CH616402A5 - Process for the preparation of analogues of somatostatin - Google Patents

Abstract

Analogues of somatostatin corresponding to the formula <IMAGE> in which R<1> and R<2> have the meaning given in Claim 1, are prepared. The process consists in oxidising a linear peptide of formula II <IMAGE> to its corresponding cyclic disulphide derivative, and in then separating the remaining protective groups. The derivatives of somatostatin obtained have an activity which is greater than or of the same order as the natural hormone as well as a duration of activity which is greater than that of somatostatin. They can be used in the treatment of acromegaly and of diabetes.

Description

The present invention relates to a process for the preparation of tetradecapeptide derivatives called somatostatin. It also relates to the preparation of the starting materials used in said process.

The term somatostatin has been proposed for the factor found in hypothalamic extracts, preventing the secretion of growth hormone (somatotropin). The structure of this factor has been demonstrated by P. Brazeau et al., "Science", 179.77 (1973) and has been assigned the following structure of tetradecapeptide:

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

The abbreviations used for the various amino acids are: Ala, alanine; Asn, asparagine; Cys, cysteine; Gly, glycine; Lys, lysine; Phe, phenylalanine; Ser, serine; Thr, threonine, and Trp, tryptophan.

The constitution of the somatostatin tetradecapeptide has been confirmed by synthesis; see, for example, D. Sarantakis and W.A. McKinley, "Biochem. Biophys. Res. Comm. ", 54 234 (1973), J. Rivier et al.," Compt. Give back. Ser. D ”, 276.2737 (1973) and H.U. Immer et al., "Helv. Chim. Acta ”, 57, 730 (1974).

The important physiological activity of this tetradecapeptide made it consider as an interesting compound in clinical pharmacology in connection with the treatment of acromegaly and diabetes;

see, for example, K. Lundbaek et al., "Lancet", 2.131 (1970) and R. Guillemin in "Chemistry and Biology of Peptides", J. Maienhofer, ed., 3rd American Peptide Symposium, Boston 60 1972, Ann Arbor Science Publications, Ann Arbor, Mich., 1972.

The linear form of somatostatin, comprising two sulhydryl groups instead of a disulphide bridge, was prepared by J.E.F. Rivier, "J. Bitter. Chem. Soc. ”, 96.2986 (1974). Rivier reported that the linear form has a potency equal to that of 65 somatostatin, with regard to the ability of these two types of compounds to paralyze the rate of secretion of growth hormone by rat pituitary cells in tissue cultures in a monomolecular layer.

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It is only recently that polypeptides, other than the natural hormone and its linear form, have been reported to have activity similar to that of somatostatin. D. Sarantakis et al., "Biochem. Biophys. Res. Comm. ”, 55, 538 (1973) recently described the synthesis of the somatostatin analog, [Ala3'14] -somatostatin, by solid phase methods. This analog showed a very small sum of activity, approximately 0.01% of the power of somatostatin. P. Brazeau et al., "Biochem. Biophys. Res. Comm. ”, 60,1202 (1974) recently described the synthesis of a number of acylated Des— [Ala1 —Gly2] -somatostatin derivatives by solid phase methods.

The present invention relates to a process for the preparation of somatostatin derivatives, which show a higher degree of activity or of the same order as the natural hormone, as well as a duration of activity which is greater than that of somatostatin. These derivatives are easily prepared by a suitable process with the following advantages: the process starts from readily available materials, it avoids harmful reagents, it develops easily and it uses protective groups which are easy to separate.

The above qualities and advantages make the peptides prepared according to the invention interesting for the treatment of diabetes and acromegaly.

The peptides prepared according to the invention are represented by formula I:

SCH2CH (R1) CO — Lys — Asn — Phe — Phe — Trp — Lys — Thr — Phe — Thr — Ser — NHCH (R2) CH2 S

In this formula:

a) R1 represents H — Gly — Gly — Ala — Gly — NH, H — Gly— Gly - Gly-Ala-Gly-NH- or H — Leu — Gly — Gly — Ala— Gly — NH and R2 represents H or COOH , or b) R1 represents H or NHR3, where R3 is the lower aliphatic acyl radical of 1 to 6 carbon atoms or benzoyl, and R2 represents H, or c) R1 represents H — Ala — Gly — NH and R2 represents COOAlk, where Alk is a straight or branched chain alkyl having from 1 to 14 carbon atoms.

The therapeutically acceptable salts of the compounds of formula I are also included within the scope of the invention.

According to the invention, the peptides are prepared by a process comprising the oxidation of a linear peptide of formula II:

.Trt - SCH2CH (R4) CO - Lys (Boc) - Asn - Phe - Phe - Trp -Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH (R2 ) CH2S — Trt (II)

in which:

a) R2 represents H or COOH and R4 represents Boc — Gly— Gly - Ala - Gly — NH, Boc - Gly — Gly - Gly - Ala - Gly - NH or Boc — Leu — Gly — Gly — Ala — Gly — NH, or b) R2 represents H and R4 represents H or NHR3, where R3 has the preceding definition, or c) R2 represents COOAlk and R4 represents Boc — Ala — Gly— NH, with iodine or thiocyanogen to obtain the disulfide derivative corresponding cyclic of formula III:

SCH2CH (R4) CO - Lys (Boc) - Asn - Phe - Phe - Trp - Lys (Boc) -

Thr (Bul) - Phe - Thr (Bu ') - Ser (Bu') -NHCH (R2) CH2S (III) in which R2 and R4 have the definition given above, followed by the hydrolytic scission of all the protective groups remaining under moderately acidic conditions to obtain the corresponding peptide of formula I.

The linear starting peptide of formula II can easily be prepared by a process which comprises the reaction, according to the azide coupling method (explained below), of a peptide of formula IV:

Trt - SCH2CH (R4) CO - Lys (Boc) - Asn - Phe - Phe - NHNH2

(IV)

with a peptide of formula V:

H - Trp - Lys (Boc) - Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') -NHCH (R2) CH2S — Trt (V)

formulas in which R2 and R4 have the definition given previously, with a view to obtaining the corresponding linear peptide of formula II, in which R2 and R4 also have the definition already given.

(I)

In general, the abbreviations used in the present description to designate the amino acids and the protective groups are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [see “Biochemistry”, II, 1726-1732 ( 1972)]. For example, Cys, Lys, Asn, Phe, Trp, Thr, and Ser representing the residues of L-cysteine, L-lysine, L-asparagine, L-phenylalanine, L-tryptophan, L-threonine and L-serine. The term “residue” denotes a radical derived from the corresponding L-amino acid by elimination of the OH portion of the carboxyl group and of the H portion of the amino group. All amino acids have the natural L configuration.

A number of processes or techniques are known to date for the preparation of peptides. By way of example, the functional groups which are not involved in the reaction for forming a peptide bond are optionally protected by one or more protective groups before the condensation reaction. By way of example, the protective groups which can be chosen for an amino function of a peptide or of an amino acid, which is not involved in the formation of the peptide bond, are: the alkoxy- carbonyls, which include benzyloxycarbonyl (represented by Z), t-butoxycarbonyl (represented by Boc), a, a-dimethyl-3,5-40 dimethoxybenzyloxycarbonyl (represented by Ddz), 2- (p-biphenyl) isopropyloxycarbonyl (represented by Bpoc), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl or ethoxycarbonyl; protecting groups of the acyl type, which include formyl, trifluoro-acetyl, phthalyl, acetyl (Ac), or toluenesulfonyl; alkyl-type protecting groups, which include triphenylmethyl or trityl (represented by Trt), or benzyl; the preferred protective groups for the process of the invention are benzyloxycarbonyl, t-butoxycarbonyl, triphenylmethyl and α, a-dimethyl-3,5-50 dimethoxybenzyloxycarbonyl. The protective groups for the hydroxyl radical of serine and tyrosine consist of acetyl, tosyl, benzoyl, tertiary butyl (represented by Bu '), trityl and benzyl; the preferred protecting group is tertiary butyl. The protective group on the sulfur atom of cysteine 55 or modified cysteine is trityl (represented by Trt). The carboxylic acid function of a peptide or an amino acid can be considered to be protected by a lower alkyl ester or lower alkoyl, in particular a methyl ester (represented by OMe), ethyl ester (represented by OEt ) or benzyl (represented 60 by OBzl), and also by substituted hydrazides including t-butoxycarbonyl hydrazide (represented by NHNH Boc), benzyloxycarbonyl hydrazide (represented by NHNH Z) or hydrazide a , cx-dimethyl-3,5-dimethoxybenzyloxycarbonyl (represented by NHNH Ddz). The abbreviation Acm represents 65 acetamidomethyl.

To promote easy condensation of the carboxyl group of a peptide with a free amino group of another peptide in order to form a new peptide bond, the carboxyl group

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terminal must be activated. Descriptions of these carboxyl radical activating groups are generally found in textbooks on peptide chemistry; see, for example, K.D. Kopple, "Peptides and Amino Acids," W.A. Benjamin, Inc., New York, 1966, pp. 45-51; E. Schröder and K. Liibke, "The Peptides", vol. I, Academic Press, New York, 1965, pp. 77-128. Examples of the activated form of the terminal carboxyl are acid chloride, anhydride, azide, activated ester or o-acylurea of a dialkylcarbodiimide. The following activated esters have been found to be particularly suitable; 2,4,5-trichlorophenyl (represented by OTcp), pentachlorophenyl (represented by OPcp), p-nitrophenyl (represented by ONp) or 1-benzo-triazolyl; the succinimido group is also of interest for such activation.

The term azide method, which is used in the present case, denotes the method of coupling two peptide fragments, which comprises reacting a peptide hydrazide with a reagent providing nitrous acid in if you. Suitable reagents for this purpose are organic nitrites (e.g. tertiary butyl nitrite, isoamyl nitrite) or alkali metal nitrites (e.g. sodium nitrite, potassium nitrite) in the presence of an acid strong, such as hydrochloric acid or sulfuric or phosphoric acid. The corresponding peptide azide thus obtained is then reacted with a peptide comprising a free amino group in order to obtain the desired peptide. Preferred conditions for the azide coupling method include the reaction of the peptide hydrazide with nitrous acid, formed in situ from an organic nitrite in the presence of a strong acid, preferably 1 hydrochloric acid (pH usually of the order of 0.1 to 2), in an anhydrous inert organic solvent, for example dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene dichloride, tetrahydrofuran , dioxane, etc., at a temperature of - 30 to 20 ° C, preferably about - 15 ° C, for 10 to 30 min, in order to obtain the corresponding azide. The peptide azide can be isolated and crystallized, and is preferably left in the reaction mixture. Then, the azide of the preceding mixture is reacted with the peptide unit comprising the free amino group at temperatures ranging from −30 ° C. to 20 ° C. for approximately 1 to 2 h, and then at a temperature from 0 to 30 ° C for 10 to 30 h. In the reaction mixture there is an acid acceptor, preferably an organic base, for example N-ethyldiisopropylamine, N-ethylmorpholine or triethylamine, to make the reaction medium slightly alkaline, with a pH preferably of the order of 7 to 7.5. See also the previously cited textbooks of Kopple or Schröder and Liibke for further descriptions of this method.

The terms peptide, tripeptide, hexapeptide, etc., which are used in the present case, should not be considered as being limited to the peptides proper, these terms also being used for the modified peptides having functionalized or protective groups. The term peptide is used when considering a peptide comprising from 2 to 17 amino acid residues. The residue SCH2CH (R1) CO denotes a modified cysteine residue, when R1 denotes hydrogen, or a cysteine residue when R1 denotes NHR3, where R3 has the definition given previously, or represents H — Gly — Gly— Ala - Gly - NH, H - Gly - Gly - Gly - Ala - Gly - NH, H — Leu — Gly — Gly — Ala — Gly — NH or H - Ala — Gly — NH. In addition, the NHCH (R2) CH2S residue denotes the cysteine residue when R2 represents COOH or COOAlk or a modified cysteine residue, when R2 represents H.

The abbreviation Me denotes a methyl group and NHNH2 denotes a hydrazide group.

The expression lower alkyl designates here hydrocarbon radicals containing 1 to 3 carbon atoms and it includes methyl, ethyl and propyl. The symbol Alk denotes a straight or modified chain alkyl group containing 1 to 14 carbon atoms.

The term acyl designates, in the present case, a lower aliphatic acyl of 1 to 6 carbon atoms and includes, under this definition, straight or branched chain acyls, in particular formyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, n-hexanoyl, etc.

The expression mineral acid denotes, in the present case, strong mineral acids and includes hydrochloric, hydrobromic, sulfuric or phosphoric acids. When this expression is used in conjunction with an anhydrous system, anhydrous hydrochloric acid is the preferred mineral acid.

The expression weakly acidic conditions designates, in the present case, conditions under which a dilute aqueous solution of an organic acid, for example an aqueous solution at 30-80% of formic, acetic or propionic acid, preferably a solution aqueous at 70-80%, or a mixture of solutions of this kind, constitutes a main component of the reaction medium.

The term moderately acidic conditions refers, in this case, to conditions under which concentrated organic acids or solutions of mineral acids are used as the main component of the reaction medium at temperatures ranging from - 30 to 30 ° C. examples of preferred conditions in this case are the use of trifluoroacetic acid at 50-100% at a temperature of 0 to 30 ° C or hydrochloric acid 0.1-12N in aqueous solution or in solution in an organic solvent, or HCl in solution in anhydrous organic solvents at a temperature of -20 to 10 ° C.

The term organic nitrite includes alkyl nitrites available on the market, for example tertiary butyl nitrite, isoamyl nitrite, etc.

The term organic base includes, in this case, triethylamine, N-ethylmorpholine, N-ethyldiisopropylamine, etc.

The expression strong base designates, in the present case, both organic bases as described above and strong mineral bases, in particular hydroxides and carbonates of sodium and potassium.

The new peptides are obtained in the form of the free base or of an acid addition salt, either directly by means of the process of the present invention, or by reacting the peptide with one or more equivalents of the appropriate acid. . Examples of preferred salts are the salts with pharmacy-acceptable organic acids, for example acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or toluenesulfonic acids, as well as salts with polymeric acids, such as tannic acid or carboxymethylcellulose, and salts with inorganic acids, such as hydrohalic acids, for example hydrochloric acid, or sulfuric acid or even phosphoric acid. It should be noted that the peptides have two basic nitrogen giving addition salts with one and perhaps up to two equivalents of acid. If desired, a particular acid addition salt is converted to another acid addition salt, for example a salt with a pharmaceutically acceptable, non-toxic acid, by treatment with the appropriate resin, exchanger d 'ions, as described by RA Boissonas et al., "Helv. Chim. Acta ”, 43.1349 (1960). Suitable ion-exchange resins are cellulose-based cation exchange resins, for example carboxymethylcellulose, or chemically modified, cross-linked dextran-based cation exchangers, for example exchangers of the Sephadex C type, and resins strongly basic anion exchangers, for example those listed by JP Greenstein and M. Winitz in "Chemistry of the Amino Acids", John Wiley and Sons, Inc., New York and London, 1961, vol. 2, p. 1456.

The somatostatin analogs of formula I give complex salts with heavy metal ions. An example of a heavy metal complex acceptable in pharmacies is a complex formed with zinc or with zinc protamine.

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The peptides produced by the process of the invention, as well as their corresponding pharmaceutically acceptable salts, are of interest because they exhibit the pharmacological activity of the tetradecapeptide formed by natural somatostatin. Their activity is easily demonstrated in pharmacological tests, for example according to the variant [A.V. Schally et al., "Biochem. Biophys. Res. Commun. ”, 52.1314 (1973); J. Rivier et al., “C.R. Acad. Sci. Paris, Ser. D ”, 276, 2737 (1973)] of the in vitro method of M. Saffran and A.V. Schally,“ Can. J. Biochem. Physiol. ”, 33.405 (1955).

The activity of the peptides of formula I prepared according to the invention has also been demonstrated in vivo using a variant of the method of increasing, caused by pentobarbital, the level of growth hormone in plasma in rats, this method having been described by Brazeau et al., above quotation. In this test, the peptides prepared according to the invention show a level of activity which is higher than that of somatostatin or of the same order of magnitude.

The peptides obtained according to the method of the invention are advantageous for the treatment of acromegaly and related states of endocrine hypersecretion, as well as for the treatment of diabetes in mammals; see, for example, P. Brazeau et al., above quotation. When the peptides or their salts are used for such treatment, they are administered to the body, preferably parenterally, in combination with a liquid vehicle, acceptable in pharmacies. The peptides of formula I have a low order of toxicity. The proportion of the peptide or its salt is determined by its solubility in the given vehicle, by the vehicle itself or by the route chosen for administration. When the peptide or a salt thereof is used in sterile aqueous solution, this solution may also contain other dissolved products, such as buffers or preservatives, as well as a sufficient quantity of pharmaceutically acceptable salts or glucose to make the solution isotonic. The dose will vary with the form of administration and with the particular case to be treated, this dose preferably being maintained at a rate of 1 (ig to 300 jxg / kg of body weight. However, to achieve effective results, use is made particularly advantageously a dose of the order of 1 ng to 50 (ig / kg of body weight.

The peptides or their salts can also be administered in one of the long-acting, slow-release or deposition forms, which are described below, preferably by intramuscular injection or by implantation. Such dosage forms are designed to release 0.1 to 50 ng / kg body weight / d.

It is often desirable to administer the therapeutic agent continuously over extended periods of time in long acting, slow release or depot forms. These forms may contain a pharmaceutically acceptable salt of the peptide, having a low degree of solubility in body fluids, for example one of the salts described above, or they may contain the peptide in the form of a salt soluble in water, along with a protective vehicle that prevents rapid release. In the latter case, for example, the peptide can be formulated with a partially hydrolyzed gelatin, non-antigen, in the form of a viscous liquid, or else the peptide can be absorbed on a solid vehicle, acceptable in pharmacy, for example zinc hydroxide, or alternatively it can be administered in suspension in a liquid vehicle which is acceptable in pharmacies; the peptide can also be formulated in gels or in suspensions with a non-antigenic, protective hydrocolloid, for example sodiumcarboxymethylcellulose, polyvinylpyrrolidone, sodium alginate, gelatin, polygalacturoic acids, for example pectin , or certain mucopolysaccharides, together with surface-active agents, preservatives and liquid vehicles acceptable in pharmacy, aqueous or non-aqueous. Examples of such formulations can be found in conventional pharmaceutical publications, for example in "Pharmaceutical Sciences" by Remington, 14th ed., Mack

Publishing Co., Easton, Pennsylvania, 1970. A long-acting, slow-release preparation of the peptide produced according to the process of the invention can also be obtained by placing in microcapsules in a coating that is acceptable in pharmacies. , for example gelatin, polyvinyl alcohol or ethyl cellulose. Other examples of coating materials and methods used for microcapsules are described by J.A. Herbig in "Encyclopedia of Chemical Technology", vol. 13.2nd ed., Wiley, New York 1967, pp. 436-456. Formulations of this kind, as well as suspensions of peptide salts, which are only sparingly soluble in body fluids, for example salts with pamoic acid or tannic acid, are designed to release about 1.0 at about 100 µg of the active compound / kg body weight / day, administration preferably by intramuscular injection. Alternatively, some of the solid dosage forms listed above, for example certain sparingly water-soluble salts or dispersions of peptide salts in solid vehicles or also adsorption products of these salts on solid vehicles, for example dispersions in a neutral hydrogel of a polymer of ethylene glycol methacrylate or of similar crosslinked monomers, as described in US Pat. No. 3,555,556, can also be presented in the form of pellets releasing to roughly the same amounts as those given above and can be implanted subcutaneously or intramuscularly.

The process according to the present invention will be illustrated by the following embodiments, in which specific peptides of formula I are prepared.

a) Compounds of formula I in which R1 = H — Gly — Gly—

Ala — Gly — NH—, H — Gly — Gly — Gly — Ala — Gly — NH -

or H - leu — Gly — Gly — Ala — Gly — NH— and R2 = H or COOH

The protected lower alkyl ester of alanyl-glycine, preferably Boc-Ala-Gly-OMe [described by H.U. Immer et al., "Helv. Chim. Acta. ”, 57, 730 (1974)] is dissolved in tri-fluoroacetic acid, and the solution is kept at 0-10 ° C for about 1 h, then providing for the evaporation of the trifluoroacetic acid to obtain the H — Ala — Gly — OMe in the form of its addition salt of trifluoroacetic acid, which can, if desired, be converted to the form of the free base and used in the latter form.

Other cleavage reagents for the removal of the Boc protecting group are hydrobromic acid in acetic acid, alcoholic solutions of hydrochloric acid, etc. The compound obtained, mentioned last, is dissolved in an inert organic solvent, preferably dimethylformamide, and the resulting solution is cooled to about 0-10 ° C. An excess, preferably 1, is added to the solution. , 1 to 1.3 equivalent, of an organic base, preferably N-ethylmorpholine; the solution then has a pH of approximately 8. An equivalent of an activated ester protected with glycine, preferably Boc — Gly — OTcp [described by J. Pless and R.A. Boissonnas, “Helv. Chim. Acta ", 46, 1609 (1963)] and the reaction mixture is kept at about 0-20 ° C for about 2 days. The solvent is evaporated and the remainder is crystallized to obtain the lower alkyl ester protected from glycyl-alanyl-glycine, preferably Boc-Gly-Ala-Gly-OMe, which, by treatment with trifluoroacetic acid as mentioned previously gives the lower alkyl ester of the glycyl-alanyl-glycine tri-peptide, preferably H-Gly-Ala-Gly-OMe, in the form of the addition salt of trifluoroacetic acid, which can, if if desired, convert to the form of the free base. The latter tripeptide is reacted with Boc-Gly-OTcp in the manner mentioned above to obtain the lower alkyl ester protected from glycyl-glycyl-alanyl-glycine, preferably Boc-Gly-Gly-Ala-Gly-OMe, which by treatment with trifluoroacetic acid as mentioned above, gives the lower alkyl ester of glycyl-glycyl-alanyl-glycine, preferably H —Gly — Gly — Ala — Gly —OMe, in the form of its salt d addition of trifluoroacetic acid, and which, if it is

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wishes, can be converted back to the free base form again. The latter tetrapeptide is reacted with an activated glycine protected ester, preferably Boc-Gly-OTcp, as previously mentioned, to give the lower alkyl ester of glycyl-glycyl-glycyl-alanyl-glycine, preferably Boc —Gly— Gly — Gly — Ala — Gly — OMe.

The tetrapeptide stripped of its protection, mentioned above, H —Gly — Gly — Ala — Gly — OMe, preferably in the form of the addition salt of trifluoroacetic acid, is reacted with an activated activated ester of leucine , preferably 1-benzotriazolyl ester of t-butyloxycarbonyl-leucine, by combining, at approximately 0-15 ° C. in an inert organic solvent, preferably dimethylformamide, the preceding tetrapeptide, stripped of its protection, approximately 1, 5 to 2 equivalents of Boc — Leu — OH,

about 1.5 to 2 equivalents of 1-hydroxybenzotriazole, about 1.5 to 2.5 equivalents of dicyclohexylcarbodiimide, and an excess of an organic base, preferably N-ethylmorpholine, to bring the pH of the solution to about 8. The resulting mixture is kept at about 0-15 ° C for 20 to 30 h. Separation of the precipitate, evaporation of the filtrate and crystallization gives the protected lower alkyl ester of leucyl-glycyl-glycyl-alanyl-glycine, preferably Boc-Leu-Gly-Gly-Ala-Gly-OMe.

The previously mentioned tetrapeptide, of formula Boc— Gly — Gly — Ala — Gly — OMe and the pentapeptides of the formulas Boc — Gly — Gly — Gly — Ala — Gly — OMe and Boc — Leu — Gly— Gly — Ala — Gly — OMe will be designated below by R5 — Gly— Gly — Ala — Gly — OMe, where R5 denotes Boc, Boc — Gly and Boc — Leu respectively.

These latter compounds of formula R5 — Gly — Gly — Ala— Gly — OMe, where R5 has the definition given, are easily transformed into the corresponding hydrazides by reaction with an excess (20 to 50 molar equivalents) of hydrazine hydrate. Preferred conditions are a treatment of the latter esters in an inert organic solvent, for example methanol, n-butanol or dimethylformamide, with 40 to 50 molar equivalents of hydrazine hydrate at a temperature of 0-30 ° C. , for approximately 2 hours to approximately 1 day. The separation of the solvent and the excess hydrazine hydrate gives the hydrazide of the corresponding amino protected peptide of formula IX:

R5 — Gly — Gly — Ala — Gly — NHNH2 (IX)

where R5 = Boc, Boc — Gly or Boc — Leu.

The previously mentioned peptide of formula IX and a penta-peptide of formula H — Cys (Trt) —Lys (Boc) —Asn - Phe — Phe— OMe (described by HU Immer et al., Above quotation) are combined according to the method of combination with the azide to give the peptide of formula R5 —Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) —Asn - Phe — Phe — OMe.

A convenient and efficient method for this operating phase comprises dissolving the peptide of formula IX in an inert organic solvent, preferably dimethylformamide or a mixture of dimethylformamide and dimethyl sulfoxide. A solution of about 2 to 5 molar equivalents, preferably 3 molar equivalents, of a strong mineral acid in an organic solvent, preferably hydrochloric acid in ethyl acetate, is added to the last solution mentioned at a temperature of -20 to -10 ° C, preferably about -15 ° C, and to the stirred solution is added an organic nitrite, preferably t-butyl nitrite (1 to 1.5 molar equivalents , preferably 1.2 equivalent). After about 15 min at a temperature of -20 to 0 ° C, the mixture is made alkaline, preferably up to a pH of 7-7.5, with an excess of an organic base, preferably N- ethyldiisopropylamine, followed by addition of approximately 1 equivalent of the penta-peptide mentioned above. A further addition of the organic base, preferably N-ethylmorpholine, can be made to make the mixture slightly alkaline. The reaction mixture is then stirred at a temperature of -10 to 0 ° C for 1 to 2 h, and then at 20 to 30 ° C for 20 to 30 h. Evaporation of the solvent,

taking up the remainder in an organic solvent, preferably methanol, adding to a solvent in which precipitation develops, preferably water or diethyl ether, and collecting the precipitate give the peptide mentioned above of the formula R5 — Gly — Gly — Ala — Gly — Cys (Trt) —Lys (Boc) - Asn — Phe — Phe — OMe.

In summary, the required pentapeptide fragment is easily obtained by reacting an activated ester of Boc-Phe-OH with H-Phe-OMe, to obtain Boc-Phe-Phe-OMe, which, after separation from the protecting group terminal (Boc) under moderately acidic conditions, gives the H — Phe — Phe — OMe. In turn, the latter compound is reacted with an activated ester of Boc-Asn-OH to obtain Boc-Asn-Phe-Phe-OMe. Subsequent separation of the amino terminal protecting group from the last mentioned compound under mildly acidic conditions gives the tripeptide H-Asn-Phe-Phe-OMe.

The latter compound is then used to obtain the desired pentapeptide fragment by reacting this tripeptide with an activated ester of Z — Lys (Boc) —OH to obtain Z — Lys (Boc) —Asn— Phe — Phe — OMe, in hydrogenating the latter compound in the presence of a noble metal catalyst to obtain H — Lys (Boc) —Asn - Phe — Phe — Orne, by condensing the latter compound with an activated ester of Trt — Cys (Trt) —OH for obtaining Trt — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — OMe, and separating the terminal N-protecting group (Trt) from the last compound mentioned under weakly acidic conditions to obtain the desired pentapeptide H— Cys (Trt) —Lys (Boc) —Asn - Phe — Phe — OMe.

Returning now to the preparation of the compounds of formulas I, the peptide ester mentioned above, R5 — Gly — Gly— Ala — Gly — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe,

where R5 has the definition given, is transformed into the corresponding hydrazide by reaction with an excess of hydrazine hydrate in the same manner as described above to obtain the peptide hydrazide of formula IVa, R5 — Gly — Gly — Ala —Gly — Cys- (Trt) —Lys (Boc) —Asn — Phe — Phe — NHNH2, where R5 has the definition given, which can also be written in the form of the peptide of formula IV, in which R4 represents Boc —Gly — Gly — Ala— Gly-NH, Boc — Gly — Gly — Gly - Ala — Gly — NH or Boc — Leu — Gly — Gly — Ala — Gly — NH.

In the synthesis of the starting material for the process of the invention, the last mentioned compound of formula IV and a peptide of formula V, H — Trp — Lys (Boc) —Thr (Bu1) —Phe — Thr (Bu ') - Ser (Bu ') - NHCH (R2) CH2S — Trt, where R2 represents H or COOH, are combined according to the azide combination method, to give the linear peptide of formula Ha, R5 —Gly — Gly — Ala - Gly — Cys (Trt) —Ly s (Boc) —Asn — Phe — Phe — Trp — Ly s (Boc) - Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - NHCH ( R2) CH2S - Trt, where R5 has the definition given and R2 represents H or COOH, which can also be written in the form of the linear peptide of formula II, in which R2 represents H or COOH and R4 represents Boc — Gly— Gly — Ala — Gly — NH, Boc-Gly-Gly-Gly-Ala-Gly — NH or Boc — Leu — Gly — Gly — Ala — Gly — NH.

A convenient and effective method for this operating phase involves dissolving the peptide hydrazide of formula IVa in dimethylformamide. To this latter solution, a solution of about 2 to 5 molar equivalents, preferably 3 molar equivalents, of a strong mineral acid in an organic solvent, preferably HCl in ethyl acetate, is added to a temperature of - 20 to - 10 ° C, preferably about - 15 ° C, and to the stirred solution is added t-butyl nitrite (1 to 1.5 mol equivalent, preferably 1.2 equivalent). After approximately 15 min at a temperature of -20 to 10 ° C, a solution of approximately 1 equivalent of the peptide of formula V and an organic base, preferably 3 to 5 equivalents of N-ethyldiisopropylamine, is added in dimethylformamide. cooled to approximately - 15 ° C.

The reaction mixture is then stirred at a temperature of -20 to 0 ° C for 1 to 2 hours, and then to 20-30 ° C for 15 to 25 hours. Evaporation of the solvent, trituration of the rest with water,

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methanol or a mixture of methanol and aqueous citric acid (2 to 5%) and the separation of the solid matter gives the previously mentioned linear peptide of formula IIa which can be used without further purification for the next phase, presented below.

In summary, the above-mentioned peptide of formula V, H — Trp - Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH (R2) CH2S — Trt, where R2 represents COOH, which can also be written in the form of the heptapeptide of formula Va, H - Trp - Lys (Boc) - Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ' ) -Cys (Trt) -OH, described by HU Immer et al., Above quotation, as well as in the patent of the United States of America N ° 3917578 granted on November 4, 1975, is easily obtained by reacting the methyl ester of Ot-butylserine with an activated ester of benzyloxycarbonyl - {0-t-butyl) threonine to obtain Z — Thr (Bu ') - Ser (Bu') —OMe. The amino terminal protecting group (Z) of the latter compound is then separated by hydrogenation in the presence of a noble metal catalyst to give the H — Thr (Bu ') - Ser (Bu') - Ser (Bu ') —Ome.

The above-mentioned methyl ester is then reacted with an activated ester of Z — Phe — OH to obtain the Z — Phe — Thr (Bu ') - Ser (Bu') - OMe, from which the amino protecting group is then separated. terminal (Z) by hydrogenation in the presence of a noble metal catalyst to obtain H — Phe — Thr (Bu ') - Ser (Bu') - OMe. The latter tripeptide ester is then reacted with an activated ester of Z — Thr (Bu ') - OH to obtain Z — ThrfBu') - Phe — Thi ^ Bu ') - Phe — Thr (Bu') - Ser ( Bu ') - OMe. Again, the amino terminal protecting group (Z) of the latter compound is separated by hydrogenation in the presence of a noble metal catalyst to give the Z-Thr (Bu ') - Phe — Thr (Bu') - Sci ^ Bu ') - OMe. The latter compound is reacted with an activated ester of Z — Lys (Boc) —OH to obtain the Z — Lys (Boc) - Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - OMe , followed by separation of the amino terminal protecting group (Z) from the latter compound by hydrogenation in the presence of a noble metal catalyst to obtain the H — Lys (Boc) —Thr (Bu ') —Phe — Thr (Bu1 ) - Ser (Bu ') - OMe. The latter compound is then reacted with an activated ester of Ddz — Trp — OH to obtain Ddz — Trp— Lys (Boc) —Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - OMe which is reacted, in turn, with hydrazine hydrate, so as to isolate the hydrazide of corresponding hexapeptide, Ddz — Trp — Lys- (Boc) - Thr (Bu ') - Phe - ThitBu ') - Ser (Bul) - NHNH2. . This hexapeptide is then combined with H —Cys (Trt) —OH according to the azide combination method to obtain the corresponding heptapeptide, Ddz — Trp - Lys (Boc) —Thr (Bu ') - Phe— Thr ( Bu ') - Ser (Bu') - Cys (Trt) —OH. Treatment of the latter compound under weakly acidic conditions gives the desired heptapeptide of formula Va or the peptide of formula V, in which R2 represents COOH.

In summary, the above-mentioned peptide of formula V, in which R 2 represents H, described in US Patent No. 3,917,581 issued November 4, 1975, is readily obtained by combining the hydrazide of previously described hexapeptide of formula Ddz — Trp — Lys (Boc) —Thr (Bu ') - Phe— Thr (Bu') - Ser (Bu ') - NHNH2 with 2-tritylthioethylamine according to the azide combination method , described above, to obtain the corresponding hexapeptide of formula Ddz - Trp - Lys (Boc) - Thr (Bu ') - Phe - Thr (Bul) - Ser (Bu') -NHCH2S — Trt. The latter compound, when reacted under weakly acidic conditions, gives the peptide of formula V, in which R2 represents H, in the form of the formic acid addition salt which is converted, if it is desire, in the form of the free base.

Alternatively, the previously described linear peptide of formula IIa is easily prepared in the following manner.

The protected lower alkyl ester of the pentapeptide of formula Trt — Cys (Trt) - Lys (Boc) - Asn - Phe — Phe — OMe, described above, is easily converted into the corresponding hydrazide by reaction with an excess of hydrate d 'hydrazine. The preferred conditions are the treatment of this ester in an inert solvent, for example methanol, butanol or dimethylformamide, with 20 to 40 molar equivalents of hydrazine hydrate at a temperature of 0 to 30 ° C for 1 to 2 d. Separation of the solvent and crystallization gives the corresponding pentapeptide hydrazide of formula VI, Trt — Cys (Trt) —Lys (Boc) —Asn — Phe— Phe — NHNH2.

The latter compound and the peptide of formula V, in which R2 represents H or COOH, are combined according to the azide combination method, as described above, to obtain the corresponding peptide of formula VII, Trt-Cys (Trt ) - Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH (R2) CH2S — Trt, where R2 represents H or COOH, with then separation of the N-protecting terminal group (Trt) of the latter compound of formula VII in order to obtain the corresponding peptide of formula VIII, H — Cys (Trt) —Lys (Boc) —Asn— Phe — Phe — Trp — Lys (Boc) - Thr (Bu ') - Ser (Bu') - NHCH (R2) CH2S — Trt, in which R2 represents H or COOH. The separation of the protective group (Trt) is easily carried out under weakly acidic conditions. The preferred conditions are the dissolution of the peptide of formula VII in a mixture of 5 to 15% of formic acid in 60 to 80% of acetic acid, leaving the solution to stand at 20-30 ° C for 3 to 10 h. Concentration of the solution gives the peptide of formula VIII, in which R2 represents H or COOH, in the form of its addition salt of formic acid, which is converted, if desired, to the form of free base.

The latter compound of formula VIII and a peptide hydrazide of formula IX, in which R5 has the definition given, are combined according to the azide combination method as described above, to give the corresponding linear peptide of formula Ha or of formula II, where R2 represents H or COOH and R4 represents Boc — Gly — Gly — Ala — Gly — NH—, Boc — Gly — Gly — Gly — Ala — Gly — NH— or Boc — Leu — Gly— Gly — Ala —Gly — NH -.

The conversion of this latter linear peptide of formula II, obtained by one or other of the methods described above, into the corresponding compound of formula I is carried out conveniently and efficiently by first submitting the linear peptide of formula II to the action of iodine, preferably in the presence of a lower alkanol or acetic acid, so that a separation of the protective groups of sulfhydryl, that is to say Trt, develops, and in at the same time the formation of the disulfide bridge, to give the corresponding cyclic disulfide derivative of formula III:

SCH2CH (R4) CO - Lys (Boc) - Asn - Phe - Phe - Trp - Lys (Boc) -

Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - NHCH (R2) CH2S (III)

where R2 = H or COOH and R4 = Boc-Gly-Gly-Ala-Gly-NH, Boc — Gly — Gly — Giy — Ala — Gly — NH or Boc — Leu — Gly— Gly — Ala — Gly — NH. The latter compound of formula III, where R2 = COOH and R4 = Boc — Gly — Gly — Ala — Gly - NH,

Boc — Gly - Gly — Gly — Ala — Gly — NH or Boc — Leu — Gly— Gly — Ala — Gly — NH can still be written under the formula Illa:

R5 — Gly — Gly — Ala — Gly — C ys - Lys (Boc) - Asn — Phe — Phe—

I!

Trp — Lys (Boc) —Thr (Bul) - Phe — Thr (Bul) —Ser (Bul — Cys - OH (Illa)

where Rs has the definition already given.

The further processing of the compound of formula III, in which R2 represents H or COOH and R4 represents Boc — Gly — Gly— Ala-Gly-NH, Boc - Gly-Gly-Gly-Ala-Gly-NH or Boc — Leu —Gly —Gly — Ala — Gly — NH under mildly acidic conditions separates the remaining protecting groups (ie Boc and Bu1) to give the corresponding compound of form

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mule I, in which R1 represents H —Gly — Gly — Ala — Gly— NH, H — Gly — Gly — Gly — Ala — Gly — NH or H — Leu — Gly— Gly — Ala-Gly-NH and R2 = H or COOH.

In a preferred embodiment of the preceding transformation, the linear peptide of formula II is dissolved in acetic acid or methanol, ethanol or another suitable lower alkanol, for example propanol, isopropanol or butanol, and the solution is added to an excess of iodine (5 to 25 molar equivalents, preferably 10) dissolved in one of the solvents mentioned above, preferably 2 to 5% of iodine in methanol. The duration and the temperature of this reaction are not critical; however, it is desirable to maintain the reaction between 0 and 30 ° C by controlling the addition of the iodine solution or by cooling the reaction mixture, or alternatively by providing a combination of these two solutions. Under these conditions, the addition usually takes 30 to 60 minutes. After the addition, the mixture is stirred at 20-30 ° C for 30 to 120 minutes, preferably for 60 minutes. The mixture is then cooled to about 0 ° C and an excess of a moderate reducing agent, preferably sodium thiosulfate in aqueous solution, is added. The mixture is concentrated and the rest is suspended in water. Harvesting the solid gives the desired corresponding cyclic disulfide derivative of formula IIIa, in which the protecting groups Boc and Bu 'are still present.

Alternatively, the linear peptide of formula Ha can be converted to the corresponding cyclic disulfide derivative mentioned above of formula Illa, by the method of R.G. Hiskey and R.J. Smith, "J. Bitter. Chem. Soc. ”, 90.2677 (1968) using thiocyanogen.

Finally, the above cyclic disulfide derivative of formula Illa is transformed into the corresponding peptide of formula I by subjecting this compound of formula Illa to moderately acidic conditions, so that the remaining protecting groups of the cyclic disulfide derivative are separated. Generally, this phase is carried out by dissolving the cyclic disulfide derivative in an aqueous reaction medium containing an organic acid at 0-20 ° C for 10 to about 60 minutes. Examples of such media are trifluoroacetic acid, 10-20% aqueous sulfuric acid, 10% phosphoric acid, 10-30% hydrobromic acid and 10-36 hydrochloric acid %. An extremely interesting medium consists of concentrated hydrochloric acid. The preferred conditions for this phase are the dissolution of the cyclic disulfide in a minimum of concentrated hydrochloric acid cooled to 0 ° C and the stirring of the mixture at 0 ° C for 5 to 10 min under a nitrogen atmosphere. Glacial acetic acid (10 volumes) is then added and the solution is cooled to about -70 ° C., then lyophilized to give the cyclic peptide of formula I. The latter compound is then purified by exchange chromatography ion, preferably using a cation exchanger based on carboxymethylcellulose and ammonium acetate as eluent. In this case, the product is obtained in the form of its acid addition salt with acetic acid. Alternatively, the product is purified by partition chromatography on chemically modified crosslinked dextran, for example Sephadex LH-20 or Sephadex G-25, using methanol or acetic acid respectively as solvent. elution. In the case where Sephadex LH-20 and methanol are used as the elution solvent, the product is obtained in the form of its hydrochloric acid addition salt. In the case where Sephadex G-25 and acetic acid are used, the product is obtained in the form of its acetic acid addition salt. Repeated lyophilization of the product in the form of its acetic acid addition salt gives an essentially pure peptide of formula I, in which R1 represents H-Gly-Gly-Ala-Gly-NH, H-Gly-Gly-Gly —Ala — Gly — NH or H — Leu — Gly— Gly — Ala — Gly — NH and R2 represents H or COOH, in the form of the free base.

b) Compounds of formula I in which R1 represents H or NHR3,

where R3 has the definition given above, and R2 represents H

The required peptide hydrazide of formula IV, Trt - SCH2CH (R4) CO - Lys (Boc) - Asn - Phe- Phe - NHNH2, in which R4 represents NHR3, where R3 has the definition given, which can still be write in the form of formula IVb, R3 - Cys (Trt) - Lys (Boc) - Asn - Phe - Phe - NHNH2, in which R3 has the definition given above, is prepared by acylation of the pentapeptide of formula H — Cys (Trt ) —Lys (Boc) - Asn — Phe — Phe — OMe, to obtain the pentapeptide of formula R3 — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe, which is subjected to hydrazinolysis to obtain the pentapeptide derivative of formula IV, in which R4 represents NHR3.

In a preferred embodiment of the preparation of the above pentapeptide IVb hydrazide, a mixture of substantially equimolar amounts of an organic base, preferably N-ethylmorpholine, and the pentapeptide of formula H — Cys is treated. (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe,

prepared as described by H.U. Immer et al., Previous quote, in an inert organic solvent, preferably dimethylformamide or tetrahydrofuran, at about 0-10 ° C, with an excess, preferably 1.2 to 2 molar equivalents, of acylate or benzoate of desired p-nitrophenyl, for example p-nitrophenyl acetate, prepared as described by FD Chattaway, "J. Chem. Soc. ”, 2495 (1931). The mixture is stored at 0-10 ° C for about 15 to 30 h and subjected to evaporation. The remainder is taken up in a polar organic solvent, preferably methanol, and added slowly to a non-polar organic solvent, preferably diethyl ether. The rest is collected and crystallized to obtain the corresponding acylated or benzoylated pentapeptide, for example the pentapeptide of formula R3 - Cys (Trt) - Lys (Boc) —Asn - Phe — Phe — OMe, in which R3 has the definition given above . The latter compound is dissolved in an inert organic solvent, for example methanol, ethanol, dimethylformamide, etc., preferably methanol. The solution is treated with an excess of hydrazide hydrate, for example 15 to 30 molar equivalents. The reaction mixture is kept at about 0-10 ° C for about 40 to 60 h. The precipitate is collected and dried to obtain the hydrazide of pentapeptide of formula IV, in which R4 represents NHR3, where R3 has the definition given, or of formula IVb, in which R3 has the definition given.

The required peptide hydrazide, namely the tetrapeptide of formula IV, Trt - S - CH2CH (R4) CO - Lys (Boc) - Asn - Phe -Phe — NHNH2, in which R4 represents hydrogen, is prepared by reacting an activated ester of 3-tritylthio-propionic acid with a tetrapeptide of formula H —LysfBocJ-Asn — Phe — Phe — OMe, to obtain the tetrapeptide of formula Trt — SCH2CH2CO — Lys (Boc) —Asn - Phe — Phe —Ome,

which is subjected to hydrazinolysis to obtain the aforementioned tetrapeptide hydrazide of formula IV, in which R4 represents hydrogen.

In a preferred embodiment of the preparation of the abovementioned tetrapeptide hydrazide, the activated ester of 3-triltylthiopropionic acid, preferably the penta-chlorophenyl ester, is prepared by combining practically equimolar amounts of acid. 3-tritylthiopropionique, pentachlorophenol and dicyclohexylcarbodiimide, in an inert organic solvent, preferably tetrahydrofuran, at approximately 0-10 ° C. The mixture is stirred at approximately 0-10 ° C for approximately 1 h and then at approximately 20- 30 ° C for approximately 1 hour. The mixture is then cooled to about 0 ° C, filtered, and the filtrate is evaporated. The remainder is crystallized to obtain pentachlorophenyl ester of 3-tritylthiopropionic acid. A solution of the latter compound and of a practically equimolar amount of the tetrapeptide of formula H — Lys (Boc) —Asn - Phe — Phe — OMe acetate, described above under a), in an inert organic solvent, preferably dimethylformamide or tetrahydrofuran, is treated with an amount

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practically equimolar of an organic base, preferably triethylamine, at approximately 20-30 ° C. The mixture is stirred for 2 to 3 days at approximately 20-30 ° C. and the solvent is evaporated under reduced pressure. The rest is triturated with cold dilute aqueous citric acid and water, dried and crystallized to obtain the tetrapeptide of formula Trt-SCH2CH2CO-Lys (Boc) - Asn-Phe-Phe-OMe. The latter compound is dissolved in an inert organic solvent, for example methanol, ethanol or, preferably, dimethylformamide. The solution is treated with an excess of hydrazine hydrate, for example 15 to 30 molar equivalents. The reaction mixture is stored at about 20-30 ° C for about 20-30 h and evaporated under reduced pressure. The rest is triturated with cold water and dried to obtain the tetrapeptide hydrazide of formula IV, in which R4 represents H.

In the synthesis of the starting materials for the process of the invention, the first peptide hydrazide above, of formula IV in which R4 represents H or NHR3, and the second peptide of formula V, H — Trp — Lys (Boc ) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH2CH (R2) S — Trt, in which R2 represents H, described above under a), are combined according to the method of combination with azide, described above, to obtain the corresponding linear peptide of formula II, in which R2 represents H and R4 represents H or NHR3.

The conversion of this last mentioned linear peptide of formula II into the corresponding compound of formula I is carried out conveniently and efficiently by first subjecting this linear peptide of formula II to the action of iodine, preferably in the presence of methanol [as described above for the preparation of the cyclic disulfide derivative of formula III, under a)], so that the separation of the sulfhydryl protecting group, i.e. Trt, occurs and the formation of the bridge of disulfide, which gives the corresponding cyclic disulfide derivative of formula III, in which R2 represents H and R4 represents H or NHR3, where R3 has the definition given previously. The latter compound of formula III, where R4 represents NHR3, can also be written in the form of the compound Illb, namely R3 — C ys — Lys (Boc) —Asn - Phe — Phe — Trp — Thr— (Bu1) -

Phe - Thr (Bu ') - Ser (Bu') ~ NH - CH (R2) CH2 S (IHb),

where R2 represents H and R3 has the definition given.

Subsequent treatment of the latter compound of formula III, in which R2 represents H and R4 represents H or NHR3, under moderately acidic conditions, that is to say preferably using concentrated hydrochloric acid cooled to about 0 ° C [as described above for the preparation of the cyclic peptide of formula I under a)], separates the remaining protective groups (that is to say Boc and Bu ') to give the corresponding peptide of formula I, in which R1 represents H or NHR3, where R3 has the definition given and R2 represents H, in the form of the free base or the acid addition salt.

c) Compounds of formula I in which R1 represents

Boc — Ala — Gly — NH and R2 = COOAlk

The first required peptide hydrazide of formula IV, in which R4 represents Boc — Ala — Gly — NH, which can also be written in the form of a heptapeptide of formula IVc, Boc — Ala— Gly — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — NHNH2, has been described by HU Immer and others, previous quote.

The second required peptide of formula V, in which R2 represents COOAlk, which can also be written in the form of a heptapeptide ester of formula Vb, H — Trp — Lys (Boc) -

Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - Cys (Trt) - COOAlk, is easily prepared in the following way.

The heptapeptide of formula Ddz — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') —Ser (Bu ') - Cys (Trt) —OH [described previously under a)] in solution in an inert solvent is reacted with a diazoalkane of the formula (Alk minus H) N2 in solution in an inert solvent at temperatures from 0 to 20 ° C over periods of

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time from 1 to 48 h, with then evaporation of the solvents and of the excess of diazoalkane, separation of the protective group Ddz, and purification to obtain the corresponding heptapeptide ester of formula Vb.

Diazoalkanes of interest for this purpose are, for example, diazomethane, diazoethane [von Pechmann, "Ber. Deutsch. Chem. Ges. ", 31, 2640 (1898)], 1-diazopropane [Niedlinger et al.," Am. Chem. J. ", 43, 378 (1910)], 2-diazopropane [Staudinger et al.," Ber. Deutsch. Chem. Ges. ”, 49,1905 (1916)], 1-diazobutane [Niedlinger et al., Above quotation, p. 380], 1-diazo-isobutane [Adamson et al., "J. Chem. Soc. ”, 1935, 286], 4-diazo-2-methylbutane [Werner,“ J. Chem. Soc. ”, 115,1101 (1919)], 1-diazopentane, 1-diazo-hexane, 1-diazoheptane or 1-diazooctane, the last four diazoalkanes being prepared as described by Adamson et al., Above quotation. The preferred inert solvents are alkanols, such as methanol, and ethers, such as diethyl ether.

As a variant, and in particular when it is desired to obtain the peptide of formula I, where R1 represents H —Ala — Gly — NH and R2 = COOAlk, where the alkyl group (Alk) contains from 9 to 14 carbon atoms, cysteine is reacted with a molar excess of the appropriate alkanol, AlkOH, where Alk has the above definition, in the presence of HCl at 25-120 ° C, preferably at 100-120 ° C, for 1 to 5 h , to obtain the corresponding cysteine alkyl ester hydrochloride. The latter compound is then treated with approximately 1 molar equivalent of triphenylcarbinol in the presence of 0.1-1 molar equivalent of boron trifluoride etherate at 10-30 ° C for 0.5-3 h, preferably with 0.1 molar equivalent at 25 ° C for 1 h, in order to obtain the corresponding alkyl ester, H — Cys (Trt) —OAlk.

The latter alkyl ester of a protected cysteine is then combined, using the azide method, with the protected hexapeptide hydrazide of formula Ddz — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu ') —Ser (Bu') - NHNH2, described above under a), to obtain, after separation of the protective group Ddz under weakly acidic conditions, the second desired heptapeptide ester of formula Vb. Preferred conditions for the above reaction sequence include the use of substantially equimolar proportions of this protected hexapeptide hydrazide and t-butyl nitrite in the presence of a mineral acid, preferably 2 to 3 molar equivalents of HCl, in an inert anhydrous solvent, preferably dimethylformamide, followed by the addition of the appropriate alkyl ester of the formula H — Cys (Trt) —OAlk, with 2 to 3 molar equivalents of an organic base, preferably of the N-ethyldiisopropylamine, in an inert anhydrous solvent, preferably dimethylformamide, at a temperature of -20 to 25 ° C for 6 to 12 hours, this operation being followed by a purification of the alkyl ester of protected heptapeptide resulting from formula Ddz — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - Cys (Trt) —OAlk.

The latter compound is then subjected to weakly acidic conditions, preferably by stirring in a mixture of acetic acid, formic acid and water (7/1/2) for 12 to 18 h at 20-25 ° C , to obtain the corresponding alkyl ester of heptapeptide of formula Vb.

In the synthesis of the starting materials for the process according to the invention, the first hydrazide of the above peptide of formula IVc and the second heptapeptide ester of formula Vb are combined according to the azide method, described above, to obtain the corresponding linear peptide of formula II, in which R4 = Boc — Ala — Gly — NH and R2 = COOAlk, which can also be written in the form of the linear tetradecapeptide of formula Ile Boc — Ala — Gly — Cys (Trt) —Lys (Boc) —Asn - Phe— Phe - Trp - Lys (Boc) - Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') -Cys (Trt) —OAlk.

The conversion of the preceding linear tetrapeptide of formula Ile to the corresponding compound of formula I, in which R1 = H — Ala — Gly — NH and R2 = COOAlk, is done conveniently and efficiently by first subjecting the last tetradeca-

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linear peptide mentioned under the action of iodine, preferably in the presence of methanol [as described above for the preparation of the cyclic disulfide derivative of formula III under a)], so that a separation of the protective group occurs of sulfhydryl, i.e. Trt, and the formation of the disulfide bridge, to form the corresponding cyclic disulfide derivative of formula III, wherein R2 = COOAlk and R4 = Boc — Ala — Gly— NH, that l '', it is also possible to write in the form of the cyclic disulfide derivative of formula IIIc:

Boc — Ala — Gly — C ys — Ly s (Boc) —Asn - Phe — Phe — Trp—

Lys (Boc) —Thr (Bu ') -phe — ThrfBu') - Ser (Bul) —Cys - OAlk.

Subsequent treatment of the latter compound under moderately acidic conditions, preferably with concentrated hydrochloric acid cooled to about 0 ° C [as described above for the preparation of the cyclic peptide of formula I under a)] separates the groups remaining protectors (i.e. Boc and Bu '), and further purification, as described under a), gives the peptide of formula I, in which

R1 = H — Ala — Gly — NH and R2 = COOAlk, which can also be written in the form of the formula:

H - Ala - Gly - Cys- Lys — Asn - Phe — Phe -

5 I!

Trp — Lys — Thr — Phe — Thr — Ser — Cys — OAlk,

and this in the form of the free base or of an acid addition salt thereof.

In the previous process described under c), the protective group 10 Bu 'for the hydroxyl radical of serine and threonine can be omitted without harmful effects, that is to say that Ser (Bu') and Thr (Bu ') are replaced by Ser and Thr respectively. As regards still the process according to c), the protective group Trt of the residues of cysteine amino acid can be replaced by the pro-tectorAcm group.

The processes described above under a) to c) are represented by the following reaction schemes:

a) R1 = H — Gly — Gly — Ala — Gly — NH, H — Gly — Gly — Gly— 20 Ala — Gly — NH and H — leu — Gly — Gly — Ala — Gly — NH and R2 = H or COOH

R5 — Gly — Gly — Ala — Gly — Cy s — Lys — Asn — Phe — Phe Trp — Lys — Thr — Phe — Thr — Ser — NHCH (R2) CH2S — Trt

IVa

Ila- »Illa-» I

b) R1 = H or NHR3 and R2 = H

Trt-SCH2CH2CH (R4) CO-Lys-Asn-Phe-Phe Trp - Lys - Thr - Phe - Thr - Ser - NHCH (R2) CH2S - Trt IV

1

II-> III-

c) R1 = H — Ala — Gly — NH and R2 = COOAlk

Boc — Ala — Gly — Cys - Lys — Asn — Phe — Phe Trp — Lys — Thr - Phe — Thr — Ser — Cys — OAlk

IVc

T

Vb

IIc-> IIIc- »I

Finally, it will be obvious to specialists in this field that equivalent amino, hydroxy or thiol protecting groups, equivalent methods of combining peptide fragments and equivalent methods of separating amino, hydroxy or protecting groups thiol, other than those described above, can be used in the embodiments of the present invention without thereby departing from the scope thereof.

The diagram following the examples illustrates the invention even more completely.

Linear peptide II /

Oxidation

Disulfide derivative Cyclic oxidation (III) ')

| Reduction

Removal of protection

Peptide (I)

Preparation 1:

Alanyl glycine methyl ester trifluoroacetate (H-Ala- Gly - OMe ■ CF3 COOH)

1) Ag + or Hg + +

2) H2S

Disulfhydryl derivative

45 Boc — Ala — Gly — OMe is dissolved [10 g, 45 mmol, described by H.U. Immer et al., "Helv. Chim. Acta ”, 57, 730 (1974)] in cold trifluoroacetic acid (ice bath) (100 ml). The solution is stirred for 1 h at 0 ° C and the solvent is evaporated. The remainder is dissolved in methanol, added to diethyl ether so and the precipitate is collected to obtain the title compound. Preparation 2:

T-butyloxycarbonyl-glycyl-alanyl-qlycine methyl ester (Boc -Gly-Ala- Gly - OMe)

J5 To a cold solution (ice bath) of H — Ala — Gly— 0Me-CH3C02H (45 mmol, described in preparation 1 in 50 ml of dimethylformamide, N-ethylmorpholine (pH approximately 8) is added, then a cold solution of Boc-Gly-OTcp (18 g, 45 mmol) in 50 ml of dimethylformamide, the solution is stored in an ice bath for 2 days, the solvent is evaporated off and the product is purified by chromatography. column on silica gel using ethyl acetate / methanol / pyridine (98/1/1) The product is crystallized from ethyl acetate / petroleum ether to obtain the compound mentioned in the heading; Mp: 98-100 ° C; 65 NMR (DMSO-dg): 1.25 5 (3H); 1.4 5 (9H); 3.68 5 (3H).

Preparation 3:

Glycyl-alanyl-glycine methyl ester trifluoroacetate (H — Gly — Ala — Gly — OMe- CF 3COOH)

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Boc — Gly — Ala — Gly — OMe (6.4 g, 20.1 mmol, described in Preparation 2) is dissolved in cold trifluoroacetic acid (ice bath) (120 ml) the solution at 0 ° C. for 1 hour The solvent is evaporated, the remainder is dissolved in methanol and the product is precipitated by the addition of diethyl ether, the precipitate is collected to obtain the compound cited in the title.

Preparation 4:

T-butyloxycarbonyl-glycyl-glycyl-alanyl-glycine methyl ester (Boc — Gly — Gly — Ala — Gly — OMe)

To a cold stirred solution (ice bath) of H —Gly — Ala— Gly — 0Me CH3CO2H (6.4 g, 20.03 mmol, described in preparation 3 in dimethylformamide (30 ml), 2.8 is added. ml of N-ethylmorpholine, then a solution of Boc-Gly-OTcp (8.5 g, 24 mmol) in 20 ml of dimethylformamide, the solution is kept in an ice bath for 2 days, the solvent is evaporated and dissolved. the remainder in methanol and the product is precipitated with diethyl ether. Crystallization from ethyl acetate gives the title compound; mp: 103-105 ° and 141 ° C (dimorphic); NMR: 1.25 8 (3H), 1.38 S (9H), 3.65 5 (3H).

Preparation 5:

T-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycine hydrazide; IX, R5 = Boc (Boc - Gly -Gly-Ala- Gly -NH- NH2)

To a stirred, cooled solution (ice bath) of Boc-Gly-Gly-Ala-Gly-OMe (2.5 g, 6.7 mmol, described in preparation 4 in methanol (50 ml), 2 is added. , 5 ml of hydrazine hydrate The solution is stirred at 0 ° C. for 3 h and at room temperature overnight. The precipitate is separated by filtration, washed with methanol and dried to obtain the compound cited. section; amino acid analysis; Gly 3, Ala 0.99.

Preparation 6:

T-butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N ^ -t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanine methyl ester (Boc — Gly — Gly — Ala — Gly— Cys (Trt) -Lys (Boc) —Asn — Phe — Phe — OMe)

To a solution of Boc-Gly-Gly-Ala-Gly-NH-NH2 (1.57 g, 4.2 mmol, described in preparation 5) in dimethyl sulfoxide (25 ml) and dimethylformamide (25 ml) , a 2.04N solution of HCl in ethyl acetate (5.15 ml) is added to −10 ° C. The solution is cooled to −12 ° C. and t-butyl nitrite (0 , 61 ml, 5.2 mmol). The solution is stored at -10 ° C for 15 min, it is cooled to -15 ° C and N-ethyldiisopropylamine (1.8 ml, pH 8) is added, followed by a solution of H — Cys (Trt ) - Lys (Boc) —Asn-Phe-Phe — OMe [4.5 g, 4.18 mmol, described by HU Immer et al., "Helv. Chim. Acta. ”, 57,730 (1974)] and N-ethyldiisopropylamine (0.72 ml) in dimethylformamide (25 ml). The mixture is stirred for 1 h at -10 ° C and overnight at room temperature. After evaporation, the remainder is dissolved in methanol, the product is precipitated with water and crystallized from methanol to obtain the title compound; amino acid analysis; Lys, 1.07; Asp, 0.97; Gly, 3.27; Ala, 1; Phe, 2.08; cysteic acid, 1.35.

Preparation 7:

T-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-Ne-t-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanine hydrazide; IV, R4 = Boc — Gly — Ala — Gly— NH (Boc — Gly — Gly — Ala — Gly — Cys (Trt) —Iys (Boc) - Asn - Phe - Phe -NH- NH2)

To a cold solution (ice bath) of Boc — Gly — Gly— Ala — Gly — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe (0.9 g, 0.66 mmol, described in Preparation 6) in dimethylformamide (25 ml), 1.5 ml of hydrazine hydrate is added. The solution is stirred at room temperature for 18 h. The solvent is evaporated off, the rest is triturated with methanol and dried over phosphorus pentoxide to obtain the compound mentioned in the heading: amino acid analysis: Lys, 1.10; Asp, 1; Gly, 3.33; Ala, 1; Phe, 2.14; cysteic acid, 1.35.

Example 1:

t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-Nt-t-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanyltryptophyl-N'-t-butyloxycarbonyl-lysyl-Ot-butylal threonyl -Ot-butyl-threonyl-Ot-butyl-seryl-S-trityl-cysteine; II, R2 = COOH and R4 — Boc — Gly - Gly— Ala-Gly - NH (Boc - Gly - Gly - Ala - Gly - Cys (Trt) -Iys (Boc) —Asn — Phe — Phe - Trp — Lys ( Boc) - Thr (Bul) - Phe - 1hr {Bu ') - Ser (Bu') - Cys (Trt) - OH)

To a solution stirred at -20 ° C of Boc — Gly — Gly — Ala— Gly — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — NHNH2 (800 mg, 0.59 mmol, described in the preparation 7) in dimethylformamide (12 ml), a 1.85N solution of HCl in ethyl acetate (0.795 ml, 1.475 mmol) is added. The mixture is brought to -17 ° C., t-butyl nitrite (0.081 ml, 0.71 mmol) is added, and the solution is stirred for 14 min. A solution of H - Trp - Lys (Boc) - Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') -Cys (Trt) —OH is cooled to -15 ° C. [816 mg, 0.59 mmol, described by HU Immer et al., "Helv. Chim. Acta ", 57,730 (1974)] and N-ethyldiisopropylamine (0.354 ml, 2.06 mmol) in dimethylformamide (6 ml), and added to the above reaction mixture. The mixture is stirred at -15 ° C for 1 h, and at 25 ° C for 18 h. The solvent is evaporated off, the rest is triturated with ice-cold citric acid, filtered, washed with water and then with methanol, and dried to obtain the compound mentioned in the heading: amino acid analysis: Lys, 1.94; Asp, 0.96; Thr, 1.64; Ser, 0.49; Gly, 2.82, Ala, 1.00; Phe, 2.80.

Example 2:

Cyclic disulfide of glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-lysylthreonyl-phenylalanyl-threonyl-seryl-cysteine; I, R '—H —Gly— Gly — Ala— Gly - NH and R2 = COOH (H — Gly — Gly — Ala — Gly— Cys — lys — Asn — Phe — Phe—

Trp — lys — Ihr — Phe —Thr — Ser —Cys — OH)

We dissolve Boc — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thi ^ Bu') - Ser (Bu ') - Cys (Trt) —OH described in Example 1 (0.871 g, 0.32 mmol) in acetic acid (150 ml) and added dropwise at room temperature to a solution of iodine in methanol (0.5%, 150 ml, 30 mmol) with stirring for 1 h. The mixture is stirred for an additional 45 min, cooled in an ice bath and a solution of sodium thiosulfate in water (IN, 6 ml) is added to destroy the excess iodine (colorless solution). The solvent is evaporated off and the rest is triturated with water, dried and the dry product is triturated with isopropyl ether to obtain the cyclic disulfide of hexadecapeptide of formula III:

Boc — Gly — Gly — Ala — Gly — C ys — Ly s (Boc) —Asn — Phe—

Phe - Trp - Lys (Boc) - Thr (Bul) - Phe - Thr (Bu ') - Ser (Bul) -

I:

Cys-OH.

To the latter compound, cold concentrated hydrochloric acid (23 ml) is added to a bairi of ice water, under a nitrogen atmosphere, with vigorous stirring. Stirring is continued for 10 min, 300 ml of glacial acetic acid are added and the solution is lyophilized. The rest is dissolved in water and lyophilized again. The remainder is dissolved in a 0.01N aqueous solution of ammonium acetate and applied to a column of carboxymethylcellulose (Whatman CM-23, 2.5 x 30 cm). The

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pure compound is eluted with a 0.06N ammonium acetate buffer. The purified material is lyophilized from water to give a white solid material constituting the compound mentioned in the heading in the form of its acetic acid addition salt;

C 282 nm (e: 5260), 289 nm (e: 4730), amino acid analysis: Lys, 2.28; Asp, 1.05; Thr, 1.95; Ser, 0.93; Cys, 2.34; Gly, 3; Ala, 1.05; Phe, 3.12. Repeated lyophilization of the latter product from water gives the title compound in the form of the free base; amino acid analysis: Lys, 2.10; Asp, 1.04; Thr, 1.80; Ser, 0.95; Cys, 2.17; Gly, 3; Ala, 1.07; Phe, 2.99.

In the same way, but using thiocyanogen according to the method of Hiskey and Smith, cited above, instead of iodine, the compound cited in the heading is also obtained.

Preparation 8:

T-butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycine methyl ester (Boc — Leu — Gly — Gly — Ala — Gly — OMe) A solution of Boc — Gly — Gly — Ala — Gly — OMe (2 g, 5 , 35 mmol, described in preparation 4) in 25 ml of trifluoroacetic acid is stirred at 0 ° C for 1 h. The solvent is evaporated off, the rest is triturated with ether, the solid is collected, and dried to obtain the tetrapeptide of formula H — Gly — Gly — Ala— Gly — OMe, isolated in the form of the addition salt trifluoroacetic acid.

A solution of the latter tetrapeptide (5.35 mmol), Boc-Leu-OH (2.31 g, 10 mmol), 1-hydroxy benzotriazole (1.35 g, 10 mmol), dicyclohexylcarbodiimide (2.27 g, 11 mmol), and N-ethylmorpholine (0.68 ml) in dimethylformamide (25 ml) is stirred at 0 ° C for 24 h. The mixture is filtered to separate the precipitate and the filtrate is added to diethyl ether (200 ml). The precipitate is collected and crystallized from methanol / isopropyl ether to obtain the compound mentioned in the heading; M.p .: 190.5-193 ° C; [a] ë5 = -15.2 ° (c = l, dimethylformamide).

Preparation 9:

T-Butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycine hydrazide; IX, RS = Boc — Leu (Boc — Leu— Gly — Gly— Ala — Gly— NHNH2)

A solution of Boc-Leu-Gly-Gly-Ala-Gly-OMe (2 g, 4 mmol, described in preparation 8) and of hydrazine hydrate (4.12 ml, 80 mmol) in 50 ml of methanol is stirred at 0 ° C. for 4 h. This solution is concentrated to 4 ml and added to diethyl ether (200 ml). The precipitate is collected, dissolved in methanol (4 ml) and add to diethyl ether (200 ml), collect the precipitate and dry to obtain the compound cited in the section; mp: 174-176.5 ° C; [a] f> 4 = ~ 12 ° (c = 1, dimethylformamide).

Preparation 10:

T-butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl Nc-t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanine methyl ester (Boc — Leu — Gly— Gly — Ala — Gly (Cys ( Trt) —Iys (Boc) —Asn — Phe - Phe — OMe) T-butyl nitrite (0.15 ml, 1.27 mmol) is added to a solution of Boc — Leu — Gly — Gly — Ala — Gly —N2H3 (0.308 g, 0.635 mmol, described in preparation 9, in dimethylformamide (5 ml) at −20 ° C., and 2.4N HCl in ethyl acetate (0.66 ml, 1, 59 mmol). After stirring at -20 ° C. for 15 min, a solution of H — Cys (Trt) —Lys (Boc) —Asn — Phe— Phe — OMe [0.62 g, 0.58 mmol) is added. , described by HU Immer et al., "Helv. Chim. Acta ”, 57, 730 (1974)] and diisopropylethylamine (0.372 ml) in dimethylformamide (6 ml). After stirring at -20 ° C for 1 h and at 0 ° C for 24 h, the solution is added to diethyl ether (200 ml). The precipitate is collected, dissolved in methanol (5 ml) and added to diethyl ether (200 ml). The precipitate is collected and dried to obtain the title compound, melting point 238-240.5 ° C.

Preparation 11:

T-Butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cisteinyl-Nz-t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanine hydrazide; IV, R4 = Boc — leu — Gly — Gly— Ala — Gly — NH (Boc — leu — Gly — Gly — Ala — Gly — Cys (Trt) - lys (Boc) —Asn — Phe — NHNH 2)

A solution of Boc — Leu — Gly — Gly — Ala — Gly — Cys- (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe (0.56 g, 0.38 mmol, described in Preparation 10) and hydrazine hydrate (0.74 ml) in dimethylformamide (10 ml) is stirred at 0 ° C for 12 h and at 25 ° C for 24 h. The solution is added to diethyl ether (100 ml). The precipitate is collected, dissolved in dimethylformamide (3 ml), and added to diethyl ether (100 ml). The precipitate is collected and dried to obtain the compound mentioned in the heading: m.p .: 239-242 ° C; amino acid analysis: Lys, 1.05; cysteic acid, 0.66; Asp, 0.99; Gly, 3; Ala, 1.1; 'ACys, 0.21; Leu, 0.96; Phe, 1.86.

Example 3:

t-Butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-Nz-t-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanylphenylalanyl-tryptophyl-N ^ -t-butyloxycarbonyl-lysyl-Ot-butyl -phenylalanyl-Ot-butyl-threonyl-Ot-butyl-seryl-S-trityl-cysteine; II, R2 = COOH and R4 = Boc — Ieu— Gly — Gly — Ala — Gly — NH (Boc — Leu — Gly — Gly — Ala— Gly — Cys (Trt) —lys (Boc) —Asn — Phe — Phe— Trp—

Iys (Boc) - 1hr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - Cys (Trt) - OH)

T-butyl nitrile (0.03 ml, 0.28 mmol) is added to a solution at -20 ° C of Boc — Leu — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) - Asn — Phe — Phe — N2H3 (0.20 g, 0.14 mmol, described in Preparation 11) in 2 ml of dimethyl sulfoxide, 4 ml of dimethylformamide and 2.21N HCl in acetate ethyl (0.16 ml, 0.35 mmol). After stirring at -20 ° C for 15 min, a solution of H — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - Cy s (Trt) is added. —OH • HC02H (0.20 g, 0.14 mmol), described by HU Immer et al., "Helv. Chim. Acta ”, 57.730 (1974) and diisopropylethylamine (0.08 ml, 0.49 mmol) in dimethylformamide (5 ml). The solution is stirred at -20 ° C for 1 h and at 0 ° C for 24 h. The solution is concentrated to 2 ml and added to diethyl ether (100 ml). The precipitate is collected, washed with water (2x5 ml), washed with methanol (2x5 ml) and dried to obtain the compound mentioned in the heading; amino acid analysis: Lys, 1.92; cysteic acid, 0.93; Asp, 0.93; Thr, 1.38; Ser, 0.66; Gly, 3; Ala, 1.02; 'At Cys, 0.39; Leu, 0.93; Phe, 2.70.

Example 4:

Cyclic leucyl-glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-lysyl-threonyl-phenyl-alanyl-threonyl-seryl-cysteine disulfide; I, R1 = H — leu— Gly — Gly — Ala — Gly — NH and R2 - COOH (H — leu — Gly — Gly — Ala — Gly — Cys — lys—

Asn — Phe — Phe— Trp — Lys — Ihr — Phe — Thr — Ser — Cys — OH) '

A solution of Boc — Leu — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu' ) —Ser (Bul) —Cys (Trt) —OH (0.23 g, 0.081 mmol, described in Example 3) in 90 ml of acetic acid is added dropwise over 1 hour to a solution 0.5% iodine in methanol (41.5 ml, 0.81 mmol). At the end of the addition, the solution is stirred at room temperature for 1 h and then cooled to 0 ° C. IN sodium thiosulfate (1.62 ml) is added until the solution becomes colorless. The solvent is collected, dried, triturated with ether and dried to obtain the cyclic heptadecapeptide disulfide of formula III; Boc — Leu — Gly — Gly — Ala — Gly — C ys — Lys (Boc) —Asn—

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Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') -

Ser (Bu ') - Cys — OH.

A solution of the cyclic heptadecapeptide disulfide (0.155 g, 0.066 mmol) in 6.87 ml of concentrated hydrochloric acid under a nitrogen atmosphere is rapidly stirred at 0 ° C. for 8 min. 69 ml of acetic acid are added and the solution is lyophilized. The remainder is lyophilized from water (50 ml). The remainder is chromatographed on a column of a crosslinked dextran, chemically modified Sephadex G-25M [3 x 50 cm, equilibration in the lower phase and then equilibration in the upper phase of n-butanol / acetic acid / water (4/1 / 5)], using the upper phase to desorb the peptide. The fractions containing the pure peptide are combined, evaporated and lyophilized from water to give the title compound in the form of its acetic acid addition salt; C 290 (e: 5000), 281 (s: 5540), 275 (e: 5205), 269 (s: 4980), 265 (e: 4625), 259 nm (s: 4085). Repeated lyophilization of the latter product from water gives the title compound in the form of the free base; amino acid analysis: Lys, 1.98; cysteic acid, 1.41; Asp, 1.26; Thr, 1.92; Ser, 0.96; Gly, 3; Ala, 1.02; Leu, 0.96; Phe, 2.76.

In the same way, but using thiocyanogen according to the method of Hiskey and Smith, above quotation, instead of iodine, the compound cited in heading is also obtained.

Preparation 12:

T-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycine methyl ester (Boc — Gly — Gly — Gly — Ala — Gly — OMe) Boc — Gly — OTcp solution (3.7 g, 10.04 mmol) , H - Gly - Gly - Ala - Gly - OMe • CF3C02H (3.12 g, 8.03 mmol, described in Preparation 8) and N-ethylmorpholine (1.1 ml) in dimethylformamide (15 ml) is stirred at 0 ° C for 20 h. The precipitate is collected and the filtrate is added to diethyl ether. The two combined precipitates are crystallized from methanol to give the compound mentioned in the heading; m.p .: 198-201 ° C; [a] ê4 = -3.9 ° (c = 1, dimethylformamide).

Preparation 13:

T-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycine hydrazide; IX, Rs - Boc — Gly (Boc — Gly - Gly — Gly— Ala-Gly-NHNH2)

A solution of Boc-Gly-Gly-Gly-Ala-Gly-OMe (1.0 g, 2.32 mmol, described in Preparation 12) and hydrazine hydrate (1 ml, 23.2 mmol) in 30 ml of methanol is stirred at 0 ° C for 4 h. After evaporation, the remainder is triturated with diethyl ether and dried to obtain the compound mentioned in the heading, with a melting point of 221-223 ° C.

Preparation 14:

N, S-ditrityl-cysteinyl-Nz-t-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanine hydrazide; VI (Trt — Cys- (Trt) —Lys (Boc) —Asn — Phe — Phe — NHNH 2)

A solution of Trt — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— OMe (1.25 g, 1 mmol, described by HU Immer et al., "Helv. Chim. Acta", 57, 730 ( 1974) and hydrazine hydrate (0.97 ml, 20 mmol) in 30 ml of methanol is stirred at 0 ° C. for 2 days, the solvent is evaporated off and the remainder is crystallized from ethanol / isopropyl ether. to obtain the compound cited in the heading; NMR (DMSO-ds): 5 1.38 (s, 9H), 7.19-7.30 (m, 40H).

Preparation 15:

N, S-Ditrityl-cysteinyl-Ne-t-butyloxycarbonyl-lysyl-asparaginyl-

phenylalanyl-phenylalanyl-tryptophyl-NE-t-butyloxycarbonyl-

lysyl-O-t-butyl-threonyl-phenylalanyl-O-t-butyl-threonyl-

O-t-butyl-seryl-S-trityl-cysteine; VII, R2 = COOH

(Trt— Cysf Trt) - Lys (Boc) —Asn — Phe — Phe — Trp—

Lys (Boc) - Thiißu ') - Phe - Thr (Bu') - Ser (Bu ') - Cj> s (Trt) —OH)

T-butyl nitrile (0.09 ml, 0.74 mmol) is added to a solution at −20 ° C. of Trt — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— NHNH2 (0, 62 g, 0.493 mmol, described in Preparation 14) in dimethylformamide (10 ml) and 2.6N HCl in ethyl acetate (0.475 ml, 1.23 mmol). After stirring at -20 ° C for 15 min, a solution of H — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - Cys (Trt) - is added. OH • HCOOH [0.70 g, 0.493 mmol, described by HU Immer et al., "Helv. Chim. Acta ”, 57.730 (1974)] and diisopropylethylamine (0.30 ml, 1.65 mmol) in dimethylformamide (10 ml). The solution is stirred at -20 ° C for 1 h and at 25 ° C for 24 h, then evaporated. The rest is triturated with water, diethyl ether, cold IN citric acid, and dried to obtain the compound mentioned in the heading; NMR (CDCl3) 1.08 and 1.13 (s, 27H), 1.37 (s, 18H), 7.28 (m, 60H), amino acid analysis: Lys, 2.23; cysteic acid, 1.10; Asp, 1; Thr, 2.12; Ser, 1.02; ViCys, 0.64; Phe, 3.18.

Preparation 16:

S-trityl-cysteinyl-Ne-t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-Nz-t-butyloxycarbonyl-lysyl-Ot-butyl-threonyl-phenylalanyl-Ot-butyl-threonyl form Otate seryl-S-trityl-cysteine;

VIII, R2 = COOH (H — cys (Trt) - Lys (Boc) - Asn — Phe— Phe - Trp - Iys (Boc) - Thr (Bu ') - Plie - Thr [Bu') - Ser (Bu ') -Cys (Trt) -OH-HCOOH)

A solution of Trt — Cys (Trt) —Lys (Boc) —Asn — Phe— Phe — Trp — Lys (Boc) —Thr (Bul) —Phe — Thr (Bu ') - Ser (Bu') - Cys (Trt ) —OH (0.50 g, 0.192 mmol, described in Preparation 15) in 6 ml of acetic acid / formic acid / water (7/1/2) is stirred at 25 ° C for 6 h. The solvent is evaporated off and the rest is triturated with diethyl ether to obtain the compound mentioned in the heading; amino acid analysis: Lys, 2.23; cysteic acid, 1.38; Asp, 1; Thr, 2.14; Ser, 0.86; Phe, 3.24.

Preparation 17:

t-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-Nz-t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-N ^ -t-butyloxy-carbonyl-lysyl-Ot-butyl -threonyl-phenylalanyl-Ot-butyl-threonyl-Ot-butyl-seryl-S-trityl-cysteine; II, R2 = COOH and Rs = Boc — Gly — Gly — Gly — Ala — Gly — NH (Boc — Gly— Gly - Gly — Ala — Gly — Cys (Trt) —fys (Boc) —Asn — Phe - Phe - Trp — Lys (Boc) - Thr (Bi /) - Phe - Thr (Bu ') - Ser (Bu') - Cys (Trt) - OH)

T-butyl nitrite (0.04 ml, 0.34 mmol) is added to a solution at −20 ° C. of Boc-Gly-Gly-Ala-Gly-NHNH2 (0.076 g, 0.176 mmol, described in the preparation 13) in dimethyl sulfoxide (1 ml), dimethylformamide (2 ml) and 2.5N HCl in ethyl acetate (0.176 ml, 0.44 mmol). After stirring at -20 ° C for 15 min, a solution of H — Cys (Trt) —Lys (Boc) —Asn - Phe — Phe — Trp — Lys (Boc) - Thr (Bu ') - Phe - Thr is added. (Bu ') - Ser (Bu') - Cys (Trt) - OH • HCOOH (0.385 g, 0.16 mmol, described in preparation 16) and diisopropylethylamine (0.12 ml) in dimethylformamide (5 ml) . The solution is stirred at -20 ° C for 1 h, at 0 ° C for 1 h at 25 ° C for 20 h. The solvent is evaporated off and the remainder is added to diethyl ether (100 ml). The precipitate is collected, washed with water and methanol, and dried to obtain the compound mentioned in the heading; amino acid analysis: Lys, 2.30; cysteic acid, 1.42; Asp, 1; Thr, 2.27; Ser, 1; Gly, 3.84; Ala, 1; Phe, 3.34.

Example 5:

Cyclic disulfide of glycyl-glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-lysylthreonyl-phenylalanyl-threonyl-seryl-cysteine; I, R '= H — Gly - Gly — Gly - Ala — Gly — NH and R2 = COOH (H - Gly -Gly-Gly-Ala- Gly - Cys - b's -

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Asn - Phe — Phe — Tpr — lys —Ihr — Phe —Thr — Ser — Cys — OH)

A solution of Boc — Gly — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — Trp - Lys (Boc) —Asn — Phe — Phe— Trp - Lys (Boc) - Thr (Bu ') - Phe - Thr (Bu') - SerfBu ') - Cys- (Trt) —OH (0.285 g, 0.103 mmol, described in preparation 17) in 200 ml of acetic acid is added to a solution of 0.5% iodine in methanol (52 ml, 1.03 mmol) over 60 min, and stirred for 60 min at room temperature. The solution is cooled to 0 ° C and IN sodium thiosulfate (2.06 mmol) is added to obtain a colorless solution. The solvent is evaporated off and the oily residue is added to water (100 ml). The precipitate is collected and dried to obtain the cyclic heptadecapeptide disulfide of formula III:

Boc — Gly — Gly — Gly — Ala — Gly — Cys — Lys (Boc) —Asn—

Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') -

!

Ser (Bul) —Cys — OH.

Stirred at 0 ° C for 10 min under nitrogen, a solution of the above-mentioned cyclic disulphide of heptadecapeptide (0.10 mmol) in 10 ml of concentrated hydrochloric acid. 100 ml of acetic acid are added and the solution is lyophilized. After another lyophilization from water (100 ml), the remainder is subjected to partition chromatography on a column of a crosslinked, chemically modified dextran (Sephadex G-25M, 3 x 50 cm, constituted in the lower phase n-butanol / acetic acid / water (4/1/5) and then balanced in the upper phase) using the upper phase to desorb the practically pure heptadecapeptide. The pure fractions are combined, evaporated and lyophilized to obtain the title compound in the form of its acetic acid addition salt;

283 (e: 6702), 289 nm (e: 6350). Repeated lyophilization of the latter product from water gives the title compound in the form of the free base; amino acid analysis: Lys, 2; cysteic acid, 1.42; Asp, 1.09; Thr, 1.89; Ser, 0.91; Gly, 3.64; Ala, 0.98; Phe, 2.95.

In the same way, but using thiocyanogen according to the method of Hiskey and Smith, above quotation, instead of iodine, the compound cited in heading is also obtained.

Preparation 18:

NS-Ditrityl-cysteinyl-N ^ -t-butoxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-N * -t-butoxycarbonyl-lysyl-Ot-butyl-threonyl-phenylalanyl-Ot-butyl-threonyl-Ot-butyl- seryl-2-tritylthioethylamide; VII, R2 = H (Trt — Cys- (Trt) —Iys (Boc) —Asn — Phe — Phe - Trp — Iys (Boc) —Thr- (Bu ') - Phe — 1hi {Bu') - Ser (Bu '-NHCH2CH2S-Trt) The pentrazide hydrazide Trt — Cys (Trt) —Lys (Boc) - Asn — Phe — Phe — NHNH2 (0.80 g, 0.637 mmol, described in preparation 14) is dissolved in dimethylformamide dry (9 ml) and cooled to -20 ° C. Hydrochloric acid is added in ethyl acetate (2.4N, 0.691 ml), then t-butyl nitrite (0.0872 ml, 0.764 mmol). The mixture is stirred for 15 min at -15 ° C. A solution of H — Trp — Lys (Boc) - Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - NHCH2CH2S - Trt is cooled (0.852 g, 0.637 mmol, prepared as described in U.S. Patent No. 3,917,581 issued November 4, 1975) in 8 ml of dimethylformamide containing N-ethyldiisopropylamine (0.272 ml, 1.59 mmol ) down to -15 ° C and added dropwise to the above reaction mixture. Stirring is continued at -15 ° C for 1 h and at room temperature overnight. The reaction mixture is evaporated under reduced pressure, the rest is triturated with water, filtered, washed with water, and dried over phosphorus pentoxide. The remainder is chromatographed on a column of silica gel (163 g) with chloroform containing methanol (3%) and pyridine (0.3%) as eluent,

and the pure product is crystallized from methanol / isopropyl ether to obtain the title compound; M.p .: 163-180 ° C (dec.).

Analysis for Ci49Hi8oNi60i9S2:

Calculated: C 69.85 H 7.04 N 8.76%

Found: C 69.24 H 7.09 N 8.90%.

Preparation 19:

S-trityl-cysteinyl-Nz-t-butyloxycarbonyl-lysyl-aspa- formate

raginyl-phenylalanyl-phenylalanyl-tryptophyl-Nz-t-butyl-

oxycarbonyl-lysyl-O-t-butyl-threonyl-phenylalanyl-O-t-

butyl-threonyl-O-t-butyl-seryl-2-tritylthioethylamide;

VIII, R2 = H (H — Cys (Trt) —Iys (Boc) —Asn — Phe — Phe—

Trp - lys (Boc) - 1hr [Bu ') - Phe - Thr (Goal) -Set {Buf) -

NHCH2CH2S - Trt-HCOOH)

The undecapeptide Trt — Cys (Trt) —Lys (Boc) —Asn — Phe— Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — ThifBu') - Ser (Bu ') - NHCH2CH2S — Trt ( 0.909 g, 0.355 mmol, described in preparation 18) is dissolved in a mixture of acetic acid / formic acid / water (7/1 / 2.10 ml) and the solution is stirred overnight at room temperature. The solvent is evaporated and the rest is triturated with water. The precipitate obtained is filtered, washed with water and dried over phosphorus pentoxide. The solid is triturated several times with petroleum ether / ether and dried to obtain the compound mentioned in the heading; amino acid analysis; Lys, 2.03; Asp, 1; Ser, 0.87; Phe, 2.97; cysteic acid, 0.90; Thr, 1.85.

Preparation 20:

t-Butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-Wt-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanyl-tryptophyl-Nc-t-butyloxycarbonyl-lysylon-Ot-phenyl Ot-butyl-threonyl-Ot-butyl-seryl-2-tritylthioethylamide; II, R2 = H and R4 = Boc — Leu - Gly — Gly — Ala — Gly — NH (Boc — Leu — Gly — Gly — Ala— Gly — Cys (Trt) —Iys (Boc) —Asn — Phe — Phe— Trp—

lys (Boc) -Thr (Bu ') - Phe- 1iir [Bu') - Ser {Bu ') - NH CH 2 CH 2S — Trt)

The pentoceptide hydrazide Boc-Leu-Gly-Gly-Ala-Gly-NHNH2 (0.066 g, 0.136 mmol, described in preparation 9) is dissolved in dry dimethylformamide (3 ml) and cooled to -20 ° C. On add hydrochloric acid in ethyl acetate (2N, 0.175 ml), then t-butyl nitrite (0.0186 ml, 0.163 mmol). The mixture is stirred at -15 ° C for 15 min. A solution of H — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) - ThrfBu ') - Phe - Thr (Bu') - Ser is cooled to -15 ° C. (Bu ') - NHCH2CH2S -Trt (0.315 g, 0.133 mmol, described in preparation 19) in dimethylformamide (4 ml) containing N-ethyldiisopropylamine (0.082 ml, 0.476 mmol) and it is added dropwise previous reaction mixture. Stirring is continued at -15 ° C for 1 h and at room temperature overnight. The reaction mixture is evaporated under reduced pressure, the rest is triturated with ice-cold citric acid (IN), filtered and washed with water. The solid residue is triturated with methanol and dried over phosphorus pentoxide to obtain the compound mentioned in the heading; amino acid analysis: Lys, 1.88;

cysteic acid, 0.84; Asp, 1; Thr, 1.94; Ser, 0.97; Gly, 2.78; Ala, 0.89; Leu 0.89; Phe, 3.11.

Example 6:

Cyclic leucyl-glycyl-glycyl-alanyl-glycyl-cysteinyl- disulfide

phenylalanyl-phenylalanyl-tryptophyl-lysyl-threonyl-

phenylalanyl-threonyl-seryl-2-thioethylamide; I, R1 = H — Ieu—

Gly-Gly-Ala-Gly-NH and R2 = H

(H — Leu — Gly — Gly — Ala — Gly — Cys — lys — Asn — Phe—

Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2S)

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A solution of Boc — Leu — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe— Thr (Bu' ) - Ser (Bu ') - NHCH2CH2S — Trt (0.230 g, 0.083 mmol, described in preparation 20) in 100 ml of acetic acid is added slowly to a vigorously stirred iodine solution (0.211 g, 0.83 mmol ) in methanol (42 ml) at room temperature. At the end of the addition, the solution is stirred at room temperature for 60 min. The solution is cooled to 0 ° C and a solution of sodium thiosulfate in water (IN) is slowly added to destroy the excess iodine (colorless solution). The solvent is evaporated to almost dryness, the rest is triturated with cold water, filtered, washed with water and dried over phosphorus pentoxide. The solid is washed with ether and dried to obtain the cyclic disulfide of formula III, Boc — Leu — Gly— Gly — Ala — Gly — Cys — Lys (Boc) —Asn — Phe — Phe — Trp—

I j

Lys (Boc) —Thr (Bu ') - Phe — Thr (Bul) —Ser (Bu') - NHCH2CH2 S.

This cyclic hexadecapeptide is vigorously stirred at 0 ° C. under a nitrogen atmosphere for 10 min in concentrated hydrochloric acid (7 ml). 90 ml of acetic acid are added and the solution is lyophilized. The remainder is taken up in 2% acetic acid in water and lyophilized. The remainder is subjected to partition chromatography on a column of crosslinked dextran, chemically modified (Sephadex G-25M, 3 x 50 cm, equilibrium in the lower phase of n-butanol / acetic acid / water (4/1/5) and then balancing in the upper phase) using the upper phase to desorb the essentially pure hexadecapeptide. The pure fractions are combined, evaporated and lyophilized to obtain the title compound in the form of its acetic acid addition salt; 290 (e: 4290), 282 (s: 4910), 273 nm (s: 4630). Repeated lyophilization of the latter product from water gives the title compound in the form of the free base; amino acid analysis; Lys, 2.01; Asp, 1.35;

Ser, 0.93; Ala, 0.99; Phe, 2.52; cysteic acid, 0.66; Thr, 1.98; Glyr3; Leu, 0.96.

Preparation 21:

t-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycyl-S-tritylcysteinyl-Nc-t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-NE-t-butyloxycarbonyl-lysyl-Otyl-butyl-threon Ot-butyl-threonyl-Ot-butyl-seryl-2-tritylthioethylamide; II, R2 = HetR4 = Boc— Gly— Gly -Ala-Gly- NH (Boc -Gly-Gly- Gly - Ala-Gly-Cys (Trt) —Lys (Boc) —Asn — Phe — Phe - Trp — Iys (Boc) - Thr (Buj-Ser (Bu ') - NHCH2CH2S-Trt)

Bape — Gly — Gly — Gly — Ala— NHNH2 pentapeptide hydrazide (0.0608 g, 0.141 mmol, described in Preparation 13) is dissolved in dry dimethylformamide (6 ml) and dimethyl sulfoxide (2 ml) , and cooled to -20 ° C. Hydrochloric acid is added in ethyl acetate (2N, 0.176 ml, 0.352 mmol), then t-butyl nitrite (0.0194 ml, 0.169 mmol). The mixture is stirred at -15 ° C for 15 min. A solution of H — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — Trp— Lys (Boc) —Thr (Bu ') - Phe - Thr (Bu') is cooled to -15 ° C. - Ser (Bu ') - NHCH2CH2S -Trt (0.327 g, 0.138 mmol, described in preparation 19) in dimethylformamide (4 ml) containing N-ethyldiisopropylamine (0.085 ml, 0.494 mmol), and it is added dropwise dropwise to the previous reaction mixture. Stirring is continued at -15 ° C for 1 h and at room temperature overnight. The reaction mixture is evaporated under reduced pressure, the rest is triturated with ice-cold citric acid (IN), filtered and washed with water. The solid residue is triturated with methanol and dried over phosphorus pentoxide to obtain the compound mentioned in the heading; amino acid analysis: Lys, 2.31; cysteic acid, 0.84; Asp, 1; Thr, 2.26; Ser, 1.19; Gly, 4; Ala, 0.84; Phe, 3.2.

Example 7:

Cyclic glycyl-glycyl-glycyl-alanyl-glycyl-cysteinyl-phenylalanyl-phenylalanyl-tryptophyl-lysyl-threonyl-phenyl-alanyl-threonyl-seryl-2-thioethylamide disulfide; I, R '- H — Gly — Gly— Gly-Ala-Gly-NH and R2 = H (H -Gly-Gly-Gly- Ala-Gly - Cys — lys — Asn — Phe — Phe - Trp — lys— Thr —Phe—

Thr - Ser - NHCH2CH2S)

A solution of Boc — Gly — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) - Asn - Phe - Phe - Trp - Lys (Boc) - Thr (Bu ') -Phe — Thr (Bul) —Ser (Bu ') - NHCH2CH2S — Trt (0.224 g, 0.083 mmol, described in preparation 21) in acetic acid (160 ml) is added slowly to a vigorously stirred iodine solution (0.211 g, 0, 83 mmol) in methanol (42 ml) at room temperature. At the end of the addition, the solution is stirred at room temperature for 60 min. The solution is cooled to 0 ° C and a solution of sodium thiosulfate in water (IN) is slowly added to destroy the excess iodine (colorless solution). The solvent is evaporated to almost dryness, the rest is triturated in cold water, filtered, washed with water, and dried over phosphorus pentoxide. The solid material is washed with ether and dried to obtain the cyclic disulfide of formula III:

Boc — Gly — Gly — Gly — Ala — Gly — Cys - Lys (Boc) - Asn— Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ' ) —NHCH2CH2 S.

This cyclic hexadecapeptide is vigorously stirred at 0 ° C. under a nitrogen atmosphere for 10 min in concentrated hydrochloric acid (7 ml). 90 ml of acetic acid are added and the solution is lyophilized. The remainder is taken up in 2% acetic acid in water and lyophilized. The remainder is subjected to partition chromatography on a column of chemically modified crosslinked dextran (Sephadex G-25M, 3 x 50 cm, equilibration in the aqueous phase of n-butanol / acetic acid / water (4/1/5). and then balancing in the upper phase), using the upper phase to desorb the substantially pure hexadecapeptide. The pure fractions are combined, evaporated and lyophilized to give the title compound in the form of its acetic acid addition salt; Xmâ? H: 288 (s: 4870), 280 (s: 5575), 274 (e: 5380), 268 (s: 5185), 265 nm (e: 4955). Repeated lyophilization of the latter product from water gives the title compound in the form of the free base; amino acid analysis: Lys, 1.72; Asp, 1; Ser, 0.73; Ala, 0.69; Phe, 2.78; cysteic acid, 0.57; Thr, 1.69; Gly, 3.59.

Example 8:

t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cystemy] -Ne-t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-Nc-t-butyloxycarbonyl-lysyl-Ot-butyl-threonyl -Ot-butyl-threonyl-Ot-butyl-seryl-2-tritylthioethylamide; II, R2 = H and R4 = Boc— Gly - Gly — Ala — Gly — NH (Boc — Gly — Gly — Ala — Gly— Cys (Trt) —Iys (Boc) —Asn — Phe — Phe — Trp — Iys ( Boc) -

Thi {Bu ') - Phe - Thr (Bu') - Ser (Bu ') - NHCH2CH2 - Trt)

To a solution of Boc — Gly — Gly — Ala — Gly — Cys (Trt) —Lys- (Boc) —Asn — Phe — Phe — NHNH2 (800 mg, 0.59 mmol, prepared as described in Preparation 7) in dimethylformamide (12 ml) at −20 ° C., a 1.85N solution of HCl in ethyl acetate (0.795 ml, 1.475 mmol) is added, with stirring. This mixture is brought to -15 ° C, tertiary butyl nitrite (0.081 ml, 0.71 mmol) is added and the solution is stirred for 15 min. A solution of H — Trp — Lys (Boc) —Thr (Bu ') - Phe - Thr (Bu') - Ser (Bu ') - NHCH2CH2S - Trt (0.852 g, 0.637 mmol, prepared) is cooled to -15 ° C. as described in the United States of America patent

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No. 3917581 issued November 4, 1975) and N-ethyldiisopropylamine (0.354 ml, 2.06 mmol) in 6 ml of dimethylformamide and added to the preceding reaction mixture. This mixture is stirred at -15 ° C for 1 h and at 25 ° C for 18 h. The solvent is evaporated off, the rest is triturated with ice-cold citric acid, filtered, washed with water, then with methanol, and dried to obtain the compound mentioned in the heading; amino acid analysis; Lys, 2.01; Asp, 0.97; Thr, 1.60; Ser, 0.65; cysteic acid, 0.87; Gly, 2.92; Ala, 1; Phe, 2.97.

Example 9:

Cyclic glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-lysyl-threonyl-phenylalanyl-threonyl-seryl-2-thioethylamide disulfide; I, R '= H — Gly — Gly — Gly — Ala — Gly — NH and R2 = H (H — Gly — Gly — Ala — Gly — Cys — lys — Asn — Phe — Phe—

Trp -lys-Ihr- Phe -Thr- Ser - NHCH2CH2 S)

We dissolve Boc — Gly — Gly — Ala — Gly — Cys (Trt) - Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH2CH2S — Trt described in Example 8 (0.860 g, 0.30 mmol) in glacial acetic acid (150 ml) and added dropwise at room temperature to a solution iodine in methanol (0.5%, 150 ml, 30 mmol) with stirring over a period of 1 h. The mixture is then stirred for an additional 45 minutes, cooled in an ice bath and a solution of sodium thiosulfate in water (IN, 6 ml) is added to destroy the excess iodine (colorless solution). The solvent is evaporated off and the rest is triturated with water, dried and the dry product is triturated with isopropyl ether to obtain the cyclic disulphide of pentadecapeptide of formula III, Boc — Gly — Gly — Ala — Gly— C ys - Lys (Boc) —Asn — Phe

Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bul) -

I

NHCH2CH2S

To the latter compound, cold concentrated hydrochloric acid (23 ml) is added in an ice-water bath, under a nitrogen atmosphere with vigorous stirring. Stirring is continued for 10 min, 300 ml of glacial acetic acid are added and the solution is lyophilized. The rest is dissolved in water and lyophilized again. The remainder is dissolved in a 0.01N aqueous ammonium acetate solution and applied to a column of carboxymethylcellulose (Whatman CM-23.2.5 x 30 cm). The pure compound is eluted with a buffer of 0.006N ammonium acetate. The purified material is lyophilized from water to obtain a white solid material constituting the compound mentioned in the heading in the form of its acetic acid addition salt; : 282 nm (s: 5120), 289 nm (e; 4610).

Repeated lyophilization of the latter product from water gives the title compound in the form of the free base; amino acid analysis: Lys, 2.06; Asp, 1.02; Thr, 1.75; Ser, 0.91; cysteic acid, 0.73; Gly, 3; Ala, 1.10; Phe, 3.11.

In the same way, but using thiocyanogen according to the method of Hiskey and Smith, above quotation, instead of iodine, the compound cited in heading is also obtained.

Preparation 22:

Acetyl- (S-trityî) cysteinyl- (Nz-t-butoxy-carbonytjlysyl-asparaginyl-phenylalanyl-phenylalanine methyl ester {Ac — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe)

A solution of p-nitrophenyl acetate (0.191 g, 1.05 mmol, prepared as described by FD Chattaway, "J. Chem. Soc.", 2495 (1931)] in dimethylformamide (4 ml) is added to a 0 ° C solution of H — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — OMe-

HOAc (0.750 g, 0.698 mmol, prepared as described by HU Immer et al., "Helv. Chim. Acta", 57, 730 (1974) and N-ethylmorpholine (0.1 ml). After stirring at 0 ° C for The solvent is evaporated off under reduced pressure for 24 hours, the remainder is dissolved in 3 ml of methanol and added slowly to 200 ml of diethyl ether, the precipitate is collected and crystallized from ethanol to give the compound cited. in heading; PF: 219.5-221 ° C; [a] os = —21.6 ° (c = 1, dimethylformamide).

Likewise, using the p-nitrophenyl esters of formic, propionic, butyric, isobutyric, pivotal, n-hexanoic or benzoic acids, instead of p-nitrophenyl acetate, the corresponding compounds are also obtained. the above formula, in which Ac is replaced by formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or benzoyl.

Preparation 23:

Acetyl- (S-trity [) cysteinyl- (Ne-t-butoxycarbonyl) -lysyl-asparaginyl-phenylalanyl-phenylalanine hydrazide; IV, R4 = NHCOCH3 {Ac — Cys (Trt) —Iys (Boc) —Asn — Phe— Phe-NHNH 2)

A solution of Ac — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— OMe (0.40 g, 0.379 mmol, described in preparation 22) and hydrazine hydrate (0.37 ml, 7.58 mmol) in 15 ml of methanol is stirred at 0 ° C for 48 h. The precipitate is collected and dried to obtain the compound mentioned in the heading; M.p .: 236-237 ° C; [a] p5 = —28.6 ° (c = 1, dimethylformamide).

Likewise, using the other suitable starting materials described in Preparation 22, the corresponding compounds of formula IV are also obtained, in which R4 represents NHR3, where R3 is formyl, propionyl, n-butanoyl, l 'isobutanoyl, pivaloyl, n-hexanoyl, or benzoyl.

Example 10:

Acetyl- (S-trityl) cysteinyI- (Ne-t-butoxycarbonyl) lysyl-asparagi-

nyl-phenylalanyl-phenylalanyl-tryptophyl- (N'-t-butoxy-

carbonyl) lysyl- (0-t-butyl] -threonyl-phenylalanyl- {0-t-butyl) -

threonyl- (0-t-butyl) seryl-2-tritylthioethylamide;

II, R2 = HetR4 = NHCOCH3 (Ac - Cys (Trt) - Lys (Boc) - Asn -

Phe — Phe —Trp — Iys (Boc) - Thr (Bu ') - Ser (Bu') -

NHCH2CH2S— Trt)

A solution of Ac — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— NHNH2 (IV) (0.240 g, 0.227 mmol, described in Preparation 23) in dry dimethylformamide (2 ml) and sodium sulfoxide dimethyl (1 ml) is cooled to -20 ° C. Hydrochloric acid is added in ethyl acetate (2, IN, 0.273 mmol), then tertiary butyl nitrite (0.0312 ml, 0.273 mmole). The mixture is stirred for 15 min at -15 ° C. A solution of H — Trp — Lys (Boc) - is added dropwise to the preceding reaction mixture.

Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') NHCH2CH2S -Trt (V, 0.304 g, 0.227 mmol, prepared as described in U.S. Patent No. 3,917,581 issued on 4 November 1975) in 3 ml of dimethylformamide containing N-ethyldiisopropylamine (0.097 ml, 0.568 mmol), cooled to -15 ° C. The mixture is stirred at -15 ° C for 1 hour at 25 ° C for 20 h. The solvent is evaporated off under reduced pressure. The rest is triturated with an ice-cold solution of citric acid (IN), filtered, washed with water and dried over phosphorus pentoxide. The solid residue is triturated with methanol, filtered and dried over phosphorus pentoxide to obtain the compound mentioned in the heading; amino acid analysis: Lys, 1.82; Asp, 1; Ser, 0.76; cysteic acid, 0.93; Thr, 1.87; Phe, 3.10.

Likewise, but using the other suitable starting materials of formula IV, in which R4 represents NHR3 as described in preparation 23, the corresponding compounds of formula II are also obtained, in which R3 represents H and R4 represents NHR3, where R3 is formyl,

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propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl, or benzoyl.

Example 11:

Cyclic acetyl-cysteinyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanyl-tryptophyl-lysyl-threonyl-phenylalanyl-threonyl-seryl-2-thioethylamide disulfide; I, R '= NHCOCHî and R2 = H

(Ac — Cys — lys — Asn — Phe — Phe — Trp — Lys — Thr — Phe— Ihr - Ser - NHCN2CH2S)

A solution of Ac — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH2CH2S— Trt (0.260 g, 0.110 mmol, described in Example 10) in 61 ml of acetic acid is added slowly to a stirred solution of iodine (0.278 g, 1.1 mmol) in 56 ml of methanol at 25 ° C At the end of the addition, the solution is stirred at 25 ° C for 1 h. The solution is cooled to 0 ° C. and a solution of IN sodium thiosulfate in water is slowly added to destroy the excess iodine (colorless solution). The solvent is evaporated off under reduced pressure and the rest is triturated with water. The precipitate is collected, washed with water and dried over phosphorus pentoxide to obtain the cyclic protected un-decapeptide of formula III, Ac — Cys — Lys (Boc)

Asn — Phe — Phe — Trp — Lys (Boc) —ThrfBu ') - Phe — Thr (Bul) -

!

Ser (Bu ') - NHCH2CH2 S.

This cyclic peptide is stirred vigorously at 0 ° C. under a nitrogen atmosphere for 10 min in concentrated hydrochloric acid (9.1 ml). 119 ml of acetic acid are added and the solution is lyophilized immediately. The rest is dissolved in water and lyophilized. The remainder is dissolved in the upper phase of the solvent system: butanol / acetic acid / water (4/1/5) and it is applied to a column of chemically modified crosslinked dextran (Sephadex G-25 M), prepared in the lower phase of the solvent system. The upper phase of the above solvent system is used to desorb the undecapeptide. The fractions containing the pure product are combined and evaporated under reduced pressure. The rest is triturated with diethyl ether, dissolved in 5% acetic acid and lyophilized to obtain the title compound in the form of the acetic acid addition salt; UV (methanol): X.max: 290 (s: 4990), 282 (s: 5455), 274 nm (s: 5095).

The latter compound, in the form of the acetic acid addition salt, is subjected to repeated lyophilization from water to give the title compound in the form of the free base; amino acid analysis: Lys, 1.93; Asp, 1; Ser, 0.84; cysteic acid, 0.80; Thr, 1.84; Phe, 2.97.

Likewise, using the other suitable starting materials of formula II, in which R2 represents H and R4 represents NHR3, as described in example 10, the corresponding compounds of formulas III and I are also obtained, in which R2 represents H, and R4 and R1 represent NHR3, where R3 is formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or benzoyl.

Preparation 24:

3-Tritylthiopropionic acid pentachlorophenyl ester (H-t-SCH2CH2COOPcp)

3-Tritylthiopropionic acid [1 g, 2.87 mmol, described by R.C. Hiskey and M.A. Harpold, “J. Org. Chem. ”, 33, 559 (1968)] in dry tetrahydrofuran (25 ml) and pentachlorophenol (0.765 g, 2.87 mmol) is added. The mixture is cooled to 0 ° C, dicyclohexylcarbodiimide (0.596 g, 2.87 mmol) is added and the reaction is stirred for 1 h at 0 ° C and for 1 h at 25 ° C. The reaction mixture is then cooled at 0 ° C, filtered, and the filtrate is evaporated under reduced pressure. The remainder is crystallized from ethyl acetate to give the title compound, with a melting point of 154-156 ° C.

Preparation 25:

3-Tritylthiopropionyl- (Nc-t-butoxycarbonyl) -lysyl-asparaginyl-phenylalanyl-phenylalanine methyl ester (Trt — SCH2CH 2CO — Iys (Boc) —Asn - Phe — Phe — OMe) Stir at 25 ° C for 3 days a solution of pentachlorophenyl ester of 3-tritylthiopropionic acid (0.597 g, 1 mmol, described in preparation 24), of H-Lys (Boc) —Asn — Phe — Phe— OMe-HOAc [1 mmol, prepared as described by HU Immer et al., "Helv. Chim. Acta ”, 57, 730 (1974)] and triethylamine (0.14 ml, 1 mmol). The solvent is evaporated off under reduced pressure. The rest is triturated with an ice-cold solution of IN citric acid, filtered, washed with water and dried over potassium hydroxide. The solid material is crystallized from methanol to obtain the title compound, with a melting point of 215-220 ° C.

Preparation 26:

3-tritylthiopropionyl- (Ns-t-butoxycarbonyl) -lysyl-asparaginyl-phenylalanyl-phenylalanine hydrazide;

IV, R4 = H (Trt — SCH2CH2CO — Lys (Boc) —Asn — Phe— Phe-NHNH 2)

A solution of Trt — SCH2CH2CO — Lys (Boc) —Asn — Phe— Phe — OMe (0.900 g, 0.9 mmol, described in preparation 25) and hydrazine hydrate (1 ml) in dimethylformamide (20 ml) is stirred at 25 ° C for 20 h. The solvent is evaporated off under reduced pressure. The rest is triturated with cold water, filtered, washed with water and dried to obtain the compound mentioned in the heading, with a melting point of 225-235 ° C.

Example 12:

3-Tritylthiopropionyl- {Nz-t-butyloxycarbonyî) lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyl- (Nc-t-butoxycarbonyl) -lysyl- (0-t-butyl) threonyl-phenylalanyl- {0-t-butyl) threonyl - (0-t-butyl) -seryl-2-tritylthioethylamide; II, R2 and R4 ~ H (Trt — SCH 2CH2CO — Iys (Boc) - Asn — Phe — Phe— Trp-Iys (Boc) - 1hr (Bu ') - Phe-1hr (Bu') - Ser (Bu ') -NHCH2CH2S-1i-t)

A solution of Trt — SCH2CH2CO— Lys (Boc) —Asn — Phe — Phe — NHNH2 (0.500 g, 0.5 mmol, described in Preparation 26) in dimethyl sulfoxide (5 ml) is cooled to -20 ° C. and dimethylformamide (20 ml). A solution of hydrochloric acid in ethyl acetate (1.4N, 0.895 ml) is added, then tertiary butyl nitrite (0.069 ml, 0.6 mmol). The mixture is stirred at -15 ° C for 15 min and a solution, cooled to -15 ° C, of H — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser is added. (Bu ') - NHCH2CH2S — Trt (0.670 g, 0.5 mmol, prepared as described in U.S. Patent No. 3,917,581, issued November 4, 1975) and N-ethyldiisopropylamine (0.214 ml, 1 , 25 mmol) in dimethylformamide (10 ml). The reaction mixture was stirred at -15 ° C for 1 h and at 25 ° C for 20 h, and evaporated under reduced pressure. The rest is triturated with an ice-cold solution of IN citric acid, filtered, washed with water and dried over phosphorus pentoxide. The solid is triturated with cold methanol and dried to obtain the compound mentioned in the heading; amino acid analysis: Lys, 1.99; Asp, 1.15; Thr, 1.73; Ser, 0.67; Phe, 3.

Example 13:

Cyclic 3-thiopropionyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanyl-tryptophyl-lysyl-threonyl-phenylalanyl-threonyl-seryl-2-thioethylamide disulfide; I, R1 and R2 = H (SCH2CH2CO -lys-Asn — Phe - Phe - Trp — lys — Thr—

Phe-1hr-Ser-NHCH2CH2S)

A solution of Trt — SCH2CH2CO — Lys (Boc) —Asn - Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') -

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Ser (Bu ') - NHCH2CH2S — Trt (0.500 g, 0.216 mmol, described in Example 12) in acetic acid (100 ml) is added slowly to a stirred solution of iodine (0.547 g, 2.16 mmol) in methanol (110 ml) at 25 ° C. At the end of the addition, the solution is stirred at 25 ° C for 1 h. This solution is cooled to 0 ° C and a solution of IN sodium thiosulfate in water is slowly added to destroy the excess iodine (colorless solution). The solvent is evaporated off under reduced pressure and the rest is triturated with water. The precipitate is collected, washed several times with water and dried over phosphorus pentoxide to obtain the cyclic protected decapeptide of formula III, SCH2CH2CO —Lys (Boc) —Asn — Phe—

Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') -

I ~

NHCH2CH2S.

The latter cyclic decapeptide is stirred vigorously at 0 ° C. under a nitrogen atmosphere for 10 min in concentrated hydrochloric acid (18 ml). 200 ml of acetic acid are added and the solution is lyophilized immediately. The rest is dissolved in water and lyophilized. The remainder is dissolved in the upper phase of the solvent system: butanol / acetic acid / water (4/1/5) and it is applied to a column of chemically modified crosslinked dextran (Sephadex G-25 M), prepared in the lower phase of the solvent system. The upper phase of this solvent system is used for the desoption of the decapeptide. The fractions containing the pure product are combined and evaporated under reduced pressure. The remainder is dissolved in 5% acetic acid and lyophilized to obtain the title compound in the form of the acetic acid addition salt; ultraviolet (methanol): X max: 290 (s: 4920), 282 nm (e: 5390).

The latter compound, in the form of the acetic acid addition salt, is subjected to repeated lyophilization from water to give the title compound in the form of the free base; amino acid analysis: Lys, 1.97; Asp, 1; Thr, 1.64; Ser, 0.65; Phe, 2.94.

Preparation 27:

CC methyl ester, a.-dimethyl-3,5-dimethoxybenzyloxycarbo-nyl-tryptophyl-Nc-t-butyloxy-carbonyl-lysyl-0-t-butyl-threonyl-phenylalanyl-Ot-butyl-threonyl-Ot-butyl- seryl-S-trityl-cysteine (Ddz— Trp — Iys (Boc) - Thr (Bu ') - Phe — Thr- (Bu') - SeiiBu ') - Cys (Trt) - OMe)

A solution of diazomethane in ether is added to a solution of Ddz — Trp — Lys (Boc) —Thr (Bu1) —Phe — Thr (Bu1) - Ser (Bu ') - Cys (Trt) - OH [ 12.6 g, 0.785 mmol, described by HU Immer et al., "Helv. Chim. Acta ", 57, 730 (1974)] in methanol (10 ml) at 0 ° C. The mixture is stirred at 0 ° C for 1 h and evaporated. The remainder is subjected to silica gel chromatography using 3% methanol and 0.5% pyridine in chloroform for elution. Evaporation of the eluate gives the compound mentioned in the heading; NMR (CDCI3); 8 1.13 (s, 18H), 1.27 (s, 9H), 1.48 (s, 9H), 1.77 (s, 6H), 3.68 (s, 3H), 3.79 ( s, 6H), 7.1-7.6 (m, 23H).

Similarly, using diazoethane, 1-diazo-propane, 2-diazopropane, 1-diazobutane, 1-diazo-isobutane, 1-diazopentane, 4-diazo-2-methylbutane, 1- di-azohexane, 1-diazoheptane or 1-diazooctane, instead of diazomethane, the ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbuty-lique, n-hexyl esters are obtained , n-heptylic and n-octyl of the title compound.

Preparation 28:

S-tritylcysteine decyl ester (H— Cys (Trt) -OCH2- (CH2) sCH3)

A solution of cysteine decyl ester hydrochloride [0.894 g, 3 mmol, prepared as described by Vouille et al., French patent of addition No. 75.157 (1961), “CA”, is stirred at ambient temperature for 1 hour. 57, 15235c], triphenylcarbinol (0.78 g, 3 mmol) and boron trifluoride etherate (0.42 ml) in acetic acid (5.2 ml). The solvent is separated under reduced pressure and the remainder is subjected to chromatography on silica gel using 40 g of ethyl acetate in hexane, containing 0.1% triethylamine as eluent. Evaporation of the eluate gives the title compound in the form of an oil; [ä] d5 = + 25.84 ° (c = 1, CHCI3); NMR (CDCI3) 8 0.89 (t, J = 5 Hz, 3H), 1.3 (s, 16H), 1.55 (m, 2H), 2.55 (m, 2H), 3.25 ( 2d, J = 5 Hz, 1H), 4.1 (t, J = 6 Hz, 2H).

In the same way, but by replacing the starting material with an equivalent quantity of hydrochloride of tetradecyl ester of cysteine (prepared as described by Vouille et al., Above quotation), the tetradecyl ester of S-tritylcysteine is obtained; [oc] ë5 = + 13.8 ° (c = 1, CHCI3); NMR (CDCI3) 5 0.89 (t, J = 5 Hz, 3H), 1.3 (s, 24H), 1.56 (m, 2H), 2.55 (m, 2H), 3.25 ( 2d, J = 5 Hz, 1H), 4.1 (t, J = 6Hz, EH).

Likewise also, the corresponding nonyl, undecylic, dodecyl and tridecyl esters of S-tritylcysteine are obtained.

The combination of the preceding esters of S-tritylcysteine with the hexapeptide hydrazide of formula Ddz — Trp — Lys (Boc) - Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHNH2 the azide method gives the nonyl, decyl, undecylic, dodecyl, tridecyl and tetradecyl esters corresponding to the compound mentioned in the heading for preparation 27.

Preparation 29:

Tryptophyl-N ^ -t-butyloxycarbonyl-lysyl-O-t-butyl-threonyl-phenylalanyl-O-t-butyl-threonyl-O-t-butyl-seryl-S-tritylcysteine methyl ester formate; V, R2 — COOCH3 (H - Trp — LysiBoc) - Thr (Bu ') - Phe— 1hr (Bu') - Ser- (Bu ') - Cys (Trt) - OMe ■ HC02H)

A solution of Ddz — Trp — Lys (Boc) —Thr (Bu ') - Phe— Thr (Bu') - Ser (Bu ') - Cys (Trt) —OMe (1.11 g, 0.685 mmol, described in preparation 27) in 10 ml of a mixture of formic acid / acetic acid / water (1/7/2) is stirred at room temperature overnight. The mixture is evaporated and the rest is triturated with water. The solid material is collected by filtration and dried under reduced pressure to obtain the compound mentioned in the heading; ÄH: 290 (s: 6335), 280 (e: 8470), 273 (3720), 216 nm (s: 78400).

In the same way, but by using the other heptapeptides described in preparations 27 and 28 as starting materials, the ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, n- esters are also obtained. hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecylic, n-dodecyl, n-tridecylic and n-tetradecyl corresponding to the compound mentioned in the heading.

Example 14:

T-butyloxycarbonyl-alanyl-glycyl-S-trityl-cysteinyl-Ne-t-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanyl-tryptophyl-N * -t-butyloxycarbonyl-lysyl-methyl ester phenylalanyl-Ot-butyl-threonyl-Ot-butyl-seryl-S-trityl-cysteine; II, R2 = COOCH3 and R4 — Boc — Ala— Gly — NH (Boc — Ala — Gly — Cys (Trt) —Iys (Boc) —Asn — Phe— Phe - Trp — lysfBoc) - Thr (Bu ') - Ser (Bu ') - Cys (Trt) —OMe) To a solution of the first hydrazide of heptapeptide IV, Boc — Ala — Gly — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— NHNH2, (0.775 g , 0.624 mmol, described by HU Immer et al., Above quotation) in dimethylformamide (9 ml) at −20 ° C., anhydrous HCl 2, IN in ethyl acetate (1.56 mmol) is added, then t-butyl nitrite (0.0863 ml, 0.749 mmol). The solution is stirred at -15 ° C for 15 min. A solution of the second methyl ester of heptapeptide V is cooled to -15 ° C.

H - Trp - Lys (Boc) - ThrfBu ') - Phe - ThifBu1) - Ser (Bu') -Cys (Trt) —0Me HC02H (0.900 g, 0.624 mmol, described in preparation 29) and N-ethyldiisopropylamine ( 0.373 ml,

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2.184 mmol) in dimethylformamide (5 ml), and added to the above solution at -15 ° C. The resulting mixture is stirred at -15 ° C for 1 h and at room temperature for 3 days. The solvent is separated under reduced pressure. The rest is triturated with IN citric acid, filtered, the precipitate is washed with water and dried over phosphorus pentoxide under reduced pressure. The dried residue is triturated with a small amount of methanol, and the rest is collected and dried to obtain the compound mentioned in the heading; amino acid analysis: Lys, 2.06; cysteic acid, 1.75; Asp, 0.94; Thr, 1.93; Ser, 0.97; Gly, 1; Ala, 0.88; Phe, 3.10.

Similarly, using the other alkyl esters of heptapeptide of formula V, described in preparation 29, the ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, n esters are also obtained. -hexylic, n-heptylic, n-octyl, n-nonyl, n-decyl, n-undecylic, n-dodecylic, n-tridecylic and n-tetradecylic corresponding to the compound in heading.

Example 15:

T-butyloxycarbonyl- methyl ester cyclic disulfide

alanyl-glycyl-cysteinyl-N ^ -t-butyloxycarbonyl-lysyl-

asparaginyl-phenylalanyl-phenylalanyl-tryptophyl-

N ^ -t-butyloxycarbonyl-lysyl-O-t-butyl-threonyl-

phenylalanyl-O-t-butyl-threonyl-O-t-butyl-seryl-cysteine;

III, R2 = COOCHi and R4 = Boc- Ala- Gly - NH (Boc -

Ala — Gly — Cys — LysiBoc) —Asn — Phe — Phe —Trp — Iys (Boc) -

Thr (Bu ') - Ser (Bu') - Cys - OMe)

The linear protected tetradecapeptide methyl ester of Example 14, Boc — Ala — Gly — Cys (Trt) —Lys (Boc) —Asn — Phe — Phe— Trp, is dissolved in hot acetic acid (200 ml). —Lys (Boc) —Thr (Bul) - Phe — Thr (Bu ') - Ser (Bu') - Cys (Trt) —OMe (0.898 g, 0.345 mmol). The solution is cooled to room temperature and added dropwise to a solution of 5% iodine in methanol (175 ml, 3.45 mmol) over a period of 1 h. The mixture is stirred for an additional 1 h, cooled to 0 ° C and a solution of IN sodium thiosulfate in water (15 ml) is added to destroy the excess iodine. The solvent is evaporated off, the rest is triturated with water and dried over phosphorus pentoxide under reduced pressure to obtain the compound mentioned in the heading.

Similarly, using the other linear protected tetradecapeptide esters of formula II, described in Example 14, the ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl esters are also obtained. n-hexyl, n-heptyl, n-octyl, n-nonyl,

616,402

n-decylic, n-undecylic, n-dodecylic, n-tridecylic and n-tetradecylic corresponding to the compound in heading.

Example 16:

Somatostatin methyl ester. Cyclic disulfide of methyl ester of alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanyl-tryptophyl-lysyl-threonyl-phenylalanyl-threonyl-seryl-cysteine; I, R1 - H — Ala — Gly — NH and

R2 = COOCH3 (H-Ala- Gly - Cys - Lys-Asn - Phe - Phe -Trp — lys — Ihr — Phe— Thr — Ser — C $ s — OMe)

A solution of the cyclic disulfide of Example 15, Boc — Ala — Gly — Cys — Lys (Boc) —Asn - Phe — Phe - is stirred under a nitrogen atmosphere for 10 min at 0 ° C.

Trp — Lys— (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') -

Cys-OMe, (0.345 mmol) in concentrated hydrochloric acid (29 ml). Glacial acetic acid (290 ml) is added and the solution is lyophilized. The remainder is dissolved in 5% acetic acid and lyophilized again. The remainder is dissolved in 0.2M ammonium acetate, applied to a column of carboxymethylcellulose (Whatman CM-23) and eluted with a 0.2M ammonium acetate buffer. The eluate is lyophilized. The remainder is subjected to partition chromatography on a column of crosslinked dextran, chemically modified (Sephadex G-25M), equilibrated in the lower phase of n-butanol / acetic acid / water (4/1/5) and then equilibrated in the upper phase, and using the upper phase for the desorption of the somatostatin methyl ester. The pure fractions are combined, evaporated and lyophilized to obtain the compound cited in the form of its acetic acid addition salt 290 (e: 4345), 287 (e: 4385), 268 (e: 4090) , 265 (e: 3900), 215 nm (e: 38305); alkaline hydrolysis and gas chromatography: calculated for methanol: 1.75%; found: 2%; NMR (DMSO-d6) 5 3.63 (s, 3H, OCH3).

Repeated lyophilization of the latter acid addition salt from water gives the title compound as the free base; amino acid analysis: Lys, 2; cysteic acid, 1.28; Asp, 0.95; Thr, 1.91; Ser, 0.97; Gly, 1; Ala, 0.94; Vi Cys, 0.30; Phe, 3.01.

Similarly, using the other cyclic disulphides of formula III, described in Example 15, the ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, n-hexyl esters are also obtained. , n-hepty-lique, n-octylic, n-nonylic, n-decylic, n-undecylic, n-dodecylic, n-tridecylic and n-tetradecylic corresponding to the compound in heading.

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Claims (2)

616402 2 1. Process for the preparation of a peptide of formula I: SCH2CH (R1) CO - Lys — Asn — Phe — Phe — Trp — Lys — Thr — Phe — Thr — Ser — NHCH (R2) CH2 S (I) formula in which: a) R1 = H - Gly — Gly - Ala - Gly - NH, H-Gly-Gly-Gly — Ala — Gly — NH or H — Leu — Gly — Gly — Ala — Gly — NH and R2 = H or COOH, or b) R1 = H or NHR3, where R3 = an aliphatic acyl of 1 to 6 carbon atoms or benzoyl, and R2 = H, or c) R1 = H — Ala — Gly — NH and R2 = COOAlk, where Alk is a straight or branched chain alkyl group comprising from 1 to 14 carbon atoms, as well as its therapeutically acceptable salts, characterized in that a protected linear peptide of formula is cyclized by oxidation with iodine or thiocyanogen Trt — S — CH2 - CH - C — Lys (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) —Thr (Bu ') - Phe — Thr (Bu') - Ser (Bu ') - NHCH- CH2S - Trt R4 ° (n) R2 in which: a) R2 = H or COOH and R4 = Boc - Gly - Gly - Ala - Gly - NH, Boc— Gly — Gly — Gly — Ala — Gly — NH or Boc — Leu — Gly— Gly — Ala — Gly — NH, or b) R2 = H and R4 = H or NHR3, where R3 has the previous definition, or c) R2 = COOAlk and R4 = Boc — Ala — Gly — NH, Alk having the above definition, to obtain the corresponding cyclic disulfide derivative of formula III: SCH2CH (R4) CO - Lys (Boc) - Asn - Phe - Phe - Trp - Lys (Boc) SCH2 - CH (R2) - NH - Ser (Bu ') - Thr (Bu') - Phe - Thr (Bu ') (III) in which R2 and R4 have the definition given above, with then hydrolytic cleavage of all the protective groups remaining under moderately acidic conditions to obtain the corresponding peptide of formula I. 3. Method according to claim 1, characterized in that a solution in acetic acid of the linear peptide of formula II is treated, with iodine in solution in a lower alkanol or acetic acid, it is treated the resulting cyclic disulfide of formula III with hydrochloric acid, and the corresponding peptide of formula I is isolated. 3. Method according to claim 1, characterized in that the linear peptide of formula II is subjected to a treatment of iodine at a temperature of 0 to 30 ° C for 30 to 180 min in a lower alkanol or l acetic acid to obtain as an intermediate the corresponding cyclic disulfide of formula III. 4. Method according to claim 1, characterized in that the intermediate product, ie the cyclic disulfide of formula III, is treated with concentrated hydrochloric acid at approximately 0 ° C for 5 to 10 minutes to obtain the corresponding peptide of formula I. 5. Application of the method according to claim 1 to a linear peptide of formula II prepared by the reaction according to the azide coupling method, in which a peptide of formula IV is reacted: Trt - SCH2CH (R4) CO - Lys (Boc) - Asn - Phe - Phe - NHNH2 (IV) with nitrous acid to obtain the corresponding azide which is then coupled under basic conditions with a peptide of formula V: 40 H - Trp - Lys (Boc) - Thr (Bul) - Phe - Thr (Bu ') - Ser (Bu') - NHCH (R2) CH2S-Trt (V) formulas in which R2 and R4 have the definition given in claim 1.