CS207570B2 - Method of making the tetradecapeptide - 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

(54) A method for producing a tetradecapeptide

The invention relates to tetradecapeptide somatostatin derivatives. More specifically, it relates to a process for the preparation of somatostatin derivatives and their salts.

The name 'somatostatin' has been suggested for a factor found in hypothalamic ex tracts that inhibits growth hormone secretion (soimatropropin). The structure of this factor was determined in P. · Brazeau et al. Science 179, 77 (1973) · and was found to have the following structure of tetradecapeptide of formula

H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-C ^; ys — OH

Abbreviations used herein for various amino acids have the following meanings: Ala, alanine; Asn, asparagine; Cys, cysteine; Gly, glycine; Lys, Lysine; Phe, phenylalanine; Ser, serine; Thr, threonine and Trp, tryptophan.

The structure of the tetradecapeptide of somatostatin has been confirmed by synthesis, e.g., see D. Sarantakis and W. A. McKinley,. Biochem. Biophys. Res. Coimm, 54,234 (1973), J. River et al., Compt. Rend. Ser. D, 276, 2737 (1973) and H. U. Immer et al., Helv. Chim. Acta, 57, 730 (1974).

The important physiological effect of this tetradecapeptide classifies it as a compound suitable for clinical pharmacology in the treatment of acromegaly and for use in diabetes. K. Lundbaek et al., Lancet, 2, 131 (1970) and R. Guillemin in "Chernistry and Biology of Peptides", J. Meinhofer, Ed., 3rd Americans Peptide Symposium Boston 1972, Ann.

Arbor Science Publications, Ann Arbor, Mich., 1972.

The linear form of somatostatin with two thiol groups instead of the disulfide bridge has been prepared and is described in J. E. F. Rivier, J. Amer. Chem. Soc., 96, 2986 (1974). It is described that the linear form is as effective as. somatostatin. Efficacy was determined by the ability of both compounds to inhibit the rate of growth hormone secretion by mucosal cells of rats grown in monolayer tissue culture.

Recently, a non-natural hormone polypeptide has been found and its linear form has somatostatin-like activity. D. Sarantakis et al., Blochiem. Biophys. Res. Coimm., 55, 538 (1973) recently reported solid phase synthesis of somatostatin analogue [Ala ³ · ¹⁴ ]. This analog has a very low reactivity, about 0.01 ·% somatostatin. P. Brazeau et al., Biochem. Blophys. Res. Comm., 60, 1202 (1974) recently reported solid phase synthesis of a number of acylated derivatives by des- [Ala ^ Gly ² ].

The invention discloses somatostatin derivatives having an effect higher than or equal to the natural hormone. order. and their effect is greater than that of somatostatin. These derivatives are readily prepared in a conventional manner having the following advantages: CH 2 CH (R 1) CO-Lys-Asn-Phe-Phe -Trp-Lys

where

a) R 1 is H-Gly-Gly-Ala-Gly-NH, H-Gly-Gly-Gly-Ala-Gly-NH or H-Leu-Gly-Gly-Ala-Gly-NH and R ² is H or COOH, or

b) R 1 is H or NHR ³ wherein R ³ is an acyl radical of a straight or branched aliphatic carboxylic acid having 1 to 6 carbon atoms or benzoyl and R ² is H, or

c). R 1 is H-Ala-Gly-NH and R 2. is COOalk, wherein κ is an unbranched or branched alkyl chain of 1 to 14 carbon atoms.

Therapeutically. suitable salts of the compounds of formula I are also included within the scope of the invention.

The peptides of the invention are prepared by a process characterized in that the linear peptide of formula (II) is oxidized from readily available materials and no harmful reagents are used, proceeds easily, and use readily cleavable protecting groups.

The foregoing advantages and properties demonstrate that the peptides of the invention are useful in diabetes and for the treatment of acromegaly.

The invention relates to peptides of the general formula I, -Thr-Phe-Thr-Ser-NHCH (R2) CH2 (I)

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

....................... (Π) where

a) R 2 is · H or COOH and R 4 is Boc-Gly-Gly-Ala-Gly-NH,

Boc-Gly-Gly-Gly-Ala-Gly-NH or Boc-Leu-Gly-Gly-Ala-Gly-NH, or

b) R 2 is H and R 4 is H or NHR ³ , wherein R 3 is as defined above, or

c) R2 is COOAlk and R4 is Boc-Ala-Gly-NH, iodine or rhodane 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 and R4 are as defined above, ·, and then cleaving off the remaining. protecting groups by reaction with 50 to 100% trifluoroacetic acid or a mineral acid solution.

Linear peptides of formula II used as starting material. the compounds are readily prepared by a process characterized in that the peptide of formula IV is reacted by the azide method

Trt — SCH 2 R ⁴ ) CO — Lys (Boc) - Asn — Phe — —Phe — NHNH2 (IV) with the peptide of formula V,

H — Trp — Lys (Boc) —Thr (Bu ⁺ ) —Phe— —Thr (Bu +) —Ser (Bu + j — NHCH (R2) CH2S — —Trt (V) where

R2 and R4 are as defined above, to form the corresponding linear peptide of formula II, wherein R2 and R4 are as defined above, or the pentapeptide of formula VI is reacted by the azide method

Trt — Cys (Trt) —Lys (Boc) - Asn — Phe— —Phe — NHNH2 (VI) with a peptide of formula V, wherein R2 is · H or COOH, then the peptide of formula VIII is cleaved by cleavage of the amino-terminal protecting group,

H — Cys (Trt) —Lys (Boc) · —Asn — Phe — Phe— —Trp — Lys (Boc) —T.hr (Bu ⁺ ) —Phe— —Thr (Bu +) —Ser (Bu +) - NHCH ( R 2) CH 2 S -Trt (VIII) wherein

· R ² is H or COOH · and · latter compound is allowed to react with the azide method · peptides of formula IX

R 5 -Gly-Gly-Ala-Gly-NHNH 2 (X)) wherein

R ⁵ is Boc, Boc-Gly or Boc-L1-eu, to give the corresponding linear peptide of formula II, wherein R ² is H or COOH and R ⁴ is Boc-Gly-Gly-Ala-Gly-NH, Boc-Gly- Gly-Gly-Ala-Gly-NH or Boc-Lleu-Gly-Gly-Ala-Gly-NH.

In general, the abbreviations in the description used to refer to amino acids and protecting groups are based on the recommendations of the IUPAC-Commission on Biochemical Ncimenclature, see Biochemistry, 11, 1726-1732 (1972). For example, Cys, Lys, Asn, Phe, Trp, Ph, Thr, and Ser are "residues" of L-cysteine,

L-lysine, L-asparagine, L-phenylalanine, L-tryptophan, L-threonine and L-serine.

The term "residues" refers to residues derived from the corresponding L-amino acid by eliminating the OH part of the carboxyl group and the IT part of the amino group. All amino acids have a natural L-configuration.

Many methods and methods for preparing peptides are well known. For example, functional groups that do not relate to peptide bond formation are preferably protected by a condensation reaction. For example, protecting groups suitable for protecting the amino function of a peptide or amino acid that is not involved in peptide bond formation are: alkoxycarbonyl, including benzyloxycarbonyl (symbolized as Z), tert-butoxycarbonyl (symbolized by Boc), α, α-dimethyl-3,5 -dimethoxybenzyloxycarbonyl (symbolized by Ddz), 2- (p-biphenylisopropyloxycarbonyl (symbolized by Rys)), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, isopropyloxycarbonyl or ethoxycarbonyl; · alkyl type that include triphenylmethyl or trityl (Trt symbolisovaný) or benzyl. Preferred protecting groups in the process according to the invention are benzyloxycarbonyl, tert-butoxycarbonyl, triphenylmethyl aa, a-dimethyl-3,5-dimethoxybenzyl ⁴ benzyloxycarbonyl. protecting groups for hydro- xylserine and tyrosine are acetyl, tosyl, benzoyl, tert-butyl (symbolized Bu ⁺ ), trityl and benzyl, the preferred protecting group * is tert-butyl. Sulfur groups on cysteine or modified cysteine are, for example, benzyl, triphenylmethyl or trityl (symbolized by Trt), benzyloxycarbonyl or acetamidomethyl (symbolized by Acm), preferred protecting groups are: trityl or acetamidomethyl. The carboxyl function of the peptide or amino acid can be protected with a lower alkyl or lower aryl ester including methyl ester (symbolized OMe), ethyl ester (symbolized OEt), benzyl ester (symbolized OBz11) as well as a substituted hydrazide that includes tert-butoxycarbonylhydrazide (NHNH Boc) , benzyloxycarboinylhydrazide (NHNHZ) or α, α: -dimethyl-3,5-dimethoxybenzyloxycarbonylhydrazide (NHNHDdz).

To facilitate condensation of a peptide carboxyl group with the free amino group of another peptide to form a new peptide bond, the terminal carboxyl group must be activated. A description of these carboxyl group activating groups can be found in general peptide chemistry textbooks such as K. D. Kopple, &quot; Peptides and Amine Acids &quot;, VV. A. Benjamin, Inc., New York. 1936, pp. 45-51 and E. Schroder and K. Lubke, "The Peptides", vol. I, Academic Press, New York, 1965, pp. 77-128. Examples of activated forms of terminal carboxyl groups are: acid chlorides, acid anhydrides, acid azides, activated esters or o-acyimines of dialkylcarbodiimide. The following activated esters are particularly useful in the process of the invention: 2,4,5-trichlorophenyl (OTcp), pentachlorophenyl (OPcp), p-nitrophenyl (ONp), or 1-benzotriazolyl, also succinimido group is suitable for this activation.

The term "azide method," as used herein, refers to the coupling of two peptide fragments, characterized in that the peptide hydrazide is reacted with an in situ nitrous acid reagent. Suitable reagents for this purpose are organic nitrites, for example tert-butyl nitrite, isoamyl nitrite, or alkali metal nitrites (e.g. sodium nitrite, potassium nitrite) in the presence of a strong acid such as hydrochloric, sulfuric or phosphoric acid. The corresponding azide of the peptide thus obtained is then not reacted with a free amino group petid to yield the desired peptide. Preferred conditions for the azide method include reacting the peptide hydrazide with nitrous acid generated in situ from an organic nitrite in the presence of a strong acid, preferably hydrochloric acid (pH normally from 0.1 to 2) in an anhydrous inert organic solvent such as dimethylformamide, dimethylsulfoxide, ethyl acetate, methylene chloride, tetrahydrofuran, dioxane and the like, at a temperature of from -30 ° C to (20 ° C, preferably about -15 ° C for about 10 to 30 minutes) to yield the corresponding azide. Then, the azide in the above reaction mixture is reacted with a free amide peptidic unit at a temperature of from -30 ° C to 20 ° C for one to two hours and then at 0 to 30 degrees Celsius. The acid acceptor, preferably an organic base, for example N-ethyldiisopropylamine, N-ethylmiorpholine or triethylamine, is added. is added to the reaction mixture such that the reaction medium is slightly alkaline, preferably pH 7.0 to 7.5. See also Kop pleho or Schroder and Lubke textbooks above for more information on this method.

The terms "peptide, tripeptide, hexapeptide" and the like, as used herein. here, they are not limited to the corresponding parent peptides, but are also meant to include modified peptides with altered! or protected groups. The term "peptides" as used herein refers to peptides with two to seventeen amino acid residues. The moiety "SCH 2 -H (R ¹ ) CO" as used herein refers to a modified cysteine residue wherein R ¹ is H, or a cysteine residue when R ¹ is NHR ³ wherein R ³ is as defined above 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 residue "NHCH (R ² ) CH ² S",. as used herein refers to a cysteine residue, if R ² is -COOH or COOalk or modified cysteine residue, if R 2 is H.

Me stands for methyl and NHNH 2 stands for hydrazide.

The term "lower alkyl" as used herein includes hydrocarbon radicals having one to three carbon atoms including methyl, ethyl and propyl. "Alk" means a straight or branched chain alkyl group having 1 to 14 carbon atoms.

The term "acyl" as used herein includes lower aliphatic acyls of 1 to 6 carbon atoms, namely straight or branched chain acyls such as formyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, nhexanoyl and the like.

The term "C 1 -C 6 aliphatic acyl" includes straight or branched chain acyl groups such as foirmyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, n-hexanoyl and the like.

The term "mineral acid" as used herein includes strong inorganic acids, such as hydrochloric, hydrobromic, sulfuric, or phosphoric acid. When used in conjunction with an anhydrous system, the preferred acid is anhydrous hydrogen chloride.

The term "weakly acidic." conditions, as used herein, especially those in which a dilute aqueous solution of an organic acid, for example 30-80% aqueous formic, acetic or propioic acid, preferably 70-80%, is used as the reaction medium.

The term "mildly acidic conditions" as used herein means conditions in which concentrated organic acids or mineral acid solutions are used as a major component of the reaction medium at a temperature of about -30 ° C. to 30 ° C. Examples of preferred. The conditions in this case are 50 to 100% trifluoroacetic acid at a temperature of 0 to 30 ° C or 0.1 to 12 N hydrochloric acid in aqueous solution or in solution in organic solvent or hydrogen chloride in solution in anhydrous. or a gain solvent at -20 to 10 ° C.

The term "organic nitrite" is used to refer to. commercially available nitrites such as t-butyl nitrite, isoamyl nitrite and the like.

The term "organic base" as used herein includes triethylamine, N-ethylmorpholine, N-ethyldiisopropylamine and the like.

As used herein, the term "strong base" means both the organic bases described above and the strong inorganic bases including sodium hydroxides and carbonates. and potassium.

The peptides of the invention are obtained in the form of the free base or as the acid salt either directly. in the procedure of. of the invention, or by reacting the peptide with one or more equivalents of the respective acid. Examples of preferred salts are those with pharmaceutically acceptable acids, such as. acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or toluenesulfonic acid, as well as polymeric acids such as tannin or carboxymethylcellulose and inorganic acid salts such as hydrohalic acids such as hydrochloric acid or sulfuric acid or phosphoric acid. It should be noted that the peptides have two basic nitrogen atoms and provide salts with one to two equivalents of acid. If desired, a particular acid salt is converted into another acid salt, for example a salt with a non-toxic pharmaceutically acceptable acid. by treatment with an appropriate ion exchange resin as described by R. A. Biossonas et al., Helv. Chim. Acta 43, 1349 (1960). Suitable ion-exchange resins are cellulose-based ion exchangers, for example, carboxymethylcellulose or a chemically modified cross-linked dextran cation exchanger, for example of the Sefadex C type, and a strongly basic ion-exchange resin, such as those disclosed in JP Greenstein and M. Winitz "Chemistry of the Amino Acids", John Wiley and Soins, Inc., New York and London, 1961, Vol. 2, pp. 1456.

The somatostatin analogs of formula I provide complex salts with. heavy metal ions. Examples of pharmaceutically acceptable heavy metal complexes. is a complex consisting of zinc or protamine. zinc.

The peptides prepared by the process of the invention, as well as their corresponding ones. pharmaceutically acceptable salts. are applicable. due to its pharmacological activity analogous to the natural tetradecapeptide somatostatin. Their activity has been proven by pharmacological tests such as. are modifications [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)] in vitro methods according to M. Saffran and A.V. Schally, Can. J. Biochem. Physiol.,. 33,405 (1955).

The effect of the peptides of formula I according to the invention has also been demonstrated in vivo by modifying a pentobarbital-induced increase in plasma growth hormone levels in rats as described in Brazeau et al., Supra. In this assay, the peptides of the invention exhibit an effect that is greater than or equal to that of somatostatin. <

The peptides of the invention are useful for the treatment of acromegaly and related hypersecretory endocrine conditions and in mammalian diabetes, see P. Brazeau et al., Supra. When the peptides or their salts are used for this treatment, they are applied systemically, preferably parenterally, in combination with pharmaceutically acceptable liquid carriers. The peptides of formula I have low toxicity. The ratio of the peptide or salt thereof is determined by the solubility in said carrier, said carrier or the mode of administration. When the peptide or a salt thereof is used in a sterile aqueous solution, the solution may also contain other solutes such as buffers or preservatives, as well as sufficient amounts of pharmaceutically acceptable salts or glucose to render the solution isotonic. The doses depend on the form of administration and, in particular, on the kind to be treated, and are preferably between 1 and 300 per kg of body weight. However, a dosage in the range of about 1 µg to about 50 µg per kg body weight is preferably used to achieve effective results.

The peptides or salts may be administered in a single long-acting slow release depot dosage form described below, preferably in the form of an intramuscular injection or implant. These dosage forms are adapted to release from about 1 µg to about 50 µg per kg of body weight per day.

It is often desirable to administer the agent continuously over a prolonged period of time, in a slow release or depot dosage form. These dosage forms may comprise either a pharmaceutically acceptable salt or a peptide with low solubility in body fluids, for example one of the salts described below, or a peptide in the form of a water-soluble salt together with a protective carrier that prevents rapid release. In the latter case, the peptide may, for example, be formulated with a non-antigenic partially hydrolysed gelatin in the form of a viscous liquid or absorbed onto a pharmaceutically acceptable solid carrier such as zinc hydroxide and applied in suspension in a pharmaceutically acceptable carrier. suspensions with a protective non-antigenic hydrocolloid, for example, sodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate, gelatin, polygalacturonic acids, for example pectineim, certain mucopolysaccharides, together with aqueous or non-aqueous pharmaceutically acceptable liquid carriers, preservatives or surfactants. Examples of these preparations can be found in standard pharmaceutical textbooks, for example, in Rerning ton's Pharmaceutical Sciences 14th Ed., Mack Publishing Co., Easton; Pennsylvania, 1970.

Long-term slow release preparations of the peptide prepared according to the invention can also be prepared by microencapsulation in pharmaceutically acceptable coatings, for example in gelatin, polyvinyl alcohol or ethylcellulose. Other examples of coating materials and microencapsulation procedures are described in J.A. Herbig &quot; Encyclo pedia of Chiemical Technology &quot;, Vol. 13, 2nd Ed., Wiley, New York 1967, pp. 436-456. These preparations, as well as suspensions or peptide salts that are only sparingly soluble in body fluids, for example pamoic acid or tannin salts, are formulated to release about 1.0 mcg to about 100 mcg of active compound per kg body weight per day and they are preferably administered by intramuscular injection. Alternatively, by intramuscular injection. Alternatively, some of the solid dosage forms mentioned above, for example certain water-soluble salts or dispersions or adsorbates on solid carriers or peptide salts, for example, dispersions in a neutral hydrogel of ethylene glycol methacrylate polymer or similar crosslinked monomers as described in U.S. Patent No. 3,551,556 may also be formulated as pills which release the same amount as described above and may be implanted subcutaneously or intramuscularly.

The process of the invention is explained in more detail in the following description in which the preparation of certain peptides of formula I is described.

a) Compounds of formula I wherein R ¹ = H- Gly-Gly-Ala-Gly-NH, H-Gly-Gly-Gly-Ala-Gly-NH or H-Leu-Gly-Gly-Ala- Gly-NH- and R ² is H or COOH,

Alanyl-glycine protected lower alkyl ester, preferably Boc-Ala-Gly-OMe [described by H. U. Immer et al., Helv. Chim. Acta., 57 730 (1974)], is dissolved in trifluoroacetic acid and the solution is maintained at 0-10 ° C for about one hour, and after evaporation of the trifluoroacetic acid, H-Ala-Gly-OMe is obtained as the trifluoroacetic acid salt, which may optionally be converted and used in the form of the free base.

Other cleavage agents for Boc deprotection are hydrogen bromide in acetic acid, an alcoholic solution of hydrogen chloride, and diiod. The latter compound is dissolved in an inert organic solvent, preferably dimethylformamide, and the solution obtained is cooled to about 0-10 ° C. An excess, preferably 1.1 to 1.3 equivalents of an organic base, preferably N-ethylmorpholine, is added to the solution, and the solution then has a pH of about 8. One equivalent of the protected activated glycine ester, preferably Boc-Gly-OTcp, is added. [described in J. Pless and RA Boissonnas Helv. Chim. Acta 46, 1609 (1963).], And the reaction mixture is allowed to stand for about two days at 0 to 20 ° C. The solvent is evaporated and the residue crystallized to give the protected lower alkyl ester of glycyl-alanyl-glycine, preferably Boc-Gly-Ala-Gly-OMe, which, by reaction with trifluoroacetic acid, yields the lower alkyl ester of glycyl-alanyl-glycine tripeptide, preferably H- Gly-Ala-Gly-OMe, in the form of the trifluoroacetic acid salt, optionally converted to the free base. The latter tripeptide is reacted with Boc-Gly-OTcp as described above to provide a protected lower alkyl ester of glycyl-glycyl-alanyl-glycine, preferably Boc-Gly-Gly-Ala-Gly-OMe, which by reaction with trifluoroacetic acid gives in the above manner, a lower alkyl ester of glycyl-glycyl-alanyl-glycine, preferably H-Gly-Gly-Ala.-Gly-OMe in the form of the trifluoroacetic acid salt, which can be converted again to the free base. The latter tetrapeptide is reacted with a protected activated glycine ester, preferably Boc-Gly-OTcp, as described above to give a lower alkyl ester of glycyl-glycyl-glycyl-alanyl-glycine, preferably Boc-Gly-Gly-Gly-Ala —Gly — OMe.

The above deprotected tetrapeptide, H-Gly-Gly-Ala-Gly-OMe, preferably in the form of a trifluoroacetic acid salt, is reacted with a protected activated leucine ester, preferably 1-benzortriazzlylesser of tert-butyloxycarbonylleucine at 0 to 0 15 ° C in an inert organic solvent, preferably dimethylformamide, by mixing the above unprotected tetrapeptide, substantially 1.5 to 2.0 equivalents of Boc-Leu-OH, 1.5 to 2.0 equivalents of 1-hydroxybenzotriazoil, 1.5 to 2.5 equivalents of dicyclohexylcarbodiimide, and an excess of an organic base, preferably N-ethylmorpholine; The solution was maintained at 0-15 ° C for 20-30 hours. Removal of the precipitate, evaporation of the filtrate and crystallization affords the protected lower alkyl ester of leucyl-glycyl-glycyl-alanyl-glycine, preferably Boc-Leu-Gly-Gly-Ala-Gly-OMe.

The above tetrapeptide of the formula Boc-Gly-Gly-Ala-Gly-OMe and pentapeptides of the formula 'Boc-Gly-Gly-Gly-Ala-Gly-OMe and Boc-Leu-Gly-Gly-Ala-Gly-OMe are referred to herein as' R ⁵ -Gly-Gly-Ala-Gly-OMe, wherein R ⁵ is Boc, Boc-Gly, and Boc-Leu.

Polsedně said compound of formula R ⁵ - Gly-Gly-Ala-Gly-OMe, wherein R ⁵ is as defined above The aforesaid is easily are converted to the corresponding hydrazide by reaction with an excess (20-50 molar equiv-ents) of hydrazine hydrate. Preferred conditions include reacting the latter esters in an inert organic solvent such as methanol, n-butanol or dimethylformamide with 40 to 50 molar equivalents of hydrazine hydrate at 0 to 30 ° C for two hours to one day. Evaporation of the solvent and excess hydrazine hydrate affords the corresponding amino-protected hydrazide of the peptide of formula IX,

R 5 -Gly-Gly-Ala-Gly-NHNH 2 (IX) wherein

R ⁵ is Boc, Boc-Gly or Boc-Leu.

The above peptide of formula IX and the pentapeptide of formula

H-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-OMe (described by H. U. Immer et al., Above) is reacted in an azide fashion to give a peptide of formula

R5-Gly-Gly-Ala-Gly-Cys (Trt) -Lys' (Boc) -Asn-Phe-Phe-OMe.

A conventional and effective method for this step involves dissolving the peptide of formula IX in an inert organic solvent, preferably dimethylformamide, or in a mixture of dimethylformamide and dimethylsulfoxide. A solution of about two to five molar equivalents, preferably three molar equivalents, of a solution of a strong mineral acid in an organic solvent, preferably hydrogen chloride, in ethyl acetate is added to the latter. to said solution at a temperature of -20 ° C to -10 ° C, preferably at -15 ° C, and to the stirred solution is added an organic nitrite, preferably a target, butyl nitrite (1.0 to 1.5 molar equivalent, preferably 12 equivalent). After 15 minutes at -20 ° C to 0 ° C, the mixture is basified, preferably to a pH of 7.0 to 7.5, by the addition of an organic base, preferably N-ethyldiisopropylamine, and then one equivalent of the above pentapeptide is added. Further addition of the organic base, preferably N-ethylmorpholine, makes the mixture slightly alkaline. The reaction mixture is then stirred at -10 to 0 ° C for one to two hours and then for 20 to 30 hours at 20 to 30 ° C. Evaporation of the solvent, dissolution of the residue in an organic solvent, preferably methanol, addition to a solution in a solvent in which precipitation occurs, preferably in water or diethyl ether, and collection of the product yields the above peptide of formula

R5-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-OMe.

The pentapeptide fragment described above is readily obtained by reacting an activated ester of Boc-Phe-OH 'with H-Phe-OMe to give Boc-Phe-Phe-OMe which, after removal of the terminal protecting group (Boc) under slightly acid of the conditions provides H-Phe-Phe-OMe. The latter compound is reacted with an activated ester of Boc-Asn-OH to give Boc-Asn-Phe-Phe-OMe. Subsequent cleavage of the amino-terminal protecting group of the latter compound under mildly acidic conditions affords the tripeptide H-Asn-Phe-Phe-OMe.

Then the latter compound is used to obtain the desired ¹ penfapeptidového fragment reacting an activated ester of the tripeptide Z-Lys (Boc) -OH to give Z-Lys (Boc) -Asn-Phe-Phe-OMe, kterj hydrogenation over a noble metal catalyst gives H-Lys (Boc) -Asn-Phe-Phe-OMe, which is condensed with activated ester Trt-Cys (Trt) -OH to give Trt-CysSTr-Lys-4-A) -Asn-Phe- Phe-OMe and cleavage of the terminal N-protecting group (Trt) in the latter compound under mildly acidic conditions affords the desired pentapeptide H-Cys (TN) -Lys (Boc) -Ann-Phe-Pe-OMe.

In the preparation of the compound of formula I above, the above peptide ester of R ⁵ —Gly — Gly— —Ala — Gly — Cys (Trt) —Lys (Boc) - Asn— —Phe — Phe — OMe, wherein R ⁵ is as defined above , converted to the corresponding hydrazide by reaction with an excess of hydrazine hydrate as described above to give the hydrazide of the peptide of formula IVa,

R5 - Gly - Gly - Ala - Gly - Cys (Trt) - Lys (Boc) - Asn - Phe - Phe - NHNH 2 (IVa) where

R5. has the meaning given herein or alternatively written as a peptide of formula IV wherein R ⁴ is Boc-Gly-Gly-Ala-Gly-NH, Boc-Gly-Gly-Gly-Ala-Gly-NH or Boc-Leu-Gly-Gly- Ala-Gly-NH.

In the next step of the process according to the invention, the latter compound of formula IV and the peptide of formula V,

H-Trp-Lys (Boc) Thr (tBu +) -Phe- Thr (tBu +) - NHCH (R 2) CH ₂ S-Trt (V) wherein

R ² is H or COOH, is reacted with azide in the manner described above for the linear peptide of formula (IIa)

R - Gly - Gly - Ala - Gly - Cys (Trt) - Lys (Boc) - Asn - Phe - Phe - Trp - Lys (Boc) - Thr (Bu ⁺ ) - Phe - Thr (Bu +) - - Ser (Bu ⁺ ) - NHCH (R 2) CH 2 S - Trt where

R 5 is as defined above and R ² is H or COOH or alternatively written as a linear peptide of formula II wherein R ² is H or COOH and R ⁴ is Bo-c-Gly-Gly-Ala-Gly-NH, Boc- Gly-Gly-Gly-Ala-Gly-NH or Boc-Leu-Gly-Gly-Ala-Gly-NH.

A conventional and efficient process for this step involves dissolving the hydrazide of the peptide of formula IVa in dimethylformamide. A solution of about two to five molar equivalents, preferably three molar equivalents, of a solution of a strong mineral acid in an organic solvent, preferably hydrogen chloride in ethyl acetate, is added to the latter solution at -20 to -10 ° C, preferably at -15 ° C. and t-butyl nitrite (1.0 to 1.5 molar equivalents, preferably 1.2 equivalents) is added to this solution with stirring. After about 15 minutes at -20 ° to 10 ° C, one equivalent of the peptide of formula V and an organic base, preferably three to five equivalents of N-ethyldiisopropylamine in dimethylformamide, are added at a temperature of about -15 ° C.

The reaction mixture is then stirred for one to two hours at -20 ° C to 0 ° C and then for 15 to 25 hours at 20 to 30 ° C. Evaporation of the solvent, trituration of the residue with water, methanol or a mixture of m-ethanol and aqueous citric acid (2-5%) and separation of the solids gave the above linear peptide of formula IIa, which can be used without further purification. see below.

The above peptide of formula V,

H — Trp — Lys (Boc) —Thr (Bu +) —Phe— —Thr (Bu ⁺ ) —Ser (Bu +) - NHNH— - (R2) CH2S — Trt where

R2 is COOH or alternatively written as a heptapeptide of formula · (Va)

H-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe ^- Thr (Bu ⁺ ) -Ser (Bu ⁺ ) -Cys (Trt) -OH, described by HU Immer et al., Supra and in U.S. Pat. 3,917,578 of 4. XI. 1975, is readily obtained by reacting methyl tert -butylserine with activated benzyloxycarbonyl- (tert -butyl) threonine ester to give Z-Thr (Bu +) - Ser (Bu +) -OMe.

The amino end-protecting group (Z) is then removed by hydrogenation in the presence of a noble metal catalyst to give H-Thr (Bu ⁺ ] -Ser (Bu +) - OMe.

The latter methyl ester is then reacted with an activated ester of Z 2 -Phe-OH to give Z-Phe-Thr (Bu +) -Ser (Bu ⁺ ) -OMe from which the terminal amino protecting group Z is removed by subsequent hydrogenation in in the presence of a noble metal catalyst, and H - Phe - Thr (Bu ⁺ ) - Ser (Bu +) - OMe is obtained. Then latter ester tripeptide is reacted with an activated · ester of Z-Thr ^(B u ⁺⁾ - OH to give Z-Thr (Bu +) - Phe-Thr (tBu +) - Ser (tBu +) - OMe. Again, the amino terminus protecting group (Z) in the latter compound is cleaved by hydrogenation in the presence of a noble metal catalyst to give H-Thr (Bu +) -Phe-Thr (Bu +) -Ser (Bu ') -

1β —OMe. This compound is reacted with an activated ester of Z-Lys (Boc) -OH to give Z-Lys (Boc) -Thr (Bu +) - Phe-Thr (Bu +) - Ser (Bu +) - OMe, followed by cleavage. the amino-terminal (Z) protecting group of the latter compound by hydrogenation in. presence of a rare catalyst. metal to give H - Lys · [Boc) - Thr [Bu +] - Phe - Thr (Bu +) - Ser (Bu ⁺ ) - OMe. This compound is now reacted with an activated ester of Ddz-Trp-OH to give Ddz-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu +) -Ser (Bu +) - OMe which is reacted with hydrazine hydrate to give the corresponding hexapeptide hydrazide, Ddz-Trp-Lys (Boc) -Thr (Bu + j-Phe-Thr (Bu +) -Ser (Bu +) -NHNH 2). H-Cys (Trr) -OH by azide method to give the corresponding heptapeptide, Ddz-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu + j-Ser (Bu + j-Cys (Trt)) ) -OH. reaction of the latter compound under mild acidic conditions yields the desired heptapeptide of the formula Va or a peptide of formula V where R ² is COOH.

The above peptide of formula V, wherein R 2 is H, described in U.S. Patent No. 3,917,581 of 4. XI. 1975, is obtained readily by reacting the described hexapeptide hydrazide of formula Ddz -Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu + j-Ser (Bu +) -NH 2 with 2-tritylthioethylamine azide method described above and yields with the corresponding hexapeptide of formula Ddz-Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu +) -Ser (Bu + j -NHCH 2 CH 2 -Trt) This compound is then reacted under mildly acidic conditions to give the peptide of formula V wherein R 2 is H in the form of a salt with formic acid and optionally converted to the free base.

Alternatively, the above-described linear peptide of formula IIa is readily prepared as follows:

The protected lower alkyl ester of the pentapeptide of the formula Trt-Cys (Trt) -Lys (Boc) -Ans -Phe-Phe-OMe, described above, is readily converted to the corresponding hydrazide by reaction with an excess of hydrazine hydrate. Preferred conditions include reacting the latter ester in an inert solvent such as methanol, butanol, or dimethylformamide with 20 to 40 molar equivalents of hydrazine hydrate for one to two days at 0 to 30 ° C. Cleavage of the solvent and crystallization yields the corresponding pentapeptide hydrazide of formula VI,

Trt — Cys (Trt) —Lys (Boc) —Asn — Phe — —Phe — NHNH2.

This latter reaction, the peptide of formula V where R ² is H or COOH · is reacted azide method as described above to give the peptide of formula VIII, Trt-Cys (Trt) -Lys (Boc) - Asn-Phe-Phe Trp-Lys (Boc) Thr (tBu + j- -Phe-Th.r (+ Bu) -Ser (tBu +) - NHCH ^(R2) C'H2S-Trt, wherein R ² is H or COOH, then after cleavage of the terminal N-protecting group (Trt) in the latter compound VII, the corresponding peptide of formula VIII is obtained,

H-Cys (Trt j Lys (Boc) - Asn-Phe-Phe-Trp-Lys (Boc) Thr (tBu + j -Ser (tBu +) - NHCH (R ²⁾ CH 2 S-Trt wherein

R 2 is H or COOH. The cleavage of this protecting group (Trt) is carried out under mildly acidic conditions. Preferred conditions include dissolving the peptide of formula VII in a mixture of 5-15% formic acid in 60-80% acetic acid, then allowing the reaction solution to stand at 20-30 ° C for 3-10 hours. Concentration of this solution gave the peptide of formula VIII, wherein R ² is H or COOH, · an acid salt which may be converted to the free base.

The latter compound of formula VIII and the hydrazide of the peptide of formula IX, wherein R ⁵ is as defined above, are reacted by the azide method described above to give the corresponding linear peptide of formula IIa or formula II wherein R2 is H or COOH and R4 is Boc-Gly —Gly — Ala — Gly— —NH—, —Boc — Gly — Gly — Gly — Ala — Gly — —NH, or · Boc — Leu — Gly — Gly — Ala — Gly— —NH—.

Conversion of the latter of the linear peptide of formula II obtained by the process described above into the corresponding compound of formula I is carried out conventionally and effectively by first reacting the linear peptide of formula II with poison, preferably in the presence of a lower alkanol or acetic acid. thiol protecting groups such as Trt to form a disulfide bridge to form the corresponding cyclic disulfide derivative of formula III,

SCH2CH (R4) CO-Lys (Boc) -Ann-Phe-Phe-Trp-Lys (Boc) -Thr (Bu +) -Phe- where -Thr (Bu +) -Ser (Bu +) -NHCH (R2) CH2S ( ΠΙ)

R ² is H or COOH and R4 is Boc-Gly-Gly-Ala-Gly-NH, Boc-Gly-Gly-Gly-Ala-Gly-NH or Boc-Leu-Gly-Gly-Ala-Gly- NH. The latter compound of formula III, wherein R 2 is

COOH and R 4 is Boc-Gly-Gly-Ala-Gly-NH, Boc-Gly-Gly-Gly-Ala-Gly-NH or Boc-Le'u-Gly-Gly-Ala-Gly-NH may alternatively write like a lilac pattern,

R ⁵ —Gly — Gly — Ala — Gly — Cys — Lys- (Boc) —Asn — Phe — Phe — Trp — Lys (Boc) - —Thr (Bu +) —Phe — Thr (Bu ⁺ ) — Ser (Bu +) —Cys — OH (IIIa) where

R ⁵ is as defined above.

Subsequent reaction of a compound of formula III wherein R 2 is H or -COOH and R 4 is Boc-Gly-Gly-Ala-Gly-NH, Boc-Gly-Gly-Gly-Ala-Gly-NH or Boc-Leu-Gly- Gly-Ala-Gly-NH, the remaining protecting groups (i.e. Boc and Bu +) are cleaved under mildly acidic conditions to give the corresponding compound of formula I wherein R 4 is H-Gly-Gly-Ala-Gly-NH, H —Gly — Gly — Gly— —Ala — Gly — NH or H — Leu — Gly — Gly— —Ala — Gly — NH and R ² is H or -COOH.

In a preferred embodiment, the above transformation of the linear peptide of formula IIa is carried out by dissolving 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, preferably 10 molar equivalents, dissolved in one of the above solvents, preferably a 2 to 5% solution of iodine in methanol). this reaction is not critical, but the reaction is required to be maintained by adding an iodine solution at 0 to 30 degrees Celsius or to cool the reaction, or to use a combination of both. At the end of the addition, the mixture is stirred for 30 to 120 minutes at 20 to 30 ° C, preferably stirred for 60 minutes, then the reaction mixture is cooled to about 0 ° C and an excess of a mild reducing agent, preferably sodium thiosulfate in The mixture is concentrated and the residue is suspended in water, collecting a solid to give the corresponding cyclic disulfide of formula III and wherein Boc and protecting groups are still present.

Alternatively, the linear peptide of formula IIIa can be converted to the aforementioned corresponding cyclic disulfide derivative of formula IIIa by the method of R. G. Hiskey and R. L. Srnith, J. Amer. Chem. Soc., 90, 2677 (1968) using rhodane.

Finally, the aforementioned cyclic disulfide derivative of formula III is converted to the corresponding peptide of formula I by reaction under mildly acidic conditions, leaving the remaining protecting groups of the cyclic disulfide derivative cleaved. Generally this step is carried out by dissolving ^one cyclic disulfide derivative in an aqueous reaction medium containing an organic acid at from 0 to 20 degrees C ^ isio for 10 to - 60 minutes. Examples of suitable reagents are trifluoroacetic acid, 10 to 20% aqueous sulfuric acid, 10% phosphoric acid, 10 to 30% hydrobromic acid and 10 to 36% hydrochloric acid. A particularly preferred medium is concentrated hydrochloric acid. Preferred conditions for the process of the invention are dissolving the cyclic disulfide in a minimum amount of concentrated hydrochloric acid, cooled to 0 ° C, and stirring the reaction mixture for 5 to 10 minutes at 0 ° C under a nitrogen atmosphere. Then glacial acetic acid (10 times the volume) is added and the solution is cooled to about -70 ° C and lyophilized to give the cyclic peptide of formula I. The latter product is further purified by chromate chromatography on an ion exchanger, preferably using a carboxymethylcellulose ion exchanger and ammonium acetate as a leaching agent. In this case, the product is obtained in the form of a salt with acetic acid. Alternatively, the product is purified by partition chromatography on a chemically modified cross-linked dextran such as Sephadex LH-20 or Sephadex G-25 using methanol or acetic acid as the eluent. When Sephadex LH-20 and methanol are used as the eluent, the product is obtained as the hydrochloride. When Sephadex G-25 and acetic acid are used as the eluent, the product is obtained as the acetic acid salt. Repeated lyophilization from water gives the acetic acid salt product to give an almost pure peptide of formula I wherein R 1 is H-Gly-Gly-Ala-Gly-NH, H-Gly-Gly-Gly-Ala-Gly-NH or H-Leu Gly-Gly-Ala-Gly-NH and R ² is H or COOH Velim free base form.

b) Compounds of formula I wherein R ¹ is -H or NiHR ³ , wherein R ³ is as defined above and R ^{2 is} -H

The desired hydrazide of the peptide of formula IV Trt-SCH 2 CH (R ⁴ ) CO-Lys (Boc) -Ann-Po-Phe-NHNH 2 wherein

R ⁴ is NHR ³ , wherein R ³ is as defined above, or alternatively-written as Formula IVb,

R ³ —Cys (Trt) —Ly s (B oc) - Asn — Phe — —Phe — NHNHz, where

R ³ is as defined above, prepared by acylating a pentapeptide of formula H-Cys (Trt) -Lys (Boc) -Ann-Phe-Phe-OMe to give a pentapeptide of formula R 3 -Cys (Three) -Lys (Boc) -Ann- Phe-Ph.e-OMe which is subjected to hydrazinolysis to give the corresponding pentapeptide hydrazide of formula IV wherein R ⁴ is NHR 3.

In a preferred embodiment of the preparation of the above pentapeptide hydrazide of formula IVb, an equimolar mixture of an organic base, preferably N-ethylmorpholine and pentapeptide of formula H-Cys [Trt] -Lys (Boc) -Ann-Phe-OMe, prepared according to II . U. Imtmer et al., Supra, in an inert organic solvent, preferably diethylformamide or tetrahydrofuran, at a temperature of about 0-10 ° C, reacted with an excess, preferably 1.1-2 molar equivalents, of the desired p-nitrophenylacylate or benzoate, for example p-nitrophenylacetate, prepared as described in F.

D. Chattaway J. Chem. Soc. 2495 (1931). The mixture is then held at 0-10 ° C for 15-30 hours and then evaporated. The residue is dissolved in a polar organic solvent, preferably methanol, and slowly added to a non-polar organic solvent, preferably diethyl ether. The residue is separated and crystallized to give the corresponding acylated or benzoylated pentapeptide, for example the pentapeptide of the formula R ³ - Cyse (Trt) Lys (Boc) - Asn - Phe - Phe - OMe, wherein R ³ is as defined above. The latter compound is dissolved in an inert organic solvent, for example methanol, ethanol, dimethylformairnide and the like, preferably methanol. The solution is then reacted with an excess of hydrazine hydrate, for example 15 to 30 molar equivalents. The reaction mixture is maintained at 0-10 ° C for 40-60 hours. · The precipitate was collected and dried to yield said pentapeptide hydrazide of formula IV wherein R 4 is NHR 3 wherein R ³ has the abovementioned meaning, or vzoirce IVb, wherein R ³ is as defined above.

According to another feature of the invention, the desired hydrazide of the peptide, the tetrapeptide of formula IV,

Trt-S-CHzCH (R ⁴⁾ CO-Lys (Boc) - Asn-Phe-Phe-NHNHz wherein

R 4 is a hydrogen atom, prepared by reaction activated; 3-tritylthiopropionic acid ester with a tetrapeptide of the formula HI-Lys (Boc) -Asn-Phe'-Phe-OMe to give the tetrapeptide of the formula Trt-SCH2CH2CO-Lys (Boc) -Asn-Phe-Phe-OMe which is hydrazed nolysed to the tetrapeptide hydrazide of formula IV, wherein R 4 is hydrogen.

In a preferred embodiment of the above tetrapeptide hydrazide, the activated ester of 3-tritylthiopropionic acid, preferably pentachlorophenyl ester, is prepared by mixing equimolar amounts of 3-tritylthiopropionic acid, pentachlorophenol and dicyclohexylcarbodiimide in an inert organic solvent, preferably tetrahydrofuran at 0 ° C. The mixture is stirred for about one hour at 0 to 10 ° C for about one hour and then for about one hour at 20 to 30 ° C. The mixture was cooled to about 0 ° C, filtered and the filtrate was evaporated. The residue is crystallized to give 3-tritylthiopropionic acid pentachlorophenyl ester. A solution of the latter compound and an equimolar amount of the tetrapeptide of formula H-Lys (Boc) -Asn-Phe-Phe-OMe, in the form of the acetate described in (a) above, in an inert organic solvent, preferably diethylformamide or tetrahydrofuran, is reacted. with an almost equimolar amount of organic base, preferably triethylamine, at a temperature of 20 to 30 degrees Celsius. The mixture was stirred at 20-30 ° C for two to three days and the solvent was evaporated under reduced pressure. The residue is triturated with cold aqueous citric acid and water, dried and crystallized to give the tetrapeptide of the formula Trt-SCH 2 CH 2 CO-Lys (Boc) -Ann-Phe-Phe-OMe. The latter compound is dissolved in an inert organic solvent, for example methanol, ethanol or preferably dimethylformamide. The solution is allowed to react with an excess of hydrazine hydrate, for example with 15 to 30 molar equivalents. The reaction mixture is kept at about 20-30 ° C for about 20 to 30 hours and then evaporated under reduced pressure. The residue is triturated with cold water, dried to give the tetrapeptide hydrazide of formula IV wherein R4 is H.

In the next step of the process of the invention, the hydrazide of the peptide of formula IV, wherein R 4 is H or NHR ³ , and the peptide of formula V

H-Trp-Lys (BON) Thr (Bu ⁺⁾ -Phe- Thr (tBu ⁺⁾ -Ser (BGR) - NHCH 2 CH (R ²⁾ -S- -Trt wherein

R2 is H · outlined above in paragraph a), · reacting the azide method described above to give the corresponding linear peptide of formula II wherein R2 is H and R4 is H or NHR ^third

Conversion of the latter linear peptide of formula II to the corresponding compound of formula I is suitably and efficiently carried out by first reacting the latter linear peptide of formula II with iodine, preferably in the presence of methanol [as previously described for example for the cyclic derivative derivative]. disulfide of formula III in a)], which deprotects the thiool group, i.e. Trt, and forms a disulfide bridge to form the corresponding cyclic disulfide derivative of formula III wherein R ² is H and R ⁴ is H or NHR ³ , wherein R ³ has the meaning as before. The latter compound of formula III, wherein R ⁴ is NHR ⁵ , may alternatively be written in the form of formula IIIb,

R 3 _-cys -Lys (Bac) -Ann-Phe-Phe-Trp-Thr (Bu ^r ) -Phe-Thr (Bu +) -Ser (Bu ⁺ ) -NH-CH (R2) CH2'S (IIIb) wherein

R2 is H and R3 is as defined above.

The following reaction of the latter compound of formula (III) wherein R ² is H and R 4 is H or NhR ³ is carried out under mildly acidic conditions, preferably with concentrated hydrochloric acid, cooled to about 0 ° C (as described above for cyclic preparation). of the peptide of formula I in a)), the remaining protecting groups are cleaved (i.e. Boc and Bu +) to give the corresponding peptide of formula I wherein R ¹ is H or NHR ³ , where R ³ is as defined above and R ² is H in free base or acid salt form.

c) Compounds of formula I wherein R ¹ is Buc-Ala-Gly-NH and R 2 is COOalk

The present first hydrazide of a peptide of formula IV, wherein R ⁴ is Boc-Ala-Gly-NH, or alternatively written as a heptapeptide of formula IVc.

Boc-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-NHNH 2 (IIc) is fully described in H. U. Immer et al., Supra.

A desired second peptide of formula V wherein R 2 is COOAlk, alternatively written as an ester of a heptapeptide of formula Vb,

H - Trp - Lys (Boc) - Thr (Bu +) - Phe - Thr (Bu +) - Ser (Bu ⁺ ) - Cys (Trt] - COOalk (Vb) is readily prepared as follows:

Heptapeptide Ddz — Trp — Lys (Boc) - —Thr (Bu +) —Phe — Thr (Bu +) —Ser (Bu +) - - C ^ s ^ ((^ 1'1)) - OH [described above in a )] in solution in an inert solvent is reacted with diazoalkane. of formula (alk minus H) in solution, inert solvent, at 0 to 20 ° C for 1 to 48 hours, the solvent and excess diazoalkane are evaporated, the Ddz protecting group is removed and purified to give the corresponding heptapeptide ester of formula Vb. Diazoalkanes useful for this purpose are, for example, diazomethane, diazoethane [von Pechmann, Ber. Deutsch. Chem ·. Ges. 31, 2460 (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-diazobutan (Niedlin ger et al., Supra, supra 380), 1-diazoisobutane (Adamson et al., J. Chem. Soc. 1935, 286), α-diazo. methylbutane [Werner, J. Chem. Soc., 115, 1101 (1919)], 1-diazopentane, 1-diazohexane, 1-diazoheptane, or 1-diazooctane, the last four diazoalkanes being prepared as described by Adamson et al., supra Preferred inert solvents are alkanols such as methanol and ethers such as diethyl ether.

Alternatively, and in particular, to obtain a peptide of formula I wherein R 1 is H-Ala-Giy-NH and R is COOalk, where the alkyl group (alk) contains 9 to 14 carbon atoms, the cysteine is reacted with a molar excess of the respective an alkanol alkOH, wherein alk is as defined above in hydrogen chloride at 25 to 120 ° C, preferably at 100 to 120 ° C, for 1 to 5 hours to give the corresponding cysteine alkyl ester hydrochloride. The latter compound is then reacted with about one molar equivalent of triphenylcarbinol in the presence of 0.1 to 1.0 molar equivalent of boron trifluoride etherate at 10 to 30 ° C for 0.5 to 3 hours, preferably in the presence of 0.1 molar equivalent at 25 ° C for one hour to give the corresponding H-Cys (Trt) -O Alk alkyl ester.

The latter alkyl ester of the protected cysteine is then reacted by the azide method with the protected hydrazide of the hexapeptide of the formula

Ddz-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu +) -Ser (Bu ⁺ ) -NHNI-I2.

as described in a) above, and after cleavage of the Ddz protecting group under mild acidic conditions, the desired second heptapeptide ester of formula Vb is obtained. Preferred conditions for the reaction sequence described above include the use of equimolar portions of said protected hexapeptide hydrazide and tert-butyl nitrite in the presence of a mineral acid, preferably 2-3 molar equivalents of hydrogen chloride in an inert anhydrous solvent, preferably DMF, followed by addition of the appropriate alkyl ester of formula H-Cys. (Trt) Oalk with 2 to 3 molar. equivalents of an organic base, preferably N-ethyldiisopropylamine, in an inert anhydrous solvent, preferably DMF, is carried out at -20 to 25 ° C for 6 to 12 hours, and purification of the resulting protected alkyl heptapeptide to give a compound of formula

Ddz — Trp — Lys (Boc) —Thr (Bu +) —Phe— —Thr (Bu +) - Ser (Bu +) —Cys (Trt] - O Alk.

The latter compound is then reacted under mildly acidic conditions, preferably by stirring in a mixture of acetic acid, formic acid and water (7: 1: 2) for ^{1 to} 12 to 18 hours at 20 to 25 degrees Celsius to give the corresponding alkyl ester. of a heptapeptide of formula Vb.

In the following step of the process according to the invention, the first peptide hydrazide of formula IVc and a second ester of the heptapeptide of the formula Vb is reacted with an azide by the method described above to provide the corresponding linear peptide of formula II wherein R ⁴ is Boc-Ala-Gly-NH ² and R · is COOalk or alternatively written as a linear tetradecapeptide in the face

Boc-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu ⁺ ) -Ser (Bu ⁺ ) - —Cys (Trt) -OAlk (Hey

Conversion of the preceding linear tetradecapeptide of formula IIc to the corresponding compound of formula I, wherein R ¹ is H-Ala-Gly-NH and R 2 is COOalk, is preferably effected by first reacting the latter linear tetradecapeptide with iodine, preferably in the presence of meth. thanol [as previously described for the preparation of the cyclic disulfide derivative of formula III in (a)], cleaving the thiol protecting groups, i.e. Trt, to form a disulfide bridge to give the corresponding cyclic disulfide derivative of formula III wherein R ² is COOalk and R ⁴ is Boc-Ala-Gly-NH or alternatively written as a cyclic disulfide derivative of formula IIIc,

Boc-Ala-Gly-Cys-Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu + Phe-Thr [Bu ⁺ ) -Ser (Bu +) - Cys-Oalk.

Subsequent reaction of the latter compound under mild acidic conditions, preferably concentrated hydrochloric acid cooled to about 0 ° C [as previously described for the preparation of the cyclic peptide of formula I in (a) ·], removes the remaining protecting groups (i.e., Boc and Bu +) and further purification as described in paragraph aj yields a peptide of formula I wherein R 1 is H-Ala-Gly-NH and R 2 is COOAlk or alternatively written as a compound of formula

HI-Ala-Gly-Cys-Lys-Asn-Phe-Plie-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OAlk in free base or acid salt form.

In the above procedure described under

c) the protecting group Bu ⁺ for the hydroxyl group of serine and threonine can leave groups i Ser (Bu ⁺ i Thr (Bu ⁺ ) can be replaced by Ser and Thr. Also with regard to procedure c) the Trt protecting group of cysteine residues amino acids may replace the Aom protecting group.

The procedures described above under a) to cj may be illustrated by the following schemes:

(a) R1H — Gly — Gly — Ala — Gly — NH;

H-Gly-Gly-Gly-Ala-Gly-NH and H-Leu-Gly-Gly-Ala-Gly-NH and R2 = H or COOH

PAe 777-Se.rNHCH (1C- ^H 2 ³ - ^Trt

Ctfs-tbfS-Asiz-Phe-Pbe Trp-Ls-Tfa-Phe-Tkr-Ser ' ^V I V! NHcm ^ x ^ ns- ^ гн 't ¹ >

⁴ Vin "

b) R'-H or NHR * R ^Z H

Trt-SCH ₂ CH 3 / Λ CO + 1 -Asa-Phe-Phe Trp-L 1 -Thr-Phe-Tkr-Ser-NHCH ² R ² } \ IV | In CKS-Trt —J --------- from 11-W-ti

c) pLH-Alz-Gltf -NH and R ² = COOaik

Boc-Al & rGltf-Ctis ~ Ltfs ~ Asn ~ Pbe ~ Phke - Trp-Lp-Thf-PtiéThr-Ser-Cys-CA tk | JVc | Vb _r-VIIc IIc-V

Finally, it is known to those skilled in the art that, without departing from the scope of the invention, equivalent amino, hydroxy or thiol protecting groups may be used, equivalent reactions for coupling peptide fragments may be used, and equivalent methods for cleaving amino, hydroxy protecting groups may be used. or thiol groups. These alternatives are within the scope of the invention.

The following diagram and examples illustrate the invention further.

Liner peptide (II)

I Oxidation l

Cyclic disulfide derivative (III)

AND

Removal of protecting groups l

Peptide (I) Example 1

Alanyl glycine methyl ester (H-Ala-Gly -OMe.CF ₃ COOH)

Boc-Ala-Gly-OMe [10 g 45 mmol, described by H. U. Immer et al. Helv. Chilm. Acta. 57, 730 (1974)] was dissolved in cold (ice bath) trifludacetic acid (100 ml). The solution was stirred at 0 ° C for 1 hour and the solvent was evaporated. The residue was dissolved in methanol, diethyl ether was added, and the precipitate was filtered off to give the title compound.

Example 2 t-Butyloxycarbonyl-glycyl-alanyl-glycine methyl ester (Boc-Gly-Ala-Gly-OMe)

N-Ethylmorpholine (pH ~ 8) was added to a cold (ice bath) solution of H-Ala-Gly-Omega (45 mmol, described in Example 1) in dimethylformamide (50 mol), then a cold solution was added. Boc-Gly-OTcp (18 g, 45 mimol) in dimethylformamide (50 mL). The solution was kept in an ice bath for two days. The solvent was evaporated and the product was purified by silica gel column chromatography using a mixture of ethyl acetate-methanol and pyridine (98: 1: 1): The product crystallized from a mixture of ethyl acetate and petroleum ether to give the title compound, mp 98-100 ° C; NMR (DMSO-d 6), 1.25 δ (3H), 1.4 <5 (9H), 3.68 δ (ЗН).

Example 3

Glycyl-alanyl-glycine methyl ester trifluoroacetate (H-Gly-Ala-Gly-OMe.CF3COOH)

Boc-Gly-Ala-Gly-OMe (6.4 g, 20.1 mmol, described in Example 2) was dissolved in cold (ice bath) trifluoroacetic acid (120 mL) and the solution was stirred at 0 ° C for one hour. . The solvent was evaporated, the residue was dissolved in methanol and the product was precipitated by addition of diethyl ether. Filtration of the precipitate affords the title compound.

Example 4 t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycine methyl ester (Boc-Gly-Gly-Ala.-Gly-OMe)

To a cold (ice bath) stirred, H-Gly-Ala-Gly-OME solution. CH 5 CO 2 H (6.4 g, 20.03 mmol, described in Example 3) in dimethylformamide (30 mL) was added N-ethylmorpholine (2.8 mL), and then a solution of Boc-Gly-OTcp (8.5 g) was added. , 24 mmol) in dimethylformamide (20 mL). The solution was kept in an ice bath for two days. The solvent was evaporated and the residue was dissolved in methanol and the product was precipitated with diethyl ether. Crystallization from ethyl acetate gave the title compound of m.p. 103-105 9 degrees Celsius and 141 ° C (dimorphic); NMR 1.25 δ (3H), 1.38 δ (9H), 3.65 δ (3H).

Example 5 t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycine hydrazide; IX, ^R5 = Boc (Boc-Gly-Gly-Ala-Gly-NH-NH2)

To a cold (ice bath) stirred solution of Boc-Gly-Gly-Ala-Gly-OMe (2.5 g, 6.7 mmol, described in Example 4) in methanol (50 mL) was added hydrazine hydrate (2.5 mL). ml). The solution was stirred at 0 ° C for three hours and then at room temperature overnight. The precipitate was filtered off, washed with methanol and dried to give the title compound. Amino Acid Analysis: Gly 3, Ala 0.99.

Example 6 it-butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N ^e -t-butyloxycarbonyl-lysyl-aspartyl-L-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-NH 2 (1.57 g, 4.2 mmol, described in Example 5) in dimethylsulfoxide (25 mL) and dimethylformamide (25 mL) was added 2.04 N solution of hydrogen chloride in ethyl acetate (5.15 mL) at -10 ° C. The solution was cooled to -12 ° C and tert-butyl nitrite (0.61 mil, 5.2 mimol) was added. The solution was kept at -10 ° C for 15 minutes, cooled to -15 ° C and N-ethyldiisopropylamine (1.8 ml, pH 8) was added first, followed by H-Cys (Trt) -Lys (Boc) solution. Asn-Phe-Phe-OMe [4.5 g, 4.19 mimol, described in HU Imimer et al., Helv. Chim. Acta, 57, 730 (1974)] and N-ethyldiisopropylamine (0.72 mL) in dimethylformamide (25 µl). The mixture was stirred for one hour at -10 ° C and overnight at room temperature. After evaporation, the residue was dissolved in methanol, the product was precipitated with water and crystallized from methanol to give the title compound. Amino-alkylene analysis: Lys, 1.07; Asp, 0.97; Gly, 3.27; Ala, 1.0, Phe, 2.08, Cysteic acid 1.35.

Example 7 t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-NM-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanine hydrazide, IV, R ⁴ = Boc-Gly-Gly-Ala-Gly-NH [Example] Boc — Gly — Gly — Ala— —Gly — Cys (trt) —Lys (Boc) —Asn — Phe— —Phe — NH — NH2]

To a cold (ice bath) solution of Boc-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-OMe (0.9 g, 0.66 mmioil, described in the example) 6] in dimethylformamide (25 ml) was added hydrazine hydrate (1.5 ml), stirred at room temperature for 18 hours, the solvent was evaporated and the residue was triturated with methanol, dried over phosphorus pentoxide to give the title compound. Amino Acid Analysis: Lys, 1.10, Asp, 1.00, Gly, 3.33, Ala, 1.0, Phe, 2.14, Cysteic Acid 1.35.

Example 8 t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N ^E- t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyll-N ^E- t-butyloxycarbonyl-lysyl threonyl-phenylalanyl-O-butyl-threonyl-O-butyl-seryl-S-trityl-cysteine, II, R ² = COOH and R ⁴ = Boc-Gly-Gly-Ala-Gly-NH [Boc-Gly-Gly - —Ala — Gly — Cys (Trt) —Lys (Boc) —Asn— —Phe — Phe — Trp — Lys (Boc) —Thr (Bu ⁺ ) - —Phe — Thr (Bu +) - Ser (Bu ⁺ ) - Cys (Trt) - —OH]

To a solution of Boc-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-NHNHz (800 mg, 0.59 mmol, described in Example 7) in dimethylformamide (12 mL) A solution of 1.85 N hydrogen chloride in ethyl acetate (0.795 mL, 1.475 mmol) was added, stirred at -20 ° C. The mixture was cooled to -15 ° C, tert-butyl nitrite (0.081 mL, 0.71 mmol) was added and the solution was stirred for 15 minutes. Solution H — Trp — Lys (Boc) —Tr (Bu ⁺ ) - —Phe — Thr (Bu ⁺ ) —Ser (Bu +) —Cys (Trt) - —OH [816 mg, 0.59 mmol, described by HU Immer et al. Helv. Chim. Acta, 57, 730 (1974)] and Ν-ethyldiisopropylamine (0.354 mL, 2.06 mmol) in dimethylformamide (6 mL) was cooled to -15 ° C and added to the above reaction mixture. The mixture was stirred at -15 ° C for one hour and at 25 ° C for 18 hours. The solvent was evaporated and the residue was triturated with ice-cooled citric acid, filtered, washed with water, then methanol to give the title compound. 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 9

Glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-phenylalanyl-tryptophyll-lysyl-threonyl-phenylala207570 nyl-threonylseryl-cysteine, I, R ¹ -H-Gly-Gly-Gly-Ala-Gly-Ala R ² == COOH (H - Gly - Gly - Ala - Gly - Cys - Lys - Asn - Phe - Phe - Trp - Lys - Thr - Phe - Thr - Ser - Cys - OH)

Boc-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-Trp-Lys (boc) -Thr (Bu +) -Phe-Thr (Bu +) -Ser The (Bu ⁺ ) - Cy - (Trt) -OH described in Example 8 (0.871 g, 0.32 mmol) was dissolved in glacial acetic acid (150 mL) and added dropwise to the iodine solution at room temperature over 1 hour. in methanol (0.5%, 150 mL, 30 mmol). The mixture was stirred for an additional minute, cooled in an ice bath, and a solution of sodium thiosulfate in water (1N, 6 mL) was added to remove excess iodine, (a deprived solution). The solvent is evaporated and the residue is triturated with water, dried, the dry product is triturated with isopropyl ether to give cyclic disulfide hexadecapeptide of formula III.

Boo-Gly-Gly-Ala-Gly-Cys-Lys (Bon) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu ⁺ ) -Ser (Bu ⁺ ) - Cys-OH (III).

Cold concentrated hydrochloric acid solution (23 ml) was added to the latter compound under nitrogen atmosphere with vigorous stirring in an ice bath. Stirring was continued for about 10 minutes, glacial acetic acid (300 mL) was added to the solution, and the solution was lyophilized. The residue is dissolved in water and lyophilized again. The residue was then dissolved in a 0.01 N aqueous ammonium acetate solution and loaded onto a carboxymethylcellulose column (Whatman CM-23 2.5 x 30 cm). The pure compound was eluted with 0.06 N ammonium acetate buffer. The purified material was lyophilized from water to give the title compound as a white solid as an acetic acid salt; A 282 nm (ε 5260), 289 max nm (ε 4730), amino acid analysis: Lys, 2.28, Asp, 1.05, Thr, 1.95, Ser, 0.93, Cys, 2.34, Gly 3.00, Ala, 1.05, Phe, 3.12. Repeated lyophilization of the latter product from water gives the title compound as the free base. Amino Acid Analysis: Lys, 2.10, Asp, 1.04, Thr, 1.80, Ser, 0.95, Cys, 2.17, Gly, 3.00, Ala, 1.07, Phe, 2.99 .

In an analogous manner but using rhodane, following the procedure of Hiskey and Smith, supra, instead of iodine, the title compound is also obtained.

Example 10 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 (2.0 g, 5.35 mmol, described in Example 4) in trifluoroacetic acid (25 mL) was stirred at 0 ° C for one hour. The solvent was evaporated, the residue was triturated in ether, the solid was filtered off, dried to give the tetrapeptide H-Gly-Gly-Ala-Gly-OMe, isolated as the trifluoroacetic acid salt.

A solution of the latter tetrapeptide (5.35 mmol), Boc-Leu-OH (2.31 g, 10 mmol), 1-hy (1.35 g, 10 mmol), dicyclohexylcarbodiimide (2.27 g, 11 mmol) and N-ethylmorpholine [0.68 mL] in dimethylformamide (25 mL) was stirred at 0 ° C for 24 h. The precipitate was filtered off and the filtrate was added to 200 ml of diethyl ether. The precipitate was filtered off and crystallized from methanol and isopropyl ether to give the title compound b.

190.5-193 ° C, [.alpha.] D @ ²⁵ = -15.2 DEG (c = 1, dimethylformamide).

Example 11 t-Butyloxycarbonyl-leucyl-glycyl-glycyl · -alanyl-glycine hydrazide, IX, R ⁵ = Boc-Leu (Boc-Leu-Gly-Gly-Ala-Gly-NHNHz)

A solution of Boc-Leu-Gly-Gly-Ala-Gly-OMe (2.0: g, 4 mmol, described in Example 10) and hydrazine hydrate (4.12 mL, 80 mmol) in methanol (50 mL) was stirred. 4 hours at 0 ° C. The solution was concentrated to 4 mL and added to diethyl ether (200 mL). The precipitate was filtered off, dissolved in methanol (4 mL) and added to diethyl ether (200 mL). The precipitate was filtered off, dried to give the title compound, m.p. 174-176.5 ° C, [α] D 24 = 12.0 ° (c = 1, dimethylformamide).

EXAMPLE 12 t-butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N ^£ -t-butyloxykarboinylllyyyl-aspartyl-L-phenylalanyl-phenylalanine methyl ester [Boc-Leu-Gly-Gly-Ala-Gly-Cys ( Trt) -Lys (Boc) -Asn-Phe-Phe-OMe] tB-dtyl nitrite (0.15 mL, 1.27 µmol) was added to a solution of Boic-Leu-Gly-Gly-Ala-Gly-N ₂ H3 (0.308 g, 0.635 mmol) described in Example 11) in dimethylformamide (5 mL) at -20 ° C and 2.4 N hydrochloric acid in ethyl acetate (0.66 mL, 1.59 mmol). After stirring for 15 minutes at -20 ° C, a solution of H-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-OMe [0.62 g, 0.58 excl., Described by HU Immer et al., Helv. . Chim. Acta. 57, 730 (1974)] and diisopropylethylamine (0.372 mL) in dimethylformamide (6 mL). After stirring for 1 hour at -20 degrees Celsius and for 24 hours at 0 ° C, the solution was added to diethyl ether (200 mL). The precipitate was filtered off, dissolved in methanol (5 ml) and added to diethyl ether (200 ml). The precipitate was filtered off, dried to give the title compound, mp 238-240.5 ° C.

EXAMPLE 13 ^{t-butyloxycarbonyl-leucyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N-e} -t butyloxykarbonyllysyl-aspartyl-L-phenylalanyl-fenylalaninhydrazid, IV, ^R4 = Boc-Leu- Gly-Gly-Ala —Gly — Nh [Boc — Leu— —Gly — Gly — Ala — Gly — Cys (Trt) - —Lys- (Boc) - Asn — Phe — Phe — NHNHz]

A solution of Boc-Leu-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Ann-Phe-Phe-OMe (0.56 g, 0.38 mmol, described in Example 12) and hydrazine hydrate (0.74 mL) in dimethylformamide (10 mL) was stirred at 0 ° C for 12 h and at 25 ° C for 24 h The solution was added to diethyl ether (100 mL), the precipitate was filtered off, dissolved in dimethylformamide (3 mL) and It was added to diethyl ether (100 mL) and the precipitate was filtered off, dried to give the title compound, mp 239-242 [deg.] C. Amino acid analysis: Lys, 1.05, Cysteine acid 0.66, Asp, 0, 99, Gly, 3.00, Ala, 1.11, 1/2 Cys, 0.21, Leu, 0.96, Phe, 1.86.

EXAMPLE 14 t-ButyloxxkaabonyHeucyl-glycyl-glycyl-alanyl-glycyl-S - ^{rityl-cysteinyl-N-E} -t-butyloxykaгbonkl lysyllasparaginyl-fenklalankl-fenklalankl ^{trkptofkl-N-E} -t-butkloxykarbonyl lkSkl-Ot-butyl-threonkl -fenklalankl-Ot-butkl-threonkl-Ot-butkl serkl-S-trityl-cysteine, II, R2 = COOH, ^R4 = Boc-Leu-Gly-Alo-Glk Glk --NH [Boc-Leu- ( Trt) - —Lys (Boc) - Asn — Phe — Phe — Trp— —Lys (Boc) —Thr (Bíu ⁺ ) —Phe — Thr (Bu ⁺ ) - —Ser (Bu +) —Cys (Trt) —OH t-Butylnitrite (0.03 mL, 0.28 mmol) was added to a solution of Boc-Leu-Gly.-Gly-Ala-Gly-Cys [Trt] -Lys (Boc) -Asn at -20 ° C. —Phe — Phe — Ν2-3 (0.20 g, 0.14 mmol, described in Example 13) in dimethylsulfoxide (2 mL), dimethylformamide (4 mL) and 2.21 N hydrogen chloride in ethyl acetate (0.16 mL, 0 , 35 mmol). After stirring at -20 ° C for 15 minutes, H-Trp solution was added.

—Lys (Boc) —Thr ^: (Bu ⁺ ) —Phe — Thr (Bu ⁺ ) - —Ser (Bu ⁺ ) —Cys (TrtJ — OH.HCO2H [0.20 g, 0.14 mmol, described by HU Immer et al. , Helv Chim Acta, 57, 730 (1974)] and diisopropylethylanrinium (0.08 mL, 0.49 mmol) in dimethylformamide (5 mL) was stirred at -20 ° C for 1 hour and at 24 ° C for 24 hours. The solution was concentrated to 2 mL and diethyl ether (100 mL) was added and the precipitate was filtered, washed with water (2 x 5 mL), methanol (2 x 5 mL) and dried to give the title compound. amino acids: Lys, 1.92, Cysteic acid, 0.93, Asp, 0.93, Thr, 1.38, Ser, 0.66, · Gly, 3.00, Ala, 1.02, 1/2 Cys 0.39, Leu, 0.93, Phe, 2.70.

Example 15

Cyclic disulfide. leucyl-glycyl-glycyl-alanyl-glycyl-cystemkl-lkeyl-aeparaginyl-phenylalankl-fenklalankl-ttptptlk-1kskl-threonkl-alanyl-threonkl-setk-ckstein, I, R ¹ = H-Leu-Gly Ala-Glk-NH and R2 = COOH [H-Leu-Glk-Glk-Ala-Glk-Cys-Lys-Asn-Phe-Phe-Trp-

—Lys — Thr — Phe — Thr — Ser — Cys — OH)

Boc-Leu-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Bdc) -Asn-Phe-Phe -Trp-Lys (Bole) -Thr (Bu ⁺ ) -Phe-Thr [Bu ⁺ ) - Ser (Bu-4-Cys (Trt) -OH (0.23 g, 0.081 mmol, described in Example 14) in acetic acid (90 mL) was added dropwise over 1 hour to a solution of 0.5% iodine in methanol (41.5 mL, 0.81 mmol) After the addition was complete, the solution was stirred at room temperature for one hour and then cooled to 0 DEG C. Sodium thiosulfate (1.62 mL) was then added until the solution remained colorless. The solvent is evaporated, the residue is triturated with ether and dried to give the cyclic heptadecapeptide III disulfide.

Boic-Leu-Glk-Gly-Ala-Gly-Cys-Lys · (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu ⁺ ] - - Ser (Bu ⁺ ) —Cys — OH (ΙΠ)

A solution of cyclic heptadecapeptide cyclic disulfide (0.155 g, 0.066 mmol) in concentrated hydrochloric acid (6.87 mL) was stirred vigorously at 0 ° C for 8 min. Acetic acid (69 mL) was added and the solution was lyophilized. The residue was lyophilized from water (50 mL). The residue is chromatographed on a column of chemically modified cross-linked dextran "Sephadex G-25 M" [3 x 50 cm, equilibrated in the lower phase and then in the upper phase of n-butanol-acetic acid-water (4: 1: 5). ], wherein the upper phase is used for desorption of the peptide. Fractions containing pure petpid were combined, evaporated and lyophilized from water to give the title compound as the acetic acid salt. λ 290 (ε 5000), 281 (ε 5540), 275 max (ε '5205), 269 (ε 4980), 265 (ε 4625), 259 nm (ε 4085). Repeated lyophilization of the last product from water gave the title compound as the free base. Amino Acid Analysis: Lys, 1.98, Cysteic Acid 1.41, Asp, 1.26, Thr, 1.92, Ser, 0.96, Gly, 3.00, Ala, 1.02, Leu, 0.96 , Phe 2.76.

In the same manner, but using rhodane, according to the method of Hiskey and Smlth, supra, instead of iodine, the title compound is also obtained.

Example 16 t-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycinmiethyl ester (Boc-Gly-Gly-Gly-Ala-Gly-OMe)

A solution of Boc-Gly-OTcp (3.7 g ;, 10.04 mmol), H-Gly-Gly-Ala-Gly-OMe. . CF 5 CO 2 H (3.12 g, 8.03 mmol, described in Example 10) and N-ethylmorpholine (1.1 mL) in dilmethylformamide (15 mL) were stirred at 0 ° C for 20 h. The precipitate was filtered off and diethyl ether was added. The combined precipitated fractions were crystallized from methanol a to give the title compound, mp 198-201 ° C, [α] ²⁴ = -3.9 ° (c = 1, dimethylformamide).

Example 17 t-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycine hydrazide, IX, R ⁵ = Boc-Gly (Boc-G-ly-Gly-Gly-Ala-Gly-NHNHz)

A solution of Boc-Gly-Gly-Gly-Ala-Gly-OMe (1.0 g, 2.32 mmol, described in Example 16) and hydrazine hydrate (1 mL, 23.2 mmol) in methanol (30 mL) was added. Stir 4 hours at 0 ° C. After evaporation, the residue is triturated with diethyl ether and dried to give the title compound, mp 221-233 ° C.

Example 18

N, S-Diirityl-cysteinyl-N 6 -t-butyloxycarbonyl-lysyl-asparaginyl-phenyl-alanyl-phenylalanine hydrazide, VI, [Trt-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-NHNH 2]

Trt-Cys (Til) -Lys (Boc) -Asn-Phfe-Phe-OMe solution [1.25 g, 1.0 mm | ol, described in H. U., Imimer et al., Helv. Chim. Acta, 57, 730 (1974)] and hydrazine hydrate (0.97 mL, 20 mmol) in methanol (30 mL) was stirred at 0 ° C for two days. The solvent was evaporated and the residue crystallized from ethanolisopropyl ether α to give title compound, NMR (DMSO-d 6): δ 1.38 (s, 9H), 7.19-7.30 (m, 40H).

Example 19

N, S-Di _: trityl-cysteinyl-N-t-butyloxycarbanyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyll-NM-biutyloxycarbonyl-lysyl-O-butyl-threo ^| nyl-phenyl-alanyl-O-t-butyl-threonyl-O-4-butyl-seryl-S-trityl-cysteine, VII, R2 = COOH [Trt-Cys (Trt) -Lys (Bo.c) - —Asn —Phe — Phe. — Trp — Lys (Boc) - —Thr (Bu ⁺ ) —P'hie — The (Bu ⁺ ) —Ser (Bu ⁺ ) - —CysfTrtJ — OH] t-Butylnitrite (0.09 ml, 0.74 mmol) was added at -20 ° C to a solution of Trt-Cys (Trt) -Lys (Boc) -Ann-Phe-Phe-NHNH 2 (0.62 g, 0.493 mmol, described in Example 18). in dimethylpharmamide (10 ml) and 2.6 N hydrogen chloride in ethyl acetate (0.475 ml, 1.23 mmol). After stirring at -20 ^° C for 15 minutes, a solution of H-Trp-Lys (Boc) -Thr '(Bu ⁺ ) -Phe-Thr (Bu +] - Ser (Bu +) -Cys (Trr) was added. - OH, HCOOH [0.70 g, 0.493m mol, described by HU Immer et al., Helv Chim Acta, 57, 730 (1974)] and diisopropylethylamine (0.30 mL, 1.65 mmol) in dimethylf The solution was stirred at -20 ° C for 1 hour and at 25 ° C for 24 hours and evaporated, and the residue was triturated with water, diethesle, cold 1N citric acid to give the title compound - NMR (CDCl3) δ 1.08 and 1.13 (s, 27H), 1.37 (s, 18H), 7.28 (m, 60H), amino acid analysis: Lys, 2.23, cssteic acid, 1.10, Asp, 1.00, Thr, 2.12, Ser, 1.02, 1/2 Cys, 0.64, Phe, 3.18.

Example 20

S-trityl-cssteinyl N ^ε -t-butsloxykarbonyl-lysyl-a'sparagmsl-fenslalansl fenylalansl-tryptophyl-N6-Bu --- sloxykarbonyl-lysyl-0-t - hreonyl-phenylalanyl-Ot-Bu-yl- -hreonyl --- O-butyl-SL-S-seгyl tritsl-CSS einformiá-, VIII, R ² = COOH [H-Gys (Trt) -Lys (Boc) - Asn-Phe-Phe-Trp- Lys (Boc) - —Thr, (Bu +) - Phe — Thr (Bu +) - Ser (Bu +) - —CyssTrt) - OH. HCOOH

Solution Trt — Cys (Trt) —Lys (Boc) —Asn— —Phe — Phe — Trp — Lys (Boc) —Thr (Bu ⁺ ) - —Phe — Thr (Bu +) - Ser (Bu +) - Cys (Trt) - OH (0.50 g, 0.192 mmol, described in Example 19) in 6 mL of a 7: 1: 2 mixture of acetic acid, formic acid and water was stirred at 25 ° C for 6 h. Evaporate, triturate the residue with diethyl ether to give the title compound: amino acid analysis: Lys, 2.23, cysteic acid,

3β

1.38, Asp, 1.00,. Thr, 2.14, Ser, 0.86, Phe, 3.24.

Example 21 t-Butyloxycarbonyl, yl-glycyl-glycyl-glycyl-alanyl-glycyl-S-tritylOysteinyl-N-t-butyloxycarbonyl-lysyllasparaginyl-phenylalanyl-phenylalanyl-tryptophyll-N-t-butyloxycarbonyl-1-t-butyl threonyl-phenylalallyl-t-butyl-threonyl-β-butyl-seryl-S-trityl-cysteine, II, R ² = COOH- and R ⁵ = Boc-Gly-Gly-Gly-Ala-Gly-NH [Boc- Gly-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu +) -Ser (Bu +) - Cys (Trr) - OH] t-Butylnitrite (0.04 mL, 0.34 mmol) was added to -20 ° C to a solution of Boc-Gly-Gly-Ala-Gly-NHNHz (0.076 g, 0.176 mimol described in Example 17] in dimethylsulfoxide (1 mL), dimethylformamide (2 mL), and 2.5 N hydrogen chloride in ethyl acetate (0.176 mil, 0.44 - mimol) After stirring for 15 minutes at -20 ° A solution of H-Cys- (Trt) -Lys (BOC) -Asn -Phe-Phe-Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu +) -Cys ( Trt) - OH, HCOOH (0.385 g 0.16 mmol (described in Example 20) and diisopropylethylamine (0.12 mL) in dimethylformamide (5 mL) was stirred at -20 ° C for 1 h. ° C. one hour at 0 ° C and 20 hours - at - 25 ° C. . The solvent was evaporated and the residue was evaporated. added to diethyl ether - (100 mL). The precipitate was filtered off, washed with water, methanol and dried to give the title compound. Amino Acid Analysis: Lys, 2.30, cysteine acid, 1.42, Asp, 1.00, -Thr, 2.27, Ser, 1.00, Gly, 3.84, Ala, 1.00, Phe , 3.34.

Example 22

Cyclic glycyl-glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-aspanaginyl-: phenylalanyl-phenylalan-4-yl-tert-butyl-lysyl-threo-4-allyl-phenylalanyl-thiophenyl-senyl-cysteine disulfide, I , R1. = - - H - Gly - Gly - Gly - Ala - Gly - NH and R 2 = COOH (H - Gly - Gly - Gly - Ala - Gly - Cys - Lys - Asn - Phe - Phe - Trp - Lys - Thr —Phe — Thr — Ser — Cys — OH)

Boc — Gly — Gly — Gly — Ala — Gly— —Cys (Trt) —Lys (Boc) —Asn — Phe — Phe — —Trp — Lys (Boc) —Asn — Phe — Phe — Trp — —Lys (BOC) Solution - Thr (Bu +) -Phe-Thr (Bu +) -Ser (Bu +) - Cys (Trt) -OH (0.285 g, 0.103 mmol, described in Example 21) in acetic acid (200 mL) was added over 60 min. minutes to a solution of 0.5% iodine in methanol (50 µl, 1.03 mole) and the mixture is stirred at room temperature for 60 minutes, cooled to 0 ° C and 1N sodium thiosulphate solution (2 ml) is added. The solvent was evaporated and the oily residue was added to water (100 ml), the precipitate was filtered off and dried to give the cyclic heptadecapeptide III disulfide.

Bo.c — Gly — Gly — Gly — Ala — Gly — Cys— —Lys- (Boc) - Asn — Phe — Phe — Trp —____ —Lys (Boc) —Thr (Bu ⁺ ) —Phe — Thr (Bu ⁺ ) - —Ser (Bu +) - Cys — OH (III)

A solution of the latter cyclic disulfide heptadecapeptide (0.10 mmol) in concentrated hydrochloric acid (10 mL) was stirred at 0 ° C for 10 min under nitrogen atmosphere. Acetic acid (100 ml) was added and the solution was lyophilized. After further freeze-drying from water (100 ml), the solution is subjected to partition chromatography on a chemically modified cross-linked dextran column ("Sephadex G-25 M", 3 x 50 cm) prepared in the lower phase of n-butanol-acetic acid-water ( 4: 1: 5) and then equilibrated in the top phase] and - using the top phase of the mixture, the almost pure heptadecapeptide is desorbed. The pure fractions were combined, evaporated and lyophilized to give the title compound as an acetic acid salt.

MeOH λ 283 (ε 6702), 289 nm (ε 6350). By opamax lyophilization of the latter product from water, the title compound is obtained as the free base. Amino Acid Analysis: Lys, 2.00, Cysteic Acid, 1.42, Asp, 1.09, Thr, 1.89, Ser, 0.91, Gly, 3.64, Ala, 0.98, Phe, 295.

In the same manner, but using rhodane, following the procedure of Hiskey - and Smith, supra, instead of iodine, the title compound is obtained.

Example 23 •

N, S-Diirityl-cysteinyl-N ^E- t-butyloxycarbonyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyll-N-t-butyloxycarbonyl-lysyl-N-butyl-threonyl-phenylalanyl-N-butyl-threonyl-O- 1 ^ ^; ^ l ^ i ^ t ^: ^^^ i ^ <^ i'yl -, ^ - ^ tt ^^ 1tyl hio ^^ hylamid, VII, R ² = H [Tt-Cys (Trt) - Lys (Boc ) —Asn — Phe — Phe — Trp— —Lys (Boc) —Thr (Bu ⁺ ) —Phe — Thr (Bu +) - —Ser (Bu +) - NHCH2CH2S — Trt]

Pentapeptide hydrazide Trt-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-NHNH 2 (0.80 g, 0.637 mmol, described in Example 18) was dissolved in anhydrous dimethylformamide (9 mL) and cooled to —20 ° C. Add hydrochloric acid in ethyl acetate (2.4 N, 0.691 mL) followed by t-butyl nitrite (0.0872 mL, 0.764 mmol). The mixture was stirred at -15 ° C for 15 minutes. Solution H-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu +) -Ser (Bu +) -NHCH 2 CH 2 -Trt (0.852 g, 0.637 mmol), prepared according to US Patent No. 3,917 581 of 4. XI. 1975, in dimethylformamide (8 mL) containing N-ethyldiisopropylamine (0.272 mL, 1.59 mmol) was cooled to -15 ° C and added dropwise to the above reaction mixture. Stirring was continued for one hour at -15 ° C and room temperature overnight. The reaction mixture is evaporated under reduced pressure, the residue is triturated with water, filtered, washed with water and dried over phosphorus pentoxide. The residue is chromatographed on a silica gel column (163 g) with chloroform containing methanol (3%) and pyridine (0.3%) as eluent and the pure product is crystallized from a mixture of methanol and isopropyl ether. The title compound is obtained, mp 163-180 degrees Celsius (dec.).

Analysis for C144H180N16O19S2 calculated:

C 69.85, H 7.04, N 8.76%; found:

C 69.24, H 7.09, N 8.90%.

Example 24

S-Trityl-cysteinyl-NE-t-butyloxycarbonyl · lysyl-as.paraginyl-phenylalanyl-phenylalanyl · tryptophyll-NM-butyloxycarbonyl-lysyl-N-butyl-threonyl-phenylalanyl-α-butyl-threonyl-O4d > ^ Butyl-Eryl -ritylthioethylamid · formate, VIII, R ² = Η [H-Cys (Trt) - Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) Thr (tBu ⁺⁾ - Phe-Thr (Bu +) - -Ser (Bu +) -NHCH ₂ CH ₂ S-Trt. HCOOH]

Undecapeptide Trt — Cys (Trt) —Lys (Boc) - —Asn — Phe — Phe — Trp — Lys (Bo-c) - —Thr (Bu +) - Phie — Thr (Bu ⁺ ) —Ser (Bu ⁺ ) - - NHCH 2 CH 2 S-Trt (0.909 g, 0.355 mmol, described in Example 23) was dissolved in a mixture of acetic acid, formic acid and water (7: 1: 2) (10 mL) and stirred at room temperature overnight. The solvent was evaporated and the residue was triturated with water. The compound obtained is filtered off, washed with water and dried over phosphorus pentoxide. The solid was triturated several times with petroleum ether / ether and dried to give the title compound. Amino Acid Analysis: Lys, 2.03, Asp, 1.00, Ser, 0.87, Phe, 2.97, cysteic acid, 0.90, Thr, 1.85.

Example 25

У BuУyloxykarbonyl · · leucyl-glycyl-glycyl · alanyl-glycyl-S-Уrityl · cysteinyl-N ^ε -t · butyl oxycarbonyl-lysyl-aspartyl-L-phenylalanyl-L-phenylalanyl-N-tryptofyI ^e -t-butylbxykarbonyl-lysyl-O4- hn} tert.butyl-ones] -keiylalaiiyl.-Ot-butyllithium · O · hreonyI --- butyl-seryl-2 · · trltyl thioethylamid II, R ² = H, R4 == Boc-Leu-Gly-Gly-Ala —Gly — NH [Boc— —Leu — Gly — Gly — Ala — Gly — Cys (Trt) - —Lys (Boc) —Asn — Phe — Phe — Trp—.

—Lys —Thr (Bu ⁺ ) —Phe — Thr (Bu ⁺ ) - —Ser (Bu +) — NHCH2CH2S — Trt]

Boc-Leu-Gly-Gly-Ala-Gly-NHNI-U pentapeptide hydrazide (0.066 g, 0.136 mmol, described in Example 11) was dissolved in dimethylformamide (3 mL) and cooled to -20 ° C. Add hydrochloric acid in ethyl acetate (2 N, 0.175 mL) followed by t-butyl nitrite (0.0186 mL, 0.163 mmol). The mixture was stirred at -15 ° C for 15 minutes. Solution H — Cys (Trr) —Lys (Boc) —Asn — Phe— —Phe — Trp — Lys (Boc) —Thr (Bu ⁺ ) - Phe — Thr (Bu +) —Ser (Bu +) —NHCH2CH2S — Trt ( 0.315 g (0.133 mmol) described in Example 24 in dimethylformamide (4 mL) containing N-ethyldiisopropylamine (0.082 mL, 0.476 mmol) was cooled to -15 ° C and added dropwise to the above reaction mixture. Stirring was continued for one hour at -15 degrees Celsius and at room temperature overnight. The reaction mixture was evaporated under reduced pressure, the residue was triturated in cold citric acid (1N), filtered and washed with water. The solid is triturated with methanol and dried over phosphorus pentoxide to give the title compound. Amino Acid Analysis: Lys, 1.88, Cysteic Acid, 0.84, Asp, 1.00, Thr, 1.94, Ser, 0.97, Gly, 2.78, Ala, 0.89, Leu, 0, 89, Phe, 3.11.

Example 26

Leucyl-glycyl-glycyl-alanyl-glycyl-cysteinyl-phenylalanyl-phenylalanyl-tryptophyll-lysyl-threonyl-phenylalanyl-threonyl-seryl-2-thioethylamide cyclic disulfide, I, R ¹ = H-Leu-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-NHCH ² CH ² S)

Boc-Leu-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe -Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu) ⁺ ) - Ser (Bu +) - NHCH 2 CH 2 - Trt (0.230 g, 0.083 mmol, described in Example 25) in acetic acid (100 mL) was slowly added to a vigorously stirred solution of iodine (0.211 g, 0.83 mmol) in methanol ( 42 ml) at room temperature. After the addition was complete, the solution was stirred at room temperature for 60 minutes. The solution was cooled to 0 ° C

4β solution of sodium thiosulfate in water (IN) is slowly added to quench the excess iodine. The solvent is evaporated to near dryness, the residue is triturated with cold water, filtered, washed with water and dried over phosphorus pentoxide. The solid was washed with ether and dried to give the cyclic disulfide of formula III.

Bac — Leu — Gly — Gly — Ala — Gly — Cys— —Lys (Boc) —Asn — Phe — Phe — Trp —__ —Lys (Boc j —hr (Bu ⁺ j —Phe —Thr (Bu ') - - Ser (Bu ⁺ ) - NHCH 2 CH 2 S (III).

This cyclic hexadecapeptide was stirred vigorously at 0 ° C under nitrogen for 10 minutes in concentrated hydrochloric acid (7 mL). Acetic acid (90 ml) was added and the solution was lyophilized. The residue is separated by column chromatography on chemically modified cross-linked dextran ("Sephadex C 25 M, 3 x 50 cm, equilibrated in the lower phase of n-butanol-acetic acid-water (4: 1: 5) and then equilibrated in the upper phase) . Using the upper phase, substantially pure hexadecapeptide is desorbed from the column. The pure fractions were combined, evaporated and lyophilized to give the title compound as a solid

MeOH salts with acetic acid, λ 290 (4,290), max

282 (4.910), 273 nm (ε 4.630). Repeated lyophilization of the latter product from water gave the title compound as 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, Gly, 3, 00, Leu, 0.96.

EXAMPLE 27 t-Butyloxycarbonyl-glycyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N -t-butyloxykarboinyl ^{e-lysyl-aspartyl-L-phenylalanyl-L-phenylalanyl-tryptophyl-NM-butyloxycarbonyl-lysyl-Ot-butyl-threonyl} phenylalanyl-Ot-butyl-threonyl-Ot-butyl-seryl-2-tritylthioethylamid, II, ^R2 = H and ^R4 = Boc-Gly-Gly-Ala-Gly-NH (Boc-Gly-Gly-Gly —Ala — Gly — Cys (Trt) - —Lys (Boc) —Asn — Phe — Phe — Trp— —Lys (Boc) —Thr (Bu ⁺ ) —Ser (Bu ⁺ ) - —NHCH2CH2S — Trt]

Boc-Gly-Gly-Gly-Ala-NHNH ₂ pentapeptide hydrazide (0.0608 g, 0.141 mmol, described in Example 17) was dissolved in anhydrous dlmethylformamide (6 mL) and dimethylsulfoxide (2 mL) and cooled to -20 ° C. degrees Celsius. Add hydrochloric acid in ethyl acetate [2N, 0.176 mL, 0.352 mmol) followed by t-butyl nitrite (0.0194 mL, 0.169 mmol). The mixture was stirred at -15 degrees Celsius for 15 minutes and a solution of H-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu +) was added dropwise to this reaction mixture. —Phe — Thr (Bu +) —Ser (Bu +) - —NHCH2CH2S — Trt (0.327 g, 0.138 mmol, described in Example 24) in dimethylformamide (4 mL) containing N-ethyldiisopropylamine (0.085 mL, 0.494 mmol), cooled to —15 ° C. Stirring was continued for one hour at -15 ° C and overnight at room temperature. The reaction mixture was evaporated under reduced pressure, the residue was triturated with ice-cooled (1 N) citric acid solution, filtered and washed with water. The solid was triturated with methanol and dried over phosphorus pentoxide to give the title compound. Amino Acid Analysis: Lys, 2.31, Cysteic Acid, 0.84, Asp, 1.00, Thr, 2.26, Ser, 1.19, Gly, 4.00, Ala, 0.84, Phe, 3, 2.

Example 28

Cyclic disulfide glycyl-glycyl-glycyl-alanyl-glycyl-cysteinyl-phenylalanyl-phenylalanyl-tryptophyll-lysyl-threonyl-phenylalanyl-threonyl-seryl-2-thioethylamide, I, R ¹ = H-Gly-Gly-Gly-Ala Gly-NH and R ² = H (H-Gly-Gly-Gly-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NHCH2CH2S)

Boc-Gly-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe -Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu ⁺ ) - Ser (Bu +) - NHCH 2 CH 2 S - Trt (0.224 g, 0.083 mmol, described in Example 27) in acetic acid (160 mL) was added slowly to a vigorously stirred solution of iodine (0.211 g, 0.83 mmol) in methanol. (42 ml) at room temperature After the addition was complete, the solution was stirred at room temperature for 60 minutes, cooled to 0 ° C, and excess iodine (colorless solution) was decomposed with a solution of sodium thiosulfate in water (1 N) added slowly. The solvent was evaporated to near dryness and the residue was triturated with cold water, filtered and dried over phosphorus pentoxide, washed with ether and dried to give the cyclic disulfide of formula III.

Boc - Gly - Gly - Gly - Ala - Gly - Cys - Lys (Boc) - Asn - Phe - Phe - Trp -

^ LyšIBSS j j —hřřřřřřřřřřř (((((((((((((((((((((2 ^{+ +} (NH NH).

(ΠΙ)

The cyclic hexadecapeptide was stirred vigorously at 0 ° C under nitrogen for 10 minutes in concentrated hydrochloric acid (7 mL). Acetic acid (90 mL) was added ^{and the} solution was lyophilized. The residue was dissolved in 2% acetic acid in water and lyophilized. The residue was separated by column chromatography on chemically modified cross-linked dextran [Sephadex G-25 M], 3 x 50 cm, equilibrated in the lower phase with n-butanol / acetic acid-water (4: 1: 5) and then equilibrated using a top phase for desorption of substantially pure hexadecapeptide). The pure fractions were combined, evaporated and lyophilized to give the title compound as an acid salt.

MeOH acetic acid, λ 288 (ε 4870), 280 (ε max

Amino Acid Analysis: Lys, 1.72, Asp, 1.00, 5575), 274 (ε 5380), 268 (ε 5185), 265 nrn (ε 4955). Repeated lyophilization of the last product from water gave the title compound as the free base. Ser, 0.73, Ala, 0.69, Phe, 2.78, Cysteic acid 0.57, Thr, 1.69, Gly, 3.59.

EXAMPLE 29 t-Butyloxycarbonyl-glycyl-glycyl-alanyl-glycyl-S-trityl-cysteinyl-N ^e -t-butyloxycarbonyl-lysyl-aspartyl-L-phenylalanyl-L-phenylalanyl-tryptophyl-NM-butyloxycarbonyl-lysyl-OI-butyl-threonyl-L-phenylalanyl butylV ol-threonyl-Ot-butyl-seryl-2-trityl-thioethylamid II, R ² = H and R4 = Boc-Gly-Gly-Ala-Gly-NH [Boc-Gly-Gly-Ala - Gly-Cys (Trt) -Lys (Boc), Asn-Phe-> Phe-Trp-Lys (Boc) Thr (tBu ⁺⁾ -Phe- Thr (tBu ⁺⁾ -Ser (Bu +) -NHCH 2 CH ₂ - Trt]

To a solution of Boc-Gly-Gly-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-2 (800 mg, 0.59 mmol, prepared according to Example 7) in dimethylforimamide (12 mL) at -20 ° C was added with stirring a 1.85 N solution of hydrogen chloride in ethyl acetate (0.795 mL, 1.475 mmol). The mixture was cooled to -15 ° C, tert-butyl nitrite (0.081 mL, 0.71 mmol) was added and the solution was stirred for 15 minutes. A solution of H-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu ⁺ ) -Ser (Bu ⁺ ) -NHCH 2 CH 2 -S-Trt (0.852 g, 0.637 mmol) was added to the reaction mixture so prepared. prepared according to the procedure of U.S. Patent No. 3,917,581 of 4. XI. 1975, and N-ethyldiisopropylamine (0.354 mL, 2.06 mmol) in dimethylformamide (6.0 mL), cooled to -15 ° C. The mixture was stirred at -15 ° C for one hour and at 25 ° C for 18 hours. The solvent was evaporated and the residue was triturated with ice-cooled citric acid, filtered, washed with water and methanol and dried to give the title compound. Amino Acid Analysis: Lys, 2.01, Asp, 0.97, Thr, 1.60, Ser, 0.65, Cysteic Acid, 0.87, Gly, 2.92, Ala, 1.00, Phe, 2, 97

Example 30

Cyclic disulfide glycyl-glycyl-alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryjitophyll-lysyl-threonyl-phenylayl-tlinionyl-seryl-thioethylamide, I, R1 = H — Gly — Gly — Gly-G —NH and R2 = H (H — Gly — Gly — Ala — Gly — Cys— —Lys — Asn — Phe — Phe — Trp — Lys — Thr— —Phe — Thr — Ser — NHCH2CH2S)

Boic — Gly — Gly — Ala — Gly — Cys (Trt) · - —Lys (Boc) - Asn — Phe — Phe — Trp— —Lys (B <rc) —Thr (Bu +) —Phe — Thr (Bu ⁺ ) Ser (Bu ⁺ ) - NHCH 2 CH 2 - Trt described in Example 29 (0.860 g, 0.30 mmol) was dissolved in glacial acetic acid (150 ml) and added dropwise to a solution of iodine in imethanol at room temperature. (0.5%, 150 mL, 30 mmol) was added and the reaction stirred for one hour. The mixture is stirred for a further 45 minutes, cooled in an ice bath and a solution of sodium thiosulphate in water (1 N, 6 ml) is added to decompose the excess iodine (colorless solution. The solution is evaporated and the residue is triturated with water, dried and the anhydrous product is triturated with iso-propyl ether to give the pentadecapeptide cyclic disulfide of formula III.

Boc — Gly — Gly — Ala — Gly — Cys — Lys (Boc) - —Asn — Phe — Phe — Trp — Lys (Boc) —_____ —Thr (Bu +) —Phe — Thr (Bu ⁺ ) — Ser (Bu +) - —NHCIWHeS. (III)

Cold concentrated hydrochloric acid (23 mL) was added to the latter compound with stirring in an ice bath and nitrogen atmosphere. Stirring is continued for 10 minutes, glacial acetic acid is added and the solution is lyophilized. The residue is dissolved in water and lyophilized again. Dissolve the residue in 0.01 N aqueous ammonium acetate and load onto a column of carbomethyl cellulose (Whatman CM-23 2.5 x 30 cm). The pure compound was eluted with 0.06 N ammonium acetate buffer. The purified material was lyophilized from water to give the title compound as a white solid as a salt with α-MeOH acid λ 282 nm (ε 5120), 289 nm max (ε 4610). Repeated lyophilization of the latter product from water gives the title compound as the free base. Amino Acid Analysis: Lys, 2.06, Asp, 1.02, Thr, 1.75, Ser, 0.91, Cysteic Acid, 0.73, Gly, 3.00, Ala, 1.10, Phe, 3, 11.

In an analogous manner, but using rhodane by the method of Hiskey and Smith, supra, instead of iodine, the title compound is also obtained.

Example 31

Acetyl- (S-trityl) cysteinyl- (N-t-butoxycarbonyl) lysyl-asparaginyl-phenylalanyl-phenylalanine methyl ester [Ac-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe-OMe]

P-Nitrophenylacetate solution [0.191 g, 1.05 mmol, prepared by FD Chattaway, J. Chem. Soc., 2495 (1931)] in dimethylformamide (4 ml) was added to a solution of H-Cys (Trt) -Lys (Boc) -Asn -Phe-Phe-OMe at 0 ° C. HOAc (0.750 g, 0.698 mmol, prepared according to HU Imimer et al., Helv. Chirn. Acta., 57, 730 (1974)) and N-ethylmorpholine (0.1 mL). After stirring at 0 ° C for 24 hours, the solvent was evaporated The residue was dissolved in methanol (3 mL) and added slowly to diethyl ether (200 mL), and the precipitate was filtered off and crystallized from ethanol to give the title compound, mp 219.5-221 ° C, [α] D [.alpha.] D @ ²⁵ = -21.6 DEG (c = 1, dimethylformamide).

In the same manner, using p-nitrophenyl esters of formic, propionic, butyric, isobutyric, pivalic, n-hexanoic or benzoic acid instead of p-nitrophenyl acetate, the corresponding compounds of the above formula are prepared wherein Ac is replaced by formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or benzoyl residues.

Example 32

Acetyl- (S-trityl) cysteinyl- (Nt-butoxycarbonyl) lysyl-asparaginyl-phenylalanyl-phenylalanine hydrazide, IV, R ⁴ -NHCOCH 3 [Ac-Cys (Trt) -Lys (Boc) -Asn-Phe-Phe- - NHNH2)

A solution of Ac-Cys (Trt) -Lys (Boc) -Asn -Phe-Phe-OMe (0.40 g, 0.379 mmol, described in Example 31) and hydrazine hydrate (0.37 mL, 7.58 mmol) in methanol (15 mL) was stirred at 0 ° C for 48 h. The precipitate was filtered off and dried to give the title compound, mp 236-237 ° C, [α] ²⁵ D = -28.6 ° (c = 1, dimethylformamide).

In the same way, using the appropriate other starting materials described in Example 31, the corresponding compounds of formula IV are obtained, wherein R ⁴ is NHR ³ , wherein R ³ is formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or benzoyl .

Example 33

Acetyl- (S-trityl) cysteinyl (N ^e -t-butyloxycarbonyl butoxykar- ₄₎ lysyl-aspartyl-phenylalanyl-L-phenylalanyl-tryptophan yl- (N ^e t-butoxycarbonyl) lysyl- (Ot-butyl) -threonyl- f enylalanyl- (Ot-butyl) Threonine 1- (Ot-butyl] -seryl-2-tritylthioethylamid II, R ² = H and R ⁴ = NHCOCH 3 [Ac-Cys (Trt) -Lys (Boc) -Asn-Phie - —Phe — Trp — Lys (Boc) - Thr (Bu ⁺ ) - —Ser (Bu ⁺ ) —Ser (Bu +) - NHCH2CH2S — Trt]

A solution of Ac-Cys (Trt) -Lys (Boc) -Asn -Phe-Phe-NHNH ₂ (IV) (0.240 g, 0.227 mmol, described in Example 32) in anhydrous dimethylformamide (2 mL) and dimethylsulfoxide (1 mL) is cooled to -20 ° C. Add hydrochloric acid in ethyl acetate (2.1 N, 0.273 mmol) then t-butylnitrite (0.0312 mL, 0.273 mmol). The mixture was stirred at -15 ° C for 15 minutes and a solution of H-Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu +) -Ser (Bu ⁺ ) -NHCH 2 CH 2 -Trt was added dropwise to this reaction mixture. (V, 0.304 g, 0.227 mmol), prepared according to the procedure of US Patent No. 3,917,581 of 4. XI. 1975, in dimethylformamide (3 mL) containing N-ethyldiisopropylamine (0.097 mL, 0.568 mmol) cooled to -15 ° C. The mixture was stirred at -15 ° C for one hour and then at 25 ° C for 20 hours. The solvent was evaporated under reduced pressure. The residue is triturated with ice-cooled citric acid (1 N), filtered, washed with water and dried over phosphorus pentoxide. The solid residue was triturated with methanol, filtered, dried over phosphorus pentoxide to give the title compound. Amino Acid Analysis: Lys, 1.82, Asp, 1.00, Ser, 0.76, cysteic acid, 0.93, Thr, 1.87, Phe, 3.10.

In the same way, but using the corresponding other starting materials of formula IV, wherein R ⁴ is NHR ³ described in Example 32, the corresponding compounds of formula II are obtained, wherein R ² is H and R ⁴ is NHR ³ , R ³ is formyl, propionyl, n -butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or benzoyl.

Example 34

Acyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptoiphenyl-lysyl-threoinyl-phenylalanyl-threonyl cyclic disulfide207570

-Seryl-2-thloethylamide, I. R ¹ -NHCOCH 3 a

R ² - H (Ac-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-S-ER- -NHCH2CH2S)

Ac-Cys (Trt) -Lys (Boc) -Ann-RЫе-Phe-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu ⁺ ) -Ser (Bu + *) -NHCH 2 CH 2 S - - Trt (0.260 g, 0.110 mmol, described in Example 33) in acetic acid (61 mL) was slowly added to a stirred solution of yoga (0.278 g, 1.1 mmol) in methanol (56 mL) at 25 ° C. After the addition was complete, the solution was stirred at 25 ° C for one hour. The solution was cooled to 0 ° C and a solution of 1 N sodium thiosulfate in water was slowly added to decompose the excess iodine (colorless solution). The solvent was evaporated under reduced pressure and the residue was triturated with water. The precipitate is filtered off, washed with water and dried over phosphorus pentoxide to give the cyclic protected undecapeptide of formula III.

Ac-Cys-Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu +) -Ser (Bu +) -NHCH 2 CH 2 S (III)

The latter cyclic peptide was vigorously stirred at 0 ° C under nitrogen for 10 minutes in concentrated hydrochloric acid (9.1 mL). Acetic acid (119 mL) was added and the solution was immediately lyophilized. The residue is dissolved in the upper phase of the solvent system butanol-acetic acid-water [4: 1: 5] and applied to a column of chemically modified cross-linked dextran (Sephadex 6-25M) prepared in the lower phase of the dissolution system.

The upper phase of the above solvent system is used after desorption of the undecapeptide. Fractions containing pure product were combined and evaporated under reduced pressure. The residue is triturated in diethyl ether, dissolved in 5% acetic acid by lyophilization to give the title compound as the acetic acid salt. UV (methanol) λ mox 290 (ε 4990 '), 282 (ε 5455), 274 nm (ε 5095).

The latter compound, as its acetic acid salt, was subjected to repeated freeze-drying from water to give the title compound as the free base. Amino Acid Analysis: Lys, 1.93, Asp, 1.00, Ser, 0.84, cysteic acid, 0.80, Thr, 1.84, Phe, 2.97.

In the same manner, using the corresponding other starting materials of formula II, wherein R ² is H and R 4 is NHR ³ described in Example 33, the corresponding compounds of formulas III-I are obtained, wherein R ² is H and R 4 and R ¹ , respectively. are NHR ³ wherein R ³ is formyl, propionyl, n-butanoyl, isobutanoyl, pivaloyl, n-hexanoyl or benzoyl.

Example 35

3-Tritylthiopropionic acid pentochlorophenyl ester (Trt — S — CH2CH2COOPCp)

3-Tritylithiopropionic acid [1, Og, 2.87 mmol, described by R. C. Hiskey of M.A. Horpold, J. Org. Chem., 33, 559 (1968)] was dissolved in tetrohydrofuron (25 mL) and pentochlorophenol (0.765 g, 2.87 mmol) was added. Cool the mixture to 0 ° C, add dicyclohexylcarbodiimide (0.596 g, 2.87 mmol) and stir the reaction mixture at 0 ° C for one hour at 25 ° C. The reaction mixture was then cooled to 0 ° C, filtered and the filtrate evaporated under reduced pressure. The residue was crystallized from ethyl acetate to give the title compound, mp 154-156 ° C.

Example 36

3-Tritylthiopropionyl- [NM-butoxycarbonyl] lysyl-asparoginyl-phenylalanyl-phenylalanine methyl ester [Trt-SCH2CH2CO-Lys (Boc) -Asn-Phe-Phe-OMe]

A solution of 3-tritylthiopropionic acid pentochlorophenyl ester (0.597 g, 1 mmol, described in Example 35), H-Lys · (Boc) -Asn-Phe-Phe-OMe. HOAc [1 mmol, prepared according to H. U. Immer et al., Helv. Chirh. Acto., 57, 730 (1974)] and triethylamine (0.14 mL, 1 mmol) was stirred at 25 ° C for three days. The solvent was evaporated under reduced pressure. The residue is triturated in an ice-cooled solution of 1 N citric acid, filtered, washed with water and dried over potassium hydroxide. The solid was crystallized from methanol to give the title compound, mp 215-220 ° C.

Leading 3 7 i ^^^^ ithiopropionyl- ^(e N -t-butoxycarbonyl-1) lysyl-phenylalanyl-asporoginyl fenylalaninhydrazid IV, R ⁴ = H [SCHzCtaCO- Trt-Lys (Boc) -Phe-Phe- -Asn- NHNHz]

A solution of Trt-SCH 2 CH 2 CO-Lys (Boc) -Asn-Phe-Phe-OMe (0.900 g, 0.9 mmol, described in Example 36) with hydrosinhydrate (1 mL ·) in dimethylformamide (20 mL) was stirred for 20 hours at Deň: 22 ° C. The solvent was evaporated under reduced pressure. The residue is triturated in cold water, filtered, washed with water and dried to give the title compound, mp 225-235 ° C.

Example 38

3-Tritylthiopropionium 1- (NM-butoxycarbonyl) lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophenyl- (NM-butoxycarbonyl) lysyl 1- (t-butyl) threonyl-phenylalanyl- (t-butyl) threonyl- [t-butyl] ) seryl-2-tritylthioethylamid II, R ² and R ⁴ = H [-SCHžCHžCO-Trt-Asn-LysfBocJ PIie- -Phe-Trp-Lys (Boc) Thr (tBu ⁺⁾ - Phe-Thr (Bu ⁺ ) —Ser (Bu ⁺ ) —NHCH2CH2S— —Trt)

A solution of Trt-SCH2CH2CO-Lys (Boc) -Asn-Phe-Phe-NHNH2 (0.500 g, 0.5 mmol, described in Example 37) in dimethylsulfoxide (5 mL) and dimethylformamide (20 mL) was cooled to -20 °. C. A solution of hydrochloric acid in ethyl acetate (1.4 N, 0.895 mL) was added followed by t-butyl nitrite (0.069 mL, 0.6 mmol). The mixture is stirred for 15 minutes at -15 ° C and a cooled solution of H-Trp-Lys (Boc) -Thr (Bu +) -Phe-Thr (Bu +) -Ser (Bu +) - Ser is added to -15 ° C. NHCH 2 CH 2 S-Trt (0.670 g, 0.5 immol, prepared according to U.S. Patent No. 3,917,581, Nov. 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 hour and at 25 ° C for 20 hours and then evaporated under reduced pressure. The residue is triturated with ice-cooled 1N citric acid solution, filtered, washed with water and dried over phosphorus pentoxide. The solid was triturated with cold methanol and dried to give the title compound. Amino Acid Analysis: Lys, 1.99, Asp, 1.15, Thr, 1.73, Ser, 0.67, Phe, 3.00.

Example 1 a d 3 9

3-Thiopropionyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyll-lysyl-threonyl-phenylalanyl-threonyl-seryl-2-thioethylamide cyclic disulfide, I, R ¹ and R ² = H (SCH ² CH ² CO-Lys-Asn-Phe-Phe-Phe) Trp — Lys — Thr — Phe — Thr — Ser — NHCH2CH2S)

Solution Trt — SCH2CH2CO — Lys (Boc) - —Asn — Phe — Phe — Trp — Lys (Boc) - —Th; r (Bu ⁺ ) —Phe — Thr (Bu +) — Ser (Bu ⁺ ) - —NHCH2CH2S — Trt (0.500 g, 0.216 mmol, described in Example 38) in acetic acid (100 mL) was slowly added to a stirred solution of iodine (0.547 g, 2.16 mmol) in methanol (110 mL) at 25 ° C. After the addition was complete, the solution was stirred at 25 ° C for one hour. The solution was cooled to 0 ° C and excess iodine (colorless solution) was decomposed by slow addition of 1N sodium thiosulfate solution in water. The solvent was evaporated under reduced pressure and the residue was triturated with water. The precipitate is filtered off, washed several times with water and dried over phosphorus pentoxide. A cyclic protected decapeptide of formula III is obtained.

SCH2CH2CO-Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thir (Bu +) -Ser (Bu +) -NHCH 2 CH 2 S.

(III)

The latter cyclic decapeptide was stirred vigorously at 0 ° C under nitrogen for 10 minutes in concentrated hydrochloric acid (18 mL). Acetic acid (200 ml) was then added and the solution immediately lyophilized. The residue is dissolved in the upper phase of a 4: 1: 5 butanol-acetic acid-water solvent mixture and applied to a column of chemically modified cross-linked dextran (Sephadex G-25 M) prepared in the lower phase of the solvent system. The upper phase of the above solvent system is used for the desorption of the decapeptide. Fractions containing pure product were combined and evaporated under reduced pressure. The residue was dissolved in 5% acetic acid and lyophilized to afford the title compound as the acetic acid salt. UV (methianol) λ _max 290 (ε 4920), 282 nm (ε 5390).

The latter compound, in the form of an acetic acid salt, is subjected to repeated lyophilization from water to give the title compound as the free base. Amino Acid Analysis: Lys, 1.97, Asp, 1.00, Thr, 1.64, Ser, 0.65, Phe, 2.94.

EXAMPLE 40 OR, ^{N-dimethyl-3,5-dimethoxybenzyloxycarbonyl-tryptophyl-N-e} -t butyloxykarboinyl-lysyl-Ot-butyl-threonyl-L-phenylalanyl-Ot-butyl-threonyl-Ot-butyl-seryl-S-trityl-cysteinmethylester [Ddz — Trp — Lys (Boc) - —Thr (Bu ⁺ ) —Phe — Thr (Bu ⁺ ) —Ser (Bu ⁺ ) - —Cys (Trt) - OMe]

A solution of diazomethane in ether was added to a solution of Ddz-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu +) -Ser (Bu +) -Sys (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 was stirred at 0 ° C for one hour and evaporated. The residue is chromatographed on silica gel using 3% methanol and 0.5% pyridine in chloroform for elution. Evaporation of the eluate gave the title compound NMR (CDCl 3), δ 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).

In an analogous manner using diazoethane, 1-diazopropane, 2-diazopropane, 1-diazobutane, 1-diazoisobutane, 1-diazopentane, 4-diazo-2-methylbutane, 1-diazohexane, 1-diazoheptane or 1-diazooctane, diazoimethane is obtained instead of diazoimethane. the corresponding ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl,. n-hexyl, n-heptyl, n-octyl esters of the title compound.

Example 41

S-tritylcysteine decyl ester [H — Cys (Trt) - - OCH2 (CH2) 8CH3]

Cysteine hydrochloride decyl ester solution [0.894 g, 3 mmol, prepared according to Voullie et al., French Supplemental Patent, 75, 157 (1961), CA 57, 15235c], triphenylcarbinol [0.78 g, 3 µmol] and boron trifluoride etherate [0 , 42 ml) in acetic acid [5.2 ml] was stirred at room temperature for one hour. The solvent was evaporated under reduced pressure and the residue was chromatographed on silica gel using 40% ethyl acetate in hexane containing 0.1% triethylamine as eluent. Evaporation of the eluate gave the title compound as an oil. [α] D ²⁵ - + 25.84 ° [c = 1, CHCl 3], NMR (CDCl 3) δ 0.89, (t, J = -5 Hz, 3 H), 1.3 [s, 16 H ), 1.55 (m, 2H),

2.55 (m, 2H), 3.25 (2d, J = 5Hz), 4.1 (t, J = 6Hz, 2H).

In the same way, but substituting the starting material for an equivalent amount of cysteine tetradecyl ester hydrochloride (prepared according to Voullie et al., Supra) gives the S-tritylcysteintetradecyl ester, [α] 25 D = + 13 ° 8 ° [c = 1, CHCl 3] 1 H (CDCl 3) δ 0.89 (t, J = 5 Hz, 3H), 1.3 (s, 24H), 1.56 (m, 2H), 2.55 (m, 2H), 3.25 (2 d, J = 5Hz, 1H); 4.1 (t, J = 6Hz, 2H).

The nonyl, undecyl, dodecyl, and tridecyl esters of S-tritylcysteine are obtained in an analogous manner.

Reaction of these S-tritylcysteine esters with hexapeptide hydrazide of formula

Ddz-Trp-Lys- (Boc) -Tr (Bu ⁺ ) -Phe-ThrjBu-Ser (Bu +) -NH 2 azide yields nonyl, decyl, undecyl, dodecyl, tridecyl and tetradecyl esters corresponding to the title compound of Example 40 .

Example 42

Tryptophyll-NM-butyloxycarbonyl-lysyl-O-butyl-threonyl-phenylalanyl-O-butyl-threonyl-O-butyl-seryl-S-trityl-cysteine methyl ester V, R ² = COOCH 3 [H-Trp-Lys ( Boc) —Thr (Bu ⁺ ) —Phe— —Thr (Bu +) —Ser (Bu +) —Cys [Trt] —OMe. . HCO2H]

Rojztolk Ddz-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe-Thr (Bu ⁺ ) -Ser (Bu ⁺ ) -Cys (Trt) -OMe- (1.11 g, 0.685 mmol, described in of Example 40) in 10 ml of formic acid-acetic acid-water (1: 7: 2) was stirred overnight at room temperature. The mixture was evaporated and the residue was triturated with water. The solid was filtered, dried under reduced pressure to give the title compound

MeOH in the title, λ 290 (ε 6,335), 280 (ε max

8,470], 273 (3,720), 216 µm (ε 78,400 ').

In analogy to the other heptapeptides described in Examples 40 and 41 as starting materials, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, n-hexyl, n-heptyl and n-octyl are obtained. n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, and n-tetradecyl esters of the corresponding title compound.

EXAMPLE 43 t-butyloxycarbonyl-alanyl-glycyl-S-trityl-cysteinyl-NM-butyloxycarbonyl-lysyl-aspartyl-L-phenylalanyl-tryptophyl-fenylalan.yl N ^e -TB (ulyloxykarbonylllysyl 0-t-butyl-but-threonyl-phenylalanyl- Tert-Butyl-threonyl-tert-butyl-seryl-S-rityl-cysteine methyl ester, II, R ² = COOCH 3 and R ⁴ = Boc-Ala-Gly-NH- [Boc-Ala-Gly-Cys) - —Lys (Boc) - As'n — Phe — Phe — Trp— —Lys (Boc) —Thir ^: (Bu ⁺ ) —Ser (Bu ⁺ ) - —Cys (Trt) —OMe]

To a solution of the first heptapeptide hydrazide (IV) Boc-Ala-Gly-Cys (Trt) -LysJBoc) -An-Phe-Phe-NHNH 2 (0.755 g, 0.624 mmol) described

HU irnmer et al., Supra) in dimethylformamide [9 mL] at -20 ° C was added a 2.1 N solution of anhydrous hydrogen chloride in ethyl acetate [1.56 mmol] and then tert-butyl nitrite (0.0863 mL) was added. , 0.749 mmol). The solution was stirred at -15 ° C for 15 minutes. To this solution at -15 ° C is added a solution of the second methyl ester of the heptapeptides V, H-Trp-Lys (Boc) -Thr (Bu ⁺ ) -Phe ^- Thr (Bu ⁺ ) -Ser (Bu +) - Cys ( Trt] —OMe.

. HCO 2 H (0.900 g, 0.624 mmol, described in Example 42) and N-ethyldiisopropylamine (0.373 mL, 2.184 mmol) in dimethylformamide (5 mL ·), cooled to -15 ° C. The resulting mixture was stirred at -15 ° C for one hour and then at room temperature for three days. The solvent was evaporated under reduced pressure. The residue is triturated with 1 N citric acid, filtered, and the precipitate is washed with water and dried over phosphorus pentoxide under reduced pressure. The dried residue was triturated with a small amount of methanol to collect the residue and dried to give the title compound. Amino Acid Analysis: Lys, 2.06, cysteine ^! acid

I, 75, Asp, 0.94. Thr, 1.93, Ser, 0.97, Gly, 1.00, Ala, 0.88, Phe, 3.10.

In the same manner, using the other alkyl esters of the heptapeptide of formula V described in Example 42, the corresponding ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, n-hexyl, n-heptyl, n-octyl are also obtained. , n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl and n-tetradecyl esters of the title compound.

Example 44

The cyclic disulfide ^{t-butyloxycarbonyl-alanyl-glycyl-cysteinyl-NM-butyloxycarbonyl-lysyl-aspartyl-L-phenylalanyl-L-phenylalanyl-tryptophyl-N-e} -t blutyloxykarbonyl-lysyl-0-t-butyl-threonyl-phenylalanyl - Ot-butyl threonyl-O-butyl-seryl-cysteine methyl ester, III, R2 = COOCHs and R4 Boc-Ala-Gly-NH [Boc-Ala-Gly-Cys-Lys (Boc) -Asn-Phe-Phe-Trp-Lys ( Boc) - —Thr (Bu +) - Ser (Bu ⁺ ) —Cys — OMe]

Linear, protected methyl ester of the tetradecapeptide of Example 43, Boc-Ala-Gly-Cys '(Trt') -Lys (Boc) -Ann-Phe-Phe-Trp-Lys (Boc) -Thr (Bu +) -Phe- -Thr (Bu ⁺ ) -Ser (Bu ⁺ ) -Cys (Trt j -OMe (0.898 g, 0.345 mmol)) was dissolved in hot acetic acid (200 mL), cooled to room temperature and stirred for 1 h. dropwise to a solution of 5% iodine in methanol (175 mL, 3.45 mmol), stirred for an additional hour, cooled to 0 ° C and, after removal of excess iodine, 1 N sodium thiosulfate in water (15 mL) was added. The solvent is evaporated, the residue is triturated with water and dried over phosphorus pentoxide under reduced pressure to give the title compound in the same manner using the other linear protected esters of the tetradecapeptide of formula II described in Example 43, the corresponding ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-meth hylbutyl, n -hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-trldecyl and n-tetradecyl esters of the title compound.

Example 45

Somatostatin methyl ester, alanyl-glycyl-cysteinyl-lysyl-asparaginyl-phenylalanyl-phenylalanyl-tryptophyll-lysyl-threonyl-phenylalanyl-threonyl-seryl-cysteine methyl ester cyclic disulfide, I, R 4 -H-Ala-Gly-NH and R 2 = R 2 —Ala — Gly — Cys — Lys— —Asn — Phe — Phe — Trp — Lys — Thr — Phe— —Thr — Ser — Cys. · —OMe)

The cyclic disulfide solution of Example 44, Boc-Ala-Gly-Cys-Lys (Boc) -Asn-Phe-Phe-Trp-Lys (Boc) '-hr (Bu + j-Phe -hr (Bu +) -Ser (Bu +) - -Cys - OMe, (0.345 mmol) in concentrated hydrochloric acid (29 mL) was stirred for 10 minutes under nitrogen at 0 ° C. Glacial acetic acid (290 mL) was added and the solution was lyophilized. Dissolve in 0.2 M ammonium acetate solution, load onto a carboxymethylcellulose column (Whatman CM-23) and elute with 0.2 M ammonium acetate buffer. M) equilibrated in the lower phase with n-butanol-acetic acid-water (4: 1: 5) and then equilibrated in the upper phase, using the upper phase for desorption of the methyl ester of soaaostatin. the title compound in FORMeOH had salts with acetic acid ovou, λ 290 max (e 4,345), 287 (ε 4,385), 268 (ε 4,090), 265 (ε 3,900), 215 nm (ε 38,305), alkaline hydrolysis and gas chromatography calculated for methanol 1,75%, found 2.0%, NMR (DMSO-d 6 3.63 (s, 3 H, OCH 3)).

Repeated lyophilization of the last acid salt from water affords the title compound as the free base: Lys amino acid analysis, 2.00, cysteic acid, 1.28, Asp, 0.95, Thr, 1.91, Ser, 0 , 97, Gly, 1.00, Ala, 0.94, 1/2 Cys, 0.30, Phe, 3.01.

In the same manner, but using the other cyclic disulfides of formula III described in Example 44, the corresponding ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, 2-methylbutyl, n-hexyl, n-heptyl, n-octyl, n are prepared. nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl and n-tetradecyl esters of the title compound.

Claims (7)

I will invent the subject A process for the preparation of a tetradecapeptide of formula I, SCH 2 CH (R ¹⁾ CO-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser -NHCH (R ²⁾ CH 2 S (I) wherein (a) R ¹ is H-Gly-Gly-Ala-Gly-NH, H-Gly-Gly-Gly-Ala-Gly-NH or H-Leu-Gly-Gly-Ala-Gly-NH and R ² is H or COOH, or b) R ¹ is H or NHR ³ where R ³ is an acyl radical neroizvětvené or branched aliphatic carboxylic acids having 1 to 6 carbon 'UhJjíku or ^1; benzoyl and R ² is H, or c) R ¹ is H-Ala-Gly-NH and R ² is COOalk, wherein alk is a straight or branched chain alkyl of 1 to 14 carbon atoms, characterized in that the linear peptide of formula II is oxidized, Trt-SCH 2 CH (R ^4} -CO-Lys (Boc) - Asn-Phe-Phe-Trp-Lys (Boc) Thr (tBu ⁺⁾ - Phe-Thr (tBu +) -Ser (Bu ^+] - - NHCH (R ²⁾ CH 2 S-Trt (II) wherein a) R ² is H or COOH and R ⁴ is Boc-Gly-Gly-Ala-Gly-NH, Boc-Gly-Gly-Gly-Ala-Gly-NH or Boc-Leu-Gly-Gly-Ala-Gly-NH or b) R ² is H and R ⁴ is H or NHR ³ wherein R ³ is as defined above, or · c) R ² is COOalk and R ⁴ is Boc-Ala-Gly-NH, iodine or rhodane to give the corresponding cyclic disulfide derivative of formula III, SCH 2 CH (R ⁴⁾ CO-Lys (Boc) -Phe-Asn-Phe-Trp-Lys (Boc) Thr (tBu +) -Phe- -ThríBu · ⁴⁾ -Ser (Bu ⁺⁾ -NHCH [R ² ) CH2S (III.) Wherein R ² and R ⁴ are as defined above, and then removing the remaining protecting groups by treatment with 50-100% trifluoroacetic acid or a mineral acid solution. 2. A process according to claim 1, wherein a solution of the linear peptide of formula II in acetic acid is reacted with iodine in solution in a C1 -C4 alkanol or acetic acid and the resulting cyclic disulfide of formula III is reacted with an acid. hydrochloride and the corresponding peptide of formula I is isolated. 3. A process according to claim 1, wherein the linear peptide of formula II is reacted with iodine at 0 DEG to 30 DEG C. for 30 to 180 minutes in a C1 -C4 alkanol or acetic acid to give the corresponding cyclic disulfide of formula III. 4. The process of claim 1, wherein the cyclic disulfide of formula III is reacted with concentrated hydrochloric acid at 0 DEG C. for five to ten minutes to give the corresponding peptide of formula I. 5. Method according to claim 1 for preparing the tetradecapeptide of formula I, as defined in paragraph 1 wherein R ¹ is H-Gly-Gly-Ala-Gly-NH, H-Gly-Gly-Gly-Ala-Gly-NH or H-Leu-Gly-Gly-Ala-Gly-NH and R ² is H or COOH, characterized in that a linear peptide of the general formula (II) given in point 1, wherein R ² is as defined above and R is oxidized ⁴ is defined as in point 1, iodine or rhodani to give the corresponding cyclic disulfide derivative of the general formula III shown in item 1, wherein R ² has the abovementioned meaning and R ⁴ has the meaning given under point 1, and then removing the remaining protecting groups by reaction with 50 to 100% trifluoroacetic acid or a mineral acid solution. 6. A method according to claim 1 for preparing the tetradecapeptide of formula I, as defined in paragraph 1 wherein R ¹ is H or NHR ³ where R ³ is an acyl residue of a straight or branched aliphatic carboxylic acid having 1-6 carbon atoms or benzoyl, and R · ² is H, characterized by oxidizing the linear peptide of formula (II ⁾ as defined in (1), wherein R2 is as defined above, and R ( ⁴⁾ is as defined in (1) by iodine or rhodane to form the corresponding cyclic disulfide derivative of formula III, wherein R ² is as defined above and R ⁴ is as defined in 1, and then the remaining protecting groups are removed by treatment with 50 to 100% trifluoroacetic acid or a mineral acid solution. 7. The process of item 1, for the preparation of the tetradecapeptide of formula I, as defined in item 1, wherein R ¹ is H-Ala-Gly-NH and R ² is COOalk, wherein alk is a straight or branched chain alkyl of 1 to 14 atoms. carbon by oxidizing the linear peptide of formula (II ⁾ as defined in (1), wherein R ² is as defined above and R ( ⁴⁾ is as defined in (1) by iodine or rhodane to form the corresponding cyclic disulphide de207570 rivate of formula (III), The composition of claim 1, wherein R ² is as defined above and R ⁴ is as defined in point 1, and then the remaining protecting groups are removed by treatment with 50 to 100% trifluoroacetic acid or a mineral acid solution.