rotary apparatus and the remaining oil is triturated several times with a solution of hexane / ether, 2/1, then it is decanted and finally pumped to obtain a gum, production of 21.3 g, 36 mmol.

B. Boc-Phe-OH (9.6 g, 36 mmol), HOBT (5.3 g, 39 mmol) and DCC (8 g, 39 mmol) are stirred in 500 ml of cold dichloromethane for 20 min. . D-Trp-Lys (Z) -OMe trifluoroacetate salt (21.3 g, 36 mmol) in 200 ml of dichloromethane is added, followed by N-methylmorpholine (4.25 ml, 39 mmol). The reaction mixture is stirred overnight while it warms up to room temperature. The mixture is filtered to separate the DCC and the filtrate is washed twice with water, then twice with 10% citric acid, twice with saturated NaHCO3, twice with water, then dried over Na2S04. After filtration and evaporation, the remainder weighs 21.4 g (82%) and is used without further purification. Thin layer chromatography: silica gel F-254 (CHCl3 / CH3OH / HAc, 9/1 / 0.5); Rf = 0.76 plus traces of small impurities.

C. Dissolve Boc-Phe-Z) -Trp-Lys (Z) -OMe (22.9 g,

31.4 mmol) in 450 ml of p-dioxane and 100 ml of methanol, and 39 ml of aqueous IN KOH are added and the mixture is stirred overnight at room temperature. The solution is evaporated on a rotary evaporator. The rest is dissolved in 500 ml of hot water, acidified to pH 3 with 10% citric acid, stirred for 30 min and filtered. After drying under vacuum overnight, 21.6 g (96%) are obtained, melting point 85-90 ° C, [a] fo = + 7.09 (c, 0.987, MeOH); Rf: 0.63 (CHCl3 / CH3OH / HAc, 9/1 / 0.5), silica gel F-254.

p) tert-Butyloxycarbonyl-L-Phenylalanyl-D-Tryptophyl-Nz -Benzy loxycarbony lL-Lysyl-O-Benzyl-L-Thréonyl-L-Phény lalany lO-Benzyl-L-Thréonyl-O-Benzyl-L-Séryl- Sp-Methoxybenzyl-L-Cysteinyl, benzy l ester (XVI)

A. Add Boc-Thr (Bzl) -Phe-Thr (Bzl) -Ser- (Bzl) -Cys (SMBzl) -OBzl (XIII) (40.5 g, 35 mmol) to a cold 400 ml solution of TFA and 40 ml of anisole, then stirred cold for 30 min. The TFA is evaporated on a rotary evaporator and the rest is triturated with 11 ether / hexane (1/1) to form a white powder. After filtration, washing with ether / hexane and drying under vacuum on P2Os and NaOH pellets, 34.6 g of material (82%) are obtained, melting point of 136-139 ° C, softening at 120 °, [a] £ 3 = —9.93 (c, 1.04, MeOH); Rf: 0.63, silica gel CHCl3 / CH3OH / HAc, 9/1 / 0.5, small more polar impurity Rf: 0.53.

B. Boc-Phe-D-Trp-Lys (Z) -OH (20.0 g, 27.9 mmol), HOBT (4.15 g, 30.8 mmol) and DCC (6, 3 g, 30.8 mmol) in 2.01 cold dichloromethane in an ice bath for 30 min. 32.6 g (27.9 mmol) of H-Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) -OBzl trifluoroacetate salt are added, then 3.35 ml of N- methylmorpholine (30.8 mmol), and continued stirring overnight while the mixture warms to room temperature. Thin layer chromatography did not show an essentially complete reaction after 2 h.

The mixture is filtered and the filtrate is washed with water, with 10% citric acid, water, saturated NaHCO3 and water, then dried. After filtration and evaporation, thin layer chromatography shows a more polar product and impurity which is separated by dissolving the crude product in 200 ml of hot DMF and addition of 11 of 2% aqueous NaHCO3. The product is filtered, washed with dilute NaHC03 and water, then dried in vacuo over P2Os, yield 36.5 g (75%), melting point 182-184 ° C

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(contraction at 150 °), [a] | f = 0 (c, 0.988, CHC13). Rf (single spot) 0.76, silica gel (CHCl3 / CH3OH / HAc, 9/1 / 0.5).

q) Preparation of H-Phe-D-Trp-Lys (Z) -Thr (Btl) -Phe-The (Bzl) -Ser (Btl) -Cys (SMBzl) -OBzl, trifluoroacetate salt (XVII)

Boc-Phe-jD-Trp-Lys (z) -Thr (Bzl) -Phe-Thr (Bzl) -Ser (Btl) -Cys (SMBzl) -OBzl (32 g) and anisole (33) are dissolved ml) in 50 ml of dichloromethane and 100 ml of trifluoroacetic acid, and the mixture is stirred for 30 min at room temperature. The solution is evaporated on a rotary evaporator at 30 ° C. The rest is triturated with anhydrous ether to form a powder which is filtered, then washed with ether and dried under vacuum on P2Os and sodium hydroxide tablets.

Thin layer chromatography on silica gel 60 in CHCl3 / CH3OH / HAc (9/1 / 0.5) shows a single spot, Rf 0.65; nuclear magnetic resonance does not show a peak for t-butyl.

r) tert-Butyloxycarbonyl-Sp-Methoxybenzyl-L-Cystênyl-L-Histidyl-L-Histidyl-L-Phênylalanyl-L-Phénylalanyl-D-Tryptophyl-N & -Benzyl-oxycarbonyl-L-Lysyl-O-Benzyl-L- Threonyl-L-Phenylalanyl-O-Ben-zyl-L-Threonyl-O-Benzyl-L-Séryl-Sp-Methoxybenzyl-L-Cysteine, benzyl ester (XVIII)

Boc-Cys (SMBzl) -His-His-PheOH (5.5 g, 7.2 mmol), 1-hydroxybenzotriazole monohydrate (1.07 g, 7.9 mmol) and dicyclohexylcarbodiimide (1.65) are agitated. g, 7.9 mmol) in 100 ml of dry dimethylformamide in an ice bath for 30 min. 12.5 g (7.2 mmol) of H-Phe-Z) -Trp-Lys (z) -Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) OBzl are added, trifluoroacetate salt and 0.855 g of N-methylmorpholine (7.65 mmol), and the mixture is allowed to reach room temperature overnight with stirring. The suspension is filtered to separate the dicyclohexylurea and the filtrate is evaporated to 50 ml, then diluted with 500 ml of a saturated aqueous solution of sodium bicarbonate. The resulting solid material is filtered, washed with water and redissolved in 200 ml of DMF. This solution is diluted with 11% 5% aqueous citric acid, then filtered. The solid material is washed with water, dissolved in 200 ml of DMF and precipitated with 11 of a saturated solution of sodium bicarbonate,

then filtered, washed with water and dried under vacuum over P2Os at room temperature, production of 15 g; amino acid analysis: His: 1.66 (2.0); Lys: 1.05 (1.0); Phe: (3.0); Ser: 1.02 (1.0); Thr: 2.2 s (2.0).

s) Cyclic disulfide (1-12) of L-Cysteinyl-L-Histidyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl- L-Cystêine (XIX)

Boc-Cys (SMBzl) -His-His-Phe-Phe-D-Trp-Lys (z) -io Thr (Bzl) -Phe-Thr (Bzl) -Ser (Bzl) -Cys (SMBzl) OBzl are mixed (5 g) with anisole (10 ml), and treat with 100 ml of liquid HF excluding air and in an ice bath for 60 min. The excess HF is separated in vacuo and the rest taken up in 10% aqueous AcOH (200 ml), then poured into 71 of deaerated water. The pH is adjusted to 7 with dilute NH40H and the mixture is stirred in the open air for 24 h, then the pH is brought to 5 with glacial AcOH, and the solutions are passed through a column of Amberlite CG 50 (form H +). The peptide material which is absorbed on the ion exchange resin is eluted with 50% aqueous acetic acid and the fractions containing the peptide material are combined and lyophilized to form 2 g of crude material. This crude dodecapeptide is purified by chromatography through a column of Sephadex G25 eluting with 10% aqueous AcOH and by partition chromatography through Sephadex G25 with the N-BuOH / water / glacial AcOH two-phase system ( 4/5/1, v / v). Thin layer chromatography, Merck AG silica gel, [“Chlorine peptide spray”, DE Nitecki et al., “Biochem.”, 5,665 (1966)], Rf (BWA, 4/1/1): 0.45 ; Rf (BWAP, 30/24/6/20): 0.64; amino acid analysis: Thr (z): 1.85; Ser (1): 0.79; Phe (3): 3; Lys (1): 1.02: His (2): 1.75; Cys and Trp, not determined.

It will be apparent to those skilled in the art that other combining agents and other protecting groups may be used in the conventional synthesis described in Example 14. In addition, it will be apparent that different sequences may be used. for the accumulation of amino acid fragments in order to arrive at the desired molecule. All the methods in question are obviously also included in the context of the present invention.

R

Claims (7)

633524 1. Therapeutically active compounds corresponding to the formula: X-Cys-XrX2-Phe-Phe-X3-Lys-Thr-Phe-Thr-Ser-X4-OH I I SA SA (I) in which A represents hydrogen, or the two symbols A signify a direct bond between the sulfur atoms; X represents hydrogen, Ala-Gly, Gly-Gly, Ala-D-Ala, Gly-Gly-Gly, acetyl or benzoyl; X represents His or Arg; X2 represents His, Glu or Asp; X3 represents Trp, D-Trp or 6-F-G-Trp, and X4 represents Cys or D-Cys, as well as their pharmaceutically acceptable salts. 2. Compounds according to claim 1, characterized in that it is cyclic disulfide (1-12) of L-Cysteinyl-L-Argynil-L-Histidyl-L-Phenylalanyl-L-phenylalanyl-D-Tryptophyl- L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl-L-Cysteine, cyclic disulfide (1-12) of L-Cysteinyl-L-Histidyl-L-Histidyl-L-Phenylalanyl-L -Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylanalyl-L-Threonyl-L-Seryl-D-Cysteine, cyclic disulfide (1-12) of L-Cysteinyl-L-Arginyl-L- Glutamyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl-L-Cysteine or cyclic disulfide (1-12) of L-Cysteinyl -L-Histidyl-L-Glutamyl-L-Phenylalanyl-L-Phenylalanyl-D-Tryptophyl-L-Lysyl-L-Threonyl-L-Phenylalanyl-L-Threonyl-L-Seryl-L-Cysteine. 2 3. Compounds according to one of claims 1 or 2, characterized in that they are in the form of pharmaceutically acceptable acid addition salts, chosen from the hydrochloride, bromhy-drate, sulfate, phosphate, polyphosphate, maleate, acetate, citrate, benzoate, succhiate, malonate and ascorbate. 4. Process for the preparation of compounds corresponding to formula I of claim 1, characterized in that it comprises the combination of amino acids necessary for the formation of polypeptides, or their activated derivatives, to form the peptide bonds, and this in any desired order of succession, the reactive groups which one does not wish to see reacted being protected during the intermediate phases. 5. Process for the preparation of compounds corresponding to formula I according to claim 1, in which the two symbols A signify a direct bond between the sulfur atoms, characterized in that it comprises the preparation of compounds corresponding to formula I of claim 1 wherein A represents hydrogen by the process according to claim 4, then the oxidation of the compound obtained. 6. Pharmaceutical compositions, characterized in that they comprise at least one compound corresponding to formula I or a pharmaceutically acceptable salt of such a compound, in combination with a pharmaceutically acceptable carrier or vehicle. 7. Compositions according to claim 6, characterized in that they also comprise insulin.