PL91607B1 - - Google Patents

Description

The patent description was published: September 15, 1977 91607 MKP C07c 103/52 Int. Cl.2 C07C 103/52 CZYISLNIA Inventor: Patent holder: Richter Gedeon Vegyeszeti Gyar RT., Budapest (Hungary) Method for the production of new peptides with ACTH activity and their amide addition salts with benzyl ester. The invention relates to a method for the preparation of new peptides with ACTH activity containing at both ends of the α-amino-oxycarboxylic acid molecules of the general formula HO-OSer -Tyr-Ser-Met-Glu-His- Phe-Arg-Trp-Gly-Lys-Pro- -Val-Gly- (Lys) nX where n is an integer of 3 or 4 and X is -OLys-OH, -O-OLys-OH or -OGly-OH as well as their acid addition salts, amides and benzyl ester. As is known, the peptide chain of adrenocorticotropic hormone (ACTH) can be modified without the fear of losing its adrenocorticotropic activity. More than a third of the molecule can be removed from the C-terminal part of the amino acid chain and this does not impair its specific activity, but the cleavage of further amino acids leads to a marked decrease in activity. The peptide with the amino acid sequence 1-9 of the ACTH hormone shows 30% of the activity of the original molecule, while the activity of the 1-16 ACTH fragment is only about 1% compared to the starting substance. Removal of the first amino acid from the N-terminal part particles lead to a 50% decrease in activity (E. Schroder, K. Lubke; The Peptides, Academie Press, New York, 1966, pp. 246-9). Fragments of ACTH in which the N-terminal group of serine is replaced D-amino acid, (a non-natural amino acid or acyl group obtained by omitting the serine functions, eg, p-hydroxypropionyl or propionyl groups, sometimes exhibit activities that exceed those of natural ACTH. This is presumed to be due to the fact that These ACTH fragments of the modified compounds are resistant to the action of aminopeptidase enzymes and the rate of their degradation in the body is lower compared to that of natural ACTH. As a result, the modified ACTH fragments show a strong adrenaline. orthicotropic activity even under conditions where the modifying group cannot completely fulfill the role of the N-terminal molecule of the natural hormone serine. The fact that the decrease in the activity of amide derivatives in the C-terminal part of the molecule is slower than in the case of peptides containing free carboxyl groups and a reduced amount of amino acids starting from the C-terminal moiety of the chain molecule are caused by the same cause, namely, most carboxypeptidase enzymes do not break down peptide amides. Therefore, it is assumed that the amide substitution of the C-terminal carboxyl group it has the same protective effect as for the substitution of the N-terminal serine molecule. However, recent studies have found that certain carboxypeptidase enzymes are capable of cleaving even the G-terminal amino acid molecules of peptide amides (J.D. Glass et al; 916,073 Proc. Nat. Acad. Sci. U.S. 63, 1426, 1969). Due to the fact that even these enzymes cannot hydrolyze the substituted amides, it can be assumed that ACTH fragments containing substituted myid groups in the C-terminal moiety of the chain molecule will degrade more slowly in the body and therefore show increased biological activity compared to simple amides. This was confirmed by German researchers, who synthesized certain and heptadecapeptidalkylamide derivatives showing a strong adrenocorticotropic effect (German Patent No. 1,954,794). It was found that in the case of replacing both two-end amino acids 1-17, 1-18 or 15 1-19 fragments of the ACTH molecule by the α-aminooxycarboxylic acids, the compounds with increased adrenocorticotropic action are obtained. Compounds of particularly high activity are obtained by replacing the N-terminal molecule of 2o serine 1-18 of the ACTH fragment with D-aminooxy acid - (3-hydroxypropionic acid with simultaneous replacement of the C-terminal arginine molecule of the same fragment with L or D-aminooxy-e-aminocaproic acid. 25 The invention thus relates to a method of producing new peptides with ACTH activity having the complete sequence of the ACTH hormone. from the N-terminal amino acid to at least the 17th amino acid but at most up to the 19th amino acid 30 and having any other am amino acids at the site of the individual amino acids of the ACTH sequence and always containing α-amino oxycarboxylic acid in place of the first and last amino acids. The invention also relates to a method for the preparation of acid addition salts as well as amides and benzyl ester. In the above-mentioned substituted peptides individual amino acids of the natural sequence may be replaced by other amino acids provided that they do not cause a marked decrease in ACTH activity. so the second amino acid (tyrosine) is replaced by phenylalanine and / or the third amino acid (serine) is replaced by glycine and or 45 the fourth amino acid (methionine) is replaced by leucine ;, norvalima, norleucine or alpha-amino butyric acid and / hib piaty amino acid (glutamic acid) is replaced by glutamine and / or the fifteenth and sixteenth (lysine) amino acids are replaced by ornithine and or the seventeenth and eighteenth (arginine) amino acids are replaced by lysine or ornithine. The most preferred compounds are due to their adrenocorticotropic saucer activities (D-OSer1.OLiz18) -a 1-18NH2-ACTH and (D-OSer ^, D-OLiz18- 55 -al-18NH2-ACTH, where OSer is a-aminoxy-p-hydroxypropionic acid rose derived from serine with only a single oxygen atom between the α-carbon and the amino group. In the description of the invention, abbreviations for eo are used to denote a-amino oxycarboxylic acids having a structure similar to amino acids, e.g. OLiz means a-aminooxy-E-aminocapronic acid, etc. According to the invention, new peptides with the activity of 05 4 ACTH, as well as their addition goals * with frwasatmi, derivatives and complexes are obtained so that the starting product is a substituted peptide of the general formula Q D-OSer-Tyr-Ser-Met-Glu - (OR) - His - Fen - Arg - Trp - Gly- -Lys (<^ - Pro-Val-Gly- [Lys (Q)] - X ', where n = 3 or 4, X' represents the groups -OLys (Q) -Y, -D-OEys ( Q) - -Y or -OGly-Y, in which Y is an -OR or -NH2 group, where R is a conventional carboxyl group, preferably a third order butyl or a benzyl group, Q is an amine group protected preferably by a benzyloxycarbony group. - low or formyl, where the Q or R groups present in the peptide may be the same or different, protecting groups are removed by one or more steps and, if necessary, by with acid addition salts, amides or a benzyl ester of the complete ACTH sequence, from the N-terminal amino acid to at least the 17th amino acid, up to the 19th amino acid, but containing any other α-amino acids in place of the individual amino acids amino acids of the ACTH sequence, always containing an α-anoxycarboxylic acid at the site of the first and last amino acids and containing protecting groups at least on the terminal oxyamino group and on the amino groups of the side chains and possibly on the terminal carboxyl groups and carboxyl groups of the side chains , the protecting groups are removed by one or more steps and optionally converted into acid addition salts, amides or benzyl ester. According to the invention, the protected peptides used as starting compounds are obtained from the corresponding α-amino oxycarboxylic acids and amino acids partially condensation or the gradual introduction of individual amino acids in the correct sequence. In the condensation processes mentioned, the following methods are used with the use of anhydrides, azides, active esters or condensations with the use of dyklohexylcarbodiimide (DCC). A solid-phase synthesis is also carried out in which the peptide attached to the polymer and its terminal carboxyl group is expanded by combining the C-terminal α-aminooxy carboxyl group with the polymer containing amino acids and the terminal α-amino oxycarboxylic acid. - xyl in the appropriate sequence. "Active groups that do not participate in the reaction are substituted with appropriate protecting groups, easily removable by hydrolysis, acid hydrolysis, hydrazinolysis or reduction. Carboxyl groups are usually protected with ester groups, for example, methyl, tert-butyl, benzene, ¬syl, p-chlorobenzyl or p-nitrobenzyl, or by a method of converting them into amide groups. The amine and oxyamino groups are preferably protected by groups - tosyl, trityl, formyl, trifluoroacetyl, o-nitrosulfenyl, phthalophenyl, benzyloxyloxy - oxycarbonyl, and particularly preferably the tertiary butoxycarbonyl group. The guanidine group of arginine molecules is protected by a nitro group or converted into a protonated farm. The histidine amide group is protected by a benzyl, trityl or dinitrophenyl group. The sideways are protected by converting them to ethers, with the formation of ethers being preferred Tertiary butyl and benzyl groups. Appropriate selection of protecting groups allows obtaining compounds which are selectively reflected, and thus deprotected in one step, or undergo one-stage reflection by generally known methods, for example by hydrolysis, acid hydrolysis, hydrazinization or reductions. According to the method of the invention, the protected α-aminooxycarboxylic acids are condensed with the methyl ester of a tripeptide of the formula Tyr-Ser-Met-OCH3, the resulting substituted methyl ester of the tetrapeptide is converted into hydrazide, and stepwise into azide, and then acylated with this compound. The decapeptide has the formula GlutOBu ^ -His-Phe-Arg-Trp-Gli-Liz (BOC) -Pro-Wal-Gli. The thus obtained substituted tetradecapeptide is condensed with an ester of the protected peptide formed of amino acids in the appropriate sequence, and the resulting substituted peptide ester is converted to hydrazide, then to an azide, and with this, an appropriately protected derivative of the C-terminal acid is acylated. The aminooxy carboxylic acid, for example, a substituted tetradecapeptide is condensed with the dipeptide ester 15-16, the tripeptide ester 15-17 or the tetrapeptide ester 15-18 to obtain an appropriately substituted heptadecapeptide, octadecapeptide or nonadecapeptide. In this synthesis, it is preferable to use a reaction with dicyclohexylcarbodiimide (DCC) or active esters, for example pentachlorophenyl or pentafluorophenyl esters, the esters also having to be separately prepared, but formed directly in the reaction environment by acting on the tetradecaphenolepeptide substituted tetradecaphenepeptide 40 or pieeiofluorophenol and DCC (British Patent No. 1,201,053). Another procedure is that a suitably protected amino-oxycarboxylic acid derivative is acylated with a suitably protected 15-16 dipeptide, 15-17 tripeptide or 15 With the tetrapeptide, the N-terminal hydrogenating group is selectively removed and the resulting compound is condensed with the substituted tetradecapeptide to give an appropriately protected peptide containing amino oxycarboxylic acids at both ends of the molecule. In the last stage of the synthesis, the Tertiary butoxycarbonyl groups, protecting the N-terminal α-aminooxycarboxyl groups and the amino groups of the side chains, the ester 55 groups of the tertiary butyl groups protecting the hydroxyl groups of the side chains, and the C-terminal carboxyl groups, if not left. substituted with amide groups, as well as tertiary-bullyl ether groups protecting the hydroxyl groups of the 60 side chains are simultaneously reflected by the acid hydrolysis method, e.g. by treatment with trifluoroacetic acid. If the original, protected peptide also contains groups that cannot be removed 65 6 by the action of trifluoroacetic acid, e.g. fermyl groups, they are removed before or after reflection of the remaining protecting groups at the appropriate stage of the synthesis.The end product, devoid of protecting groups, is purified by means of anti-current extraction or by column chromatography, e.g. on ion exchange resins with the use of different types of carboxymethylcehilose.Depending on the synthesis method and Under conditions of the reaction, the new compounds are obtained in the form of free bases or salts, these salts being converted into the free bases by generally known methods. The free base compounds are converted into medicinally acceptable acid addition salts by the action of organic or mineral acids, for example hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, lactic acid, tartaric acid, citric acid, higher fatty acids and others. The derivatives of the new compounds obtained according to the invention are esters, amides, N-substituted amides and particularly peptide amides containing an amide group on the Cn-terminal carboxyl group and having free hydroxyl groups in the side chain. The therapeutically acceptable complexes of new peptides obtained by the method according to the invention are compounds resulting from the treatment of a peptide by organic or mineral substances which prolong its action. The organic compounds used are certain types of gelatine, various types of carboxymethyl cellulose, alginates, polyphlorethine phosphates, polymerized amino acids, and other polymers or copolymers. Among the mineral compounds, metal derivatives are used, especially hydroxides and sparingly soluble salts, for example zinc phosphates and pyrophosphates. In order to prolong the action, certain silicates are used, which form with peptides insoluble compounds of hitherto unknown structure. New peptides and their salts, derivatives or complexes are used in medicine in the form of pharmaceutical forms containing biologically active compounds in admixture with organic or mineral carriers, preferably enterally or parenterally. Some formulations use continuously lyophilized active compounds in admixture with mannitol, lactose or starch. The forms of suspensions or emulsions, in addition to the active ingredient and carriers, also contain inert protective and stabilizing agents. The most preferred pharmaceutical forms are the above-mentioned complexes which exhibit a prolonged action of the active compounds. The pharmaceutical preparations contain the active substances in the amounts generally used for adrenocorticotropic hormones, ie 0.1 mg / ml. They are administered 1-7 times daily subcutaneously, intramuscularly or parenterally. For the determination of amino acid and peptide derivatives, the abbreviations recommended by IUPAC-IUB (J. Biol. Chem. 247, 977, 1972) are used in the following examples. The α-α-aminooxycarboxylic acids are designated by OSer, OLiz and unless otherwise specified, these abbreviations refer to the L-configuration with the exception of glycine. The following abbreviations were also used in the examples: PCPOH - pentachlorophenol, PFPOH - pentafluorophenol, DCC - dicyclohexylcarbodiimide, DCU, - dicyclohexylurea, BOC - third order-butoxycarbonyl, Bu * - For III form-butyl, , ONSu - N-oxysuccinimidyl group. Melting points were determined using a Dr Tottoly type apparatus (Buchli, Switzerland). Tests by thin layer chromatography were carried out with the use of silica gel according to Stan with the following solvent mixtures: 1) ethyl acetate: (pyridine: acetic acid: water - 15: 6: 11) = 99: 1 2) ethyl acetate: (pyridine: acetic acid: water -: 6:11) = 8: 2 3) ethyl acetate: (pyridine: acetic acid: water -: 6:11) = 6: 4 20 The method according to the invention is particularly illustrated by the following examples Example I. D-OSer-Tyr-Ser-Met-Glu-His- -Fen-Arg-TRp-Gli-Liz-Pro-Wal-Gli-Liz-Liz-OLiz- -NH2. 0.75 g (0.267 mole) BOC-D-OSer-Tyr-Ser- 25 -Met-Glu - (OBu *) - His-Fen-Arg - Trp - Gli - Liz- (BOC) -Liz (BOC) - Lys (BOC) -OLIz (For) -NH2 is dissolved in a mixture of 6 ml of trifluoroacetic acid, 0.75 ml of water and 0.75 ml of anisole, and after one hour it is diluted with 150 ml of ether. "The precipitate formed is filtered off, washed with ether and dried in vacuo over phosphorus pentoxide and potassium hydroxide. The obtained 0.7 g (86%) of the substituted form-looktapeptide trifluoroacetate is dissolved in 10 ml of water and 0 is added to the solution. 25 ml of mercaptomethanol, then the pH of the reaction mixture is adjusted to 5.0 with 1N sodium hydroxide solution. Then 11.5 ml of a 2 molar solution of hydrazine acetate are added and the mixture is heated in a water bath at 95 ° C for 2 hours. After this time, the solution is concentrated under reduced pressure and then applied to a column of Whatman CM-52 ion exchange resin. The substances are eluted with a buffer solution made of 0.2 molar ammonium acetate solution of pH - 6.7 and 0.5 molar ammonium acetate solution with pH - 6.7 The collected appropriate fractions are subjected to the freeze-drying process and 0.275 g (38%) of the appropriately substituted octadexpeptide amide is obtained. the substituted octadexpeptide amide used as a starting compound was prepared as follows: Step 1. L-α-t-butoxycarbonylaminoxy-55-formylamimocaproic acid (BOC-OLiz / For). 23.2 g (60 mmol) of Ne-formyl-D-lysine and 43.2 g (423 mmol) of sodium bromide are dissolved in 248 ml of 2.5 N sulfuric acid, and the solution is cooled to 0 ° C and T3.3 g (192 mmol) of sodium nitrite are added in small portions within 30 minutes while stirring. The reaction mixture is stirred for an hour at 0 ° C. and for a further hour at room temperature. After this time, it was extracted three times with 200 ml. Portions of ether, and the combined ether extracts were dried and evaporated to dryness under reduced pressure. The obtained crude precipitate of D-bromo-e-formylaminocaproic acid, 18.4 g, is dissolved in 190 ml of ethanol, and 8.4 g (150 mmol) of potassium hydroxide and 10 g (76 mmol) are added to the solution. butoxycarbonylhydroxylamine. The reaction mixture was refluxed for 2 hours and then allowed to stand at room temperature over the top day. After this time, the solvent is evaporated, the residue is dissolved in 80 ml of water, the pH is adjusted to 3 with 10% hydrochloric acid, the solution is saturated with sodium chloride, and then the mixture is extracted three times with 150 ml portions of ether and 150 ml of ethyl acetate at a time. The combined organic extracts are dried and evaporated to dryness. The crystalline residue of 16 g is recrystallized from 25 ml of ethyl acetate to give 10.4 g (27%) of Lat-butoxycarbonyl-aminooxy-E-formylaminocaproic acid, mp 114-116 ° C, RF (2) = 0.31 and (a) D = -52.5 ° (c = 1 in methanol). Elemental analysis: for the formula C12H22N2O6 (290.32) Calculated: 0 ^ 19.6%; H - 7.65% Found: C - 49.9%; H - 7.6%. Step 2. L-a-t-butoxycarbonyl-aminooxy-e-formylaminocaproic acid amide (BOC-OLiz (For) -NH2). 5.0 g (17.2 mmol) of BOC-OLiz (For) are dissolved in 107 ml of dry tetrahydrofuran and 1.9 ml (17.2 mmol) of N-methylmorpholine are added to the solution and the reaction mixture is cooled to temperature of -10 ° C, and 2.25 ml (17.2 mmol) of isobutyl chloroformate are added dropwise with stirring. After stirring at this temperature for 10 minutes, it is cooled to -20 ° C, and 10.7 ml (142 mmol) of concentrated ammonium hydroxide are carefully added dropwise to the solution and then stirred for one hour in room temperature. The tetrahydrofuran is then evaporated off and the partially crystalline residue is diluted with 20 ml of a 900: 54: 16: 30 mixture of ethyl acetate, pyridine, acetic acid and water and transferred to a column filled with 250 ml of silica gel. The reaction is carried out with the solvent mixture alone and the collected fractions containing the product are evaporated to give 4.3 g (86%) of slightly yellow BOC-OLiz (For) -NH2 as amorphous RF (2) = 0.54 Stage. 3. Third order-butoxycarbonyl-D-α-aminooxy-P-benzoxypropionic acid. A mixture of 48.4 g (0.364 mol) of third tert-butoxycarbonylhydroxylamine, 40.7 g (0.728 mol) of potassium hydroxide and 94.0 g (0.364 mol) of DL-α-bromo-β-benzoxypropioic acid. in 360 ml of water is stirred at room temperature for 3 hours, then the solution is acidified to pH -3 and extracted three times with 720 ml of ether each time. The combined ether extracts are washed with 720 ml of water, dried and mixed with 78 ml (0.40 mol) of dicyclohexylcarbodiimide. 43.61 g of the dicyclohe-9 xylamino salt of tertiary-butoxycarbony-1q-DL-α - ainoxy-O-benzoxypropionic acid, mp 144-146 ° C, are obtained, suspended in 500 ml of ether and shaken five times with 100 ml by portions of 0.2 N sulfuric acid. The separated ether solution is dried and evaporated to dryness. The residue obtained is dissolved in 700 ml of ethanol, 6.56 g (48.5 mmol) of (+) - ammethamine base are added and the solution is allowed to cool to a smooth temperature. day, then the separated crystals are drained. The (+) - amphetamine salt of n-butoxycarbonyl-D-aminooxy-β-benzoxypropionic acid is recrystallized from ethanol to give 13.27 g of pure product, mp 188-191 ° C, ) D26 = + 54 ° (c = 0.5 in molar tamol). After decomposing the salt, the usual method yielded 7.9 g (14%) of tert-butoxycarbonyl-D-aminooxy-p-beinzoxypropionic acid, mp 96-98 ° C, (a) D80 = + 38 ° (e = 1 of vetanol). Step 4. Tertiary-butoxycarbonyl-D-α-aminooxy- β-hydroxypropionic acid (BOC-D-OSer). 5.5 g <17.7 mmol) of D-tert-butoxycarbonyl-aminooxy-P-benzoxypropionic acid are dissolved in 110 ml of methanol and the resulting solution is hydrogenated against 2.75 g of 10% palladium on carbon as a catalyst for 3 hours, the catalyst is then filtered off and the solvent is evaporated, and the oily residue is poured over with petroleum ether and left for several hours. The separated crystalline material is filtered off and dried. There are obtained 3.65 g (96%) of D-tert-butoxycarbonyl-aminooxy-β-hydroxypropionic acid with a melting point of 94-95 ° C and RF (1) = 0.27. Step 5. BOC-D -OSer-Tyr-Ser-Met-N2H3. 4.4 g 35 <20 mmol) BOC-D-OSer and 9.0 g (20 mmol) Tyr-Ser-Met-OCH3 are dissolved in 30 ml dimethylformamide, the solution is cooled to 0 ° C, 2.22 ml (20 mmol) of N-methylmorpholine and 3.8 g (18.4 mmol) of DCC are added, and then stirred for one hour at 0 ° C. and left at room temperature until the next day. The separated DCU is filtered off, the filtrate is evaporated to dryness and the residue is dissolved in 400 ml of ethyl acetate. The acetate solution is washed twice with 200 ml portions of 1N hydrochloric acid and twice with 200 ml portions of 8% aqueous sodium bicarbonate solution, then dried and evaporated to dryness. 8.1 ester of the protected, substituted tetra-peptide is obtained, which is dissolved in 85 ml of methanol, 2.2 ml (46 mmol) of hydrazine hydrate are added and the mixture is allowed to stand at room temperature until the next day. After this time, the solvent is evaporated, the residue is triturated with ethyl acetate, filtered, washed with ethyl acetate and ether, and then dried in a vacuum desiccator with concentrated sulfuric acid. There is obtained 7.21 g <63%) of the protected-substituted tetrapeptide hydrazide with the melting point after recrystallization from water 174-175 ° C and RF (1) = 0.30. Elemental analysis: for the formula C25H4oN6010S <616, 69) Calculated: C, 48.7%; H - 6.55%; N - 13.6%. Found: C - 48.3%; H - 6.6%; N - 13.8% 65 Step 6. BOC-D-OSer / tyr-Ser-Met-Glu (OBut) -His- -Fen-Arg ^ Trp-Gli-Liz (BOC) - Pro - Wal - Gli. HCl. 1.25 g (2.13 mmol) of BOC-D-OSer-Tyr-Ser-Met -N2H3 are dissolved in 15 ml of dimethylformamide, the solution is cooled to - 20 ° C, then added dropwise with stirring. 2.08 ml (8.32 mmol) of a 4N solution of hydrochloric acid in ethyl acetate and 0.32 ml (2.7 mmol) of tert-butyl nitrite. The reaction mixture is stirred for 5 minutes at -10 ° C, cooled to -20 ° C, and then a solution of 2.78 g of Glu (OBut) -His-Phe-Arg-Trp-Gli-Liz BOC- is added. Pro-Wal-Gli and 1.4 ml (8.13 mmol) of diisopropylethylamine in 23 ml of dimethylformamide, then stirred for an hour at -5 to 0 ° C and then left at 0 ° C to 0 ° C. next day. After evaporation to dryness, the residue is triturated with 8% aqueous sodium hydrogen carbonate solution, the precipitate is filtered off, washed with water and dried. 3.49 g (94%) of the protected substituted tetrodecapeptide is obtained, which is suspended in 24 ml of methanol and the resulting suspension is added to a mixture of 4.75 ml of concentrated hydrochloric acid and 4.75 ml of pyridine. The resulting solution is diluted with 240 ml of water, the precipitate formed is filtered off, washed with water and dried. This gives 2.95 g (73%) of the protected, substituted tetradecapeptide hydrochloride, mp 200-202 ° C and Rf (2) = 0.25. Step 7. Lys oxalate (BOC) -Liz (BOC) -OCHs. 11.0 g (17.7 mmol) of Z-Liz (BOC) -Liz (BOC) -OCH3. (K. Hofman et al. J. Am. Chem. Soc. 86, 4991, 1964) are dissolved in 200 ml methanol, 2.0 g (22.2 mmol) of anhydrous oxalic acid are added and the reaction mixture is hydrogenated in the presence of 2.0 g of 10% strength palladium on carbon as a catalyst. After the reaction has ended, the catalyst is filtered off, the filtrate is evaporated and the residue obtained is triturated with ether. 9.1 g (89%) of the dipeptide ester oxalate are obtained, which, after recrystallization from a mixture of isopropanol and diisopropyl ether, has a melting point of 65-67 ° C, RF (2) = 0.34 and (a) D = + 3.79 ° (c = 1 in methanol). Elemental analysis: for the formula C25H48N4On (578.65). Calculated: C, 51.9%; H - 8.9%; N - 9.7% Found: C - 51.5%; H - 7.9%; N - 9.9% Step 8. Z - Liz (BOC) - Liz (BOC) - OCH3. 8.1 g (14.0 mmoles) of Lys (BOC) -Liz (BOC) -OCH3 in oxalate form are suspended in 60 ml of ethyl acetate, 3.1 ml (28.0 mmoles) of N-methyl is added to the suspension. morpholine and 6.7 g (14.0 mmol) of Z-Lys (BOC) -ONSu and then stirred at room temperature for 2 hours. The reaction mixture is then diluted with 200 ml of ethyl acetate and 40 ml of water, the aqueous phase is separated and the organic solution is washed twice with 40 ml portions of In hydrochloric acid and twice with 40 ml portions of 8% aqueous sodium hydrogen carbonate solution and then dried. with sodium sulphate and evaporated, the residue obtained is triturated with diisopropyl ether. The obtained crude product is recrystallized from a mixture of isopropanol and diisopropyl ether to give 8.0 g (9.11 (67%) of the methyl ester of the protected tripeptide, mp 117 118 ° C, RF (1) = 0.58 and (<*) D = —17.47 ° (c = 1 in methanol). Elemental analysis: for the formula C42H7 # N6012 (851.03) 5 Calculated: C —59.3%; H —8.3% Determined: C - 59.7%; H - 8.2% Step 9. BOC-D-OSer-Tyr-Ser-Met-Glu (OBut) - -His-Fen-Arg-Trp-Gli-Liz (BOC) -Pro - Wal - Gli- -Liz (BOC) - Liz (BOC) - Liz (BOC) - OCH3. HC1 1.25 g 10 Z-Liz (BOC) -Liz (BOC) -Liz (BOC) - OCH3 is dissolved in 25 ml of methanol and the reaction mixture are hydrogenated in the presence of 0.2 g of 10% palladium on carbon as the catalyst.After the reaction is complete, the catalyst is filtered off, the solvent is evaporated from the filtrate, and the remaining ester of the tripeptide and 2.42 g ( 1.24mmol) BOC-D-Ser-Tyr-Ser-Met-Glu (OBu *) - His-Fen-Arg-Trp-Gli-Liz (BOC) - Pro-Wal- -Gli. HCl is dissolved in 12 ml of dimethylformamide, then 0.97 is added to the solution g of 20 (3.64 mmol) PCPOH and 0.255 g (1.24 mmol) of DCC and leave the reaction mixture at room temperature for two days. After this time, the separated DCU precipitate is filtered off, the filtrate concentrates under reduced pressure and the product is isolated by adding ether. The precipitate is filtered off and dried. 3.4 g of a crude product with a decomposition of 177 ° C. are obtained, which are purified by precipitation from methanol with ether. 2.6 g (80%) of the methyl ester hydrochloride of the protected, substituted heptadecapeptide are obtained. with a melting point of 191 ° C with decomposition and RF (3) = 0.54. Step 10. BOC-D-OSer-Tyr-Ser-Met-Glu (ONut) - -His-Fen-ARG-Trp-Gli-Liz (BOC) - Pro - Wal-Gli- 35 -Liz (BOC) - Liz (BOC) -Liz (BOC) -N2H3. 2.5 g (0.93 mmol) BOC-D-OSer-Tyr-Ser-Met-Glu (OBut) -His- -Fen-Arg-Trp-Gli- Liz (BOC) - Pro - Wal- Gli - Liz (BOC) -Liz (BOC) -Liz in 75 ml of hot methanol, the solution is cooled to room temperature and 0.91 ml (18.7 mmole) of hydrazine hydrate is added, and the reaction mixture is allowed to stand at room temperature. room for 2 days. After this time, the methanol was distilled off and the residue was triturated with ether. This gives 2.15 g of crude product, m.p. 199 ° C, with a decomposition which is purified by precipitation with dimethylformamide. 2.0 g (81%) of a protected hydrazide, a substituted heptadecapeptide with a melting point of 205 ° C is obtained. C decomposed and RF (3) = 0.49 Step 11. BOC-D-OSer-Tyr-Ser-Met-Glu (OBut) - -His-Fen-Arg-Trp-Gli-Liz (BOC) - Pro -Wal-Gli-Liz (BOC) Liz- (BOC) -Liz (BOC) -OLiz (For) -NH2. a) 0.57 g (1.97 mmol) of BOC-OLiz (For) NH2 is dissolved in 7.5 ml of trifluoroacetic acid, and after a standing time of 0.5 hours, the solution is evaporated under reduced pressure and the resulting residue is The solid is dissolved in 4 ml of dimethylformamide and the solution is neutralized with diisopropylethylamine. 60 b) 1.0 g (0.377 mmol) BOC-D-OSer-Tyr-Ser- -Met-Glu (OBut) - His-Fen-Arg-Trp-Gli-Liz (BOC) - -Pro-Wal-Gli - Lys (BOC) - Lys (BOC) - Lys (BOC) - -N2H3 is dissolved in 5 ml of dimethylformamide with gentle heating, then the solution of M12 is cooled to -20 ° C and 0 , 54 ml (1.52 µmol), 2.8 N hydrochloric acid in ethyl acetate, and 0.05 ml (0.42 mmol) tert-butyl nitrite. After stirring for 5 minutes at -10 ° C, the solution is cooled to -20 ° C, and an inert solution of OLiz (For) -NH2 trifluoroacetate prepared above is added (together with 0.2 ml (1.17 mmol)). Diisopropylethylamine After stirring for one hour at -5 to 0 ° C., the reaction mixture is left at 0 ° C. overnight, then 50% of the solvent is distilled off and the resulting concentrated solution is diluted with 20 ml of water. A substance which solidifies after some time is filtered off and dried after it is ground in an aqueous slurry. 0.80 g (75.5%) of a protected substituted octadecapeptide amide, mp 140-145 ° C, are obtained. C and RF (3) = 0.47. Example II D-OSer-Tyr-Ser-Met-Glu-His- -Fen-Arg-TRp-Gli-Liz-Pro-Wal-Gli-Liz-Liz-Liz -D- -OLiz-NH2. 0.75 g (0.267 mmol) BOC-D-OSer-T ^ r- -Ser-Met-Glu (OBut) -His-Phe-Arg - Trp - Gli-Liz (BOC) -Pro-Wal-Gli-Liz (BOC) - Liz (BOC) -Liz (BOC) -. -D-OLiz (For) - NH2 dissolve It is combined with a mixture of 6 ml of trifluoroacetic acid, 0.75 ml of water and 0.75 ml of anisole, and after one hour's standing the solution is diluted with 150 ml of ether. The separated precipitate is filtered off, washed with ether and dried in a vacuum desiccator under reduced pressure over phosphorus pentoxide and potassium hydroxide. The thus obtained 0.65 g (80%) of the formyloctadexpeptides substituted trifluoroacetate is dissolved in ml of water and 0.25 ml of mercaptoethanol is added. The solution is neutralized with 1 N sodium hydroxide solution to pH -5 and then added 10.6 ml of a 2N molar solution of hydrazine acetate and the reaction mixture is heated for 2 hours in a water bath at 95 ° C., then concentrated under reduced pressure and the concentrated solution is loaded onto an exchanger column. Whatman CM-52 Ion. The substances are eluted with a buffer solution made of 0.2 molar (pH-6.7) and 0.5 molar (pH-6.7) ammonium acetate, and the collected appropriate fractions are pooled and lyophilized. tions. 0.28 g (41%) of the substituted octadecapeptide amide is obtained. The protected, substituted octadecapeptide amide used as a starting product is obtained as follows: Step 1. Da-III r-butoxycarbonylami-noxy-e-formylaminocaproic acid ( BOC) - D - OLiz (For)). This product is obtained from Ne-formyl-L-Lysine by the method of example I, step 1, the compound has a melting point of 114-116 ° C, RF (2) = 0.31 and (a) D = +58.6 ° (c = 1 in methanol). Elemental analysis: for the formula C12H22N2O3 (290.32) Calculated: C - 49.6%; H, 7.65%. Found C, 50.0%; H - 7.6%. Step 2. D-α-III r-butoxycarbonylaminooxy-e-formylaminocaproic acid amide (BOC-D-OLiz (For) -NH2). This product is obtained from 5.0 g of 91 * 87 13 (17.2 mmol) BOC-D-OLiz (For) according to the procedure of example I, step 2. 4.4 g (88%) of the compound with Rf (2 ) = 0.54 Step 3 BOC-D-OSer-Tyr- Ser - Met - Glu (OBut) - -His-Fen-Arg-TRp-Gli-Liz (BOC) - Pro - Wal - Gli- 5 - Lys This product is obtained from 0.57 g (1.97 mmol) of BOC-D-OLiz (For) -NH2 by the method of Example I, step 11. 0.90 g (85%) of amide is obtained. protected substituted octadecapeptide mp 140-145 ° C and RF (3) = 0.47. Example III. H-D-OSer-Tyr-Ser-Met-Glu- -His-Fen-Srg-Trp-Gly-Lys-Pro - Val-Gly-Lys / Lys- -Lys-OLys-NH2. 1.2 g (0.417 mmol) BOC-D-OSer- -Tyr-Ser-Met-GlutOBu ^ His-Fen - Srg - Trp - Gly- 15 -Lys (BOC) -Pro - Val - Gly - Lys (BOC) - Lys (BOC) -Lys (BOC) -OLys (BOC) -NH 2 is dissolved in a mixture of 9.6 ml of trifluoroacetic acid, 1.2 ml of water and 1.2 ml of anisel. The solution is allowed to stand for 1 hour, then diluted with 240 ml of ether. The resulting precipitate is filtered off, washed with ether and dried in a vacuum over phosphorus pentoxide and potassium hydroxide. 1.10 g (84%) of octadecapeptamide trifluoroacetate substituted with amino oxycarboxylic acid are obtained. The crude trifluoroacetate is dissolved in ml of water and the trifluoroacetate ions are exchanged using the Dowex Brand 1X8 Brand Acetate Cyclic Ion Exchanger (Rohm and Haas Company). The solution thus obtained is then introduced into a column filled with an ion exchanger of the Khatman CM-52 brand (4.5 × 65 cm). Elute with a linear gradient buffer prepared from 2 1 0.2 n (pH = 6.7) and 2 1 0.5 m (pH = 6.7) ammonium acetate buffer at 80 ml / hr, collecting the contents product fractions 98-115 and lyophilizes them. 0.49 g (41%) of octadecapeptidamide substituted with amino oxycarboxylic acid is obtained. Peptide content: 78% (HV spectrum) met-sulfoxide content: 2.3%; ratio Tyr / Trp 1: 10.40 Amino acid analysis: Lys 3.5 (4), His 0.9 (1), Srg 1.0 (1), Ser 0.85 (1), Glu 1.0 (1) ), Pro 1.0 (1), Gly 1.9 (2), Val 0.9 (1), Met 0.7 (1), Tyr 0.9 (1), Fen 1.0 (1), Rt5 = 0.18. The aminooxycarboxylic acid protecting groups substituted octadeca-peptidamides used as starting products are prepared as follows: Stage 1: Dicyclohexylammonium salt of La - IH-order / - butyloxycarbonyl-aminoxy-E-50-benzyloxy-carbonyl- Amino-caproic acid (BOC-OLys (Z) -OH DCHA) is obtained as described in step 1 from Ne-benzyloxycarbonyl-D-lysine. The obtained oily La-III-tert-butyloxycarbonyl-aminoxy-e-benzyloxycarbonyl-amino-caproic acid is dissolved in ether and reacted with dicyclohexylamime in order to obtain the dicyclohexylamine salt. Melting point: 156-161 ° C. (after recrystallization from ethanol-water mixture), Rt! = 60 = 0.1 [a] u = -34.6 ° (c = 1, ethanol). Stage 2. Pre-butyloxycarbonylaminooxy-e-benzyloxycarbonylamino-kaiproic acid La-III amide ( BOC-OLys (Z) -NH2). Dicyclohexylammonium salt of La-III 65 tert-butyloxycarbonyl-aminoxy-β-benzyl-xycarbonyl-amino-caproic acid is shaken with ether and 1N sulfuric acid and the ether solution is evaporated. The acid obtained is converted to the amide as described in step 2 of Example I. After evaporation of the reaction mixture, the crude product is purified, instead of by column chromatography, by dissolving in ethyl acetate and washing with water, 1 N sulfuric acid and finally 8% solution of acidic sodium carbonate. The ethyl acetate phase is then evaporated. The acid amide La-III of the pre-butyloxycarbonyl-aminoxy-e-benzylxycarbonylamino-caproic acid is obtained in the form of a thick oil. Melting point: 93-95 ° C (after recrystallization from methanol-water mixture) Rt1 = 0.42 [a] D = -27.5 ° (c = 1, methanol). Stage 3. La-III-pre-butyloxy-carbonyl-aminoxy-e-amino-caproic acid amidooxalate (BOC- OLys-NH2 oxalate). 12.0 g (30.4 mmoles) of BOC-OLys (Z) -NH2 are dissolved in 240 ml of methanol, mixed with 3.0 g (33.3 mmoles) of anhydrous oxalic acid and reduced in the presence of 2.0 g of 10% palladium on activated carbon as a catalyst. After the reaction is complete, the catalyst is filtered off, the filtrate is evaporated and the residue is triturated with ether. After recrystallization of the crude oxalate (9.3 g) from a mixture of 45 ml of methanol and 95 ml of ether, 8.7 g (81.5%) of the tert-butyloxycarbonyl-aminoxy La-III amide oxalate are obtained. - Amino-capronium. Melting point: 153-155 ° C, Rt = 0.30 [a] D = -38.1 ° (c = 1, methanol). Step 4: La-Aminoxy-E- amide III of the pre-butyloxycarbonylamino-caproic (H-OLys (BOC) -NH2). 8.0 g (22.8 mmol) of BOC-OLys-NH oxalate are dissolved in 40 ml of trifluoroacetic acid and the solution after 1 hour is evaporated under reduced pressure. The oily residue is washed with ether, then 24 ml of dioxane and 2N sodium hydroxide are added to the effect that a weak base is formed (pH 8-9). After addition of 6.6 g (45.6 mmol) of tert-butyloxycarbonyl azide III and 2.3 g (57.5 mmol) of magnesium oxide, the reaction was stirred for 16 hours at room temperature, then filtered and the filtrate was evaporated. ¬ rows. The residue is dissolved in 50 ml of water and shaken three times with 70 ml of ethyl acetate. After evaporation of the ethyl acetate phase, the residue is triturated with ether, filtered, and the crude product (6.35 g) is recrystallized from ethyl acetate. 3.6 g (60.5%) of the La-amino-amide are obtained. e - 3rd order - butyloxycarbonyl-amino-kaepone. Melting point: 118-120 ° C Rt4 = 0.30 [a] D = -39.5 ° (c = 1, methanol). Step 5: Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -N2Ha, 15 g (29.5 mmol) Z-Lys (BOC) -Lys (BOC) -Lys (BOC) -OCH3 is dissolved in 500 ml of methanol and hydrogenated in the presence of 5.0 g of a 10% palladium catalyst on activated carbon. After the reaction is complete, the catalyst is filtered off, the filtrate is evaporated and the residue is dissolved in 180 ml of dichloromethane. 5.72 ml (41.0 mmol) of triethylamine and 10.26 g (36.8 mmol) of trityl chloride are added to the solution. The reaction mixture is allowed to stand at room temperature for 16 hours, then the solution is filtered off and the residue is dissolved in 300 ml of ethyl acetate. The solution is shaken with 50 ml of water, three times with 50 ml of 5% citric acid solution and three times with 50 ml of 8% sodium carbonate acid solution, then dried and finally evaporated. The residue powders under the oil pan. After filtering and drying, 28.0 g of crude Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -OCH3 are obtained. The crude material is dissolved without further purification in 170 ml of methanol. 7.25 ml (150 mmol) of hydrazine hydrate are added and the reaction mixture is allowed to stand at room temperature. After three days, the methanol is filtered off and the residue is triturated with ether. The crude product (17.65 g) is recrystallized from a mixture of 35 ml of methanol and 350 ml of ether. 13.85 g (49%) of the protected tetrapeptide-hydrazide are obtained. Melting point: 197-199 ° C Rt1 = 0.13 [a] D = -7.1 (c = 1, methanol). Step 6: Tri-Lys ( BOC) -L, ys- (BOC) -Lys (BOC) -OLys (BOC) -NH2 4.1 g (4.27 mmol) Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -N2H3 it is dissolved in 14 ml of dimethylformamide. The solution is cooled to -20 ° C. and, while stirring, first 6.5 ml (17.0 mmol) of a 2.611 ml of hydrochloric acid solution in ethyl acetate, and then 0.65 ml (5.45 mmol) of 3 before butyl nitrite are added dropwise. . The reaction mixture is stirred for 5 minutes at -10 ° C, then cooled to -20 ° C and this solution is added 1.42 g (5.45 mmol) of H-OLys- (BOC) -NH 2 and 2.2 ml. (12.8 mmol) diisopropylethylamine in 7 ml dimethylformamide. The reaction mixture is stirred for an hour at -5 ° -0 ° C, then left to stand at 0 ° C for the next day. The solution is then evaporated, the residue is dissolved in 140 ml of ethyl acetate and the solution is shaken with water. 40 ml of water, three times with 40 ml of a 5% strength citric acid solution, then twice with 40 ml of an 8% strength sodium carbonate solution. After drying, the ethyl acetate is distilled off and the solid residue is triturated with ether. After filtration and drying, 2.75 g (54%) of the protected, aminoxycarboxylic acid-substituted tetrapeptidamide is obtained. Melting point 137-140 ° C Rt1 = 0.26 [o] D = -24.4 ° (c = 1, methanol) Step 7. H-Lys (BOC) -Lys (BOC) -Lys (BOC) -OLys (BOC) -NH2HCl 5.0 g (4.22 mmol) Tri-Lys (BOC) - Lys ( BOC) -Lys (BOC) -OLys (BOC) -NH2 is dissolved in 50 ml of methanol and treated with a solution of pyridine hydrochloride obtained from 1.7 ml of pyridine and 1.7 ml of concentrated hydrochloric acid. The mixture is allowed to stand for 20 hours at room temperature, then the methanol is distilled off and the remaining dense mass is kneaded with 150 ml of water, dried in vacuo after pouring off the water and pulverizing under ether. After filtering and drying, 3.7 g of 16 (82%) H-Lys (BOC) -Lys (BOC) -Lys (BOC) OLys (BOC) -NH2 are obtained. Melting point 120—123 ° C Rt3 = 0, 72 [a] D = + 17 ° C (c = 1, methanol). Step 8: BOC - D - OSer-Tyr-Ser-Met-GlufOBu *) - -His-Fen-Srg-Trp-Gly-Lys ( BOC) - Pro - Val-Gly- -Lys (BOC) -Lys (BOC) -Lys (BOC) -OLys (BOC) - NH2. 0.9995 g (0.5 mmol) of BOC-D-OSer-Tyr-Ser-Met- GlutOBu ^ -His-Fen-Srg-Trp-Gly-Lys (BOC) -Pro- -Val-Gly-OH. HCl, 0.491 g (0.5 mmol) H-Lys (BOC) - -Lys (BOC) - Lys (BOC) - OLys (BOC) - NH2. 1a and 0.800 g (3.0 mmol) of PCPOM are dissolved in ml of dimethylformamide and first 0.055 ml (0.5 mmol) of N-methylmorpholine and then 0.210 g (1.0 mmol) of DCC are added. The reaction mixture is allowed to stand for one day at room temperature, then the DCV is filtered off and the filtrate is diluted with 100 ml of ether. The separated product is filtered off, washed with ether and dried. 1.30 g (90%) of octadecapeptidamide-protected with amino-carboxylic acid Rt6 = 0.50 is obtained. Example IV. D-OSer-Tyr-Ser-Met-Glu-His-Fen- -SRg-Trp-Gly-Lys-Pro-Val-Gly-Lys- Lys - Lys- -D-OLys-NH2. 1.2 g (0.417 mmol) BOC-D-OSer-Tyr-Ser-Met- -Glu (OBut) -His-Fen-Srg-Trp-Gly - Lys (BOC) -Pro- -Val - Gly - Lys ( BOC) - Lys (BOC) - Lys (BOC) - D -OLys (BOC) -NH2 is dissolved in a mixture of 9.6 ml of trifluoroacetic acid, 1.2 ml of water and 1.2 ml of anisole. The solution is allowed to stand for an hour, then it is diluted with 240 ml of ether. The resulting precipitate is filtered off, washed with ether and dried in a vacuum over phosphorus pentoxide and potassium hydroxide. 1.05 g (80%) of substituted octadecapeptidamide trifluoroacetate are obtained. The crude trifluoroacetate is dissolved in 20 ml of water and the trifluoroacetate ions are exchanged for acetate ions using a Dowex 1X8 cyclic acetate ion exchanger (Rohm and Haas). The solution thus obtained was then applied to a column filled with Whatman CM-52 ion exchanger (4.5 × 65 cm). It was eluted with a linear gradient buffer obtained from 2 1 0.2 n (pH = 6.7) and 2 1 0.5 m (pH = 6.7) of ammonium acetate buffer at a rate of 80 ml / hour collecting the product fractions 103-112 and lyophilizing them. 0.42 g (37%) of octadecarboxylic acid-substituted octadecarboxylic acid is obtained. Peptide content: 82% (in the U.V spectrum); Met-sulfoxide content 1.8%: Tyr / Trp ratio 1.07. Amino acid analysis: Lys 3.4 (4), His 1.0 (1), Arg 0.9 (1), Ser 0.8 (1) , Glu 0.9 (1), Pro 1.0 (1), Gly 2.05 (2), Val 0.9 (1), Met 0.75 (1), Tyr 0.9 (1), Fen 1.0 (1), Rt5 = 0.18. Used as starting product, octadecapeptidamide substituted with amino oxycarboxylic acid is obtained as follows: Stage 1: D- -a-III dicyclohexylammonium salt of tert-butyloxycarbonyl-aminoxy-e- -benzyloxycarbonyl-amino-caproic acid (BOC-D-OLys (Z) -OH DEHA) is prepared as described in step 1 of example I from NE-benzyloxy-carbonyl-L-lysine. The obtained oily D-α-III tert-butyloxy-carbonyl-aminooxy-e-benzyloxycarbonylamino-caproic acid is dissolved in ether and reacted with dicyclohexylamine to give the dicyclohexylamine salt. ~~ 5 Melting point 159—162 ° C (after recrystallization from ethanol-water mixture) Rt1 = 0.15 [a] D = + 35.4 ° (c = 1, ethanol). Stage 2: acid amide Da-III of the order-butyloxycarbonyl-aminoxy-e-benzyloxycarbonyl-ami- * no-caproic acid (BOC-D-OLys (Z) -NH2 is obtained as described in step 2 of example III from the dicyclohexylamine salt of Da-III in the order - butyloxycarbonyl / aminoxy - e - benzyloxycarbonylamino-caproic Rt1 = 0.42 Melting point 95-97 ° C [a] D = + 28.9 ° (c = 1, methanol) Step 3: The amino acid oxalate Da-III of the tert-butyloxycarbonyl-aminoxy-e-amino-caproonic acid (BOC-D-OLys-NH2 oxalate is prepared as described in step 3 of example III from the Da-III tert-butyloxycarbonyl-aminoxy amide) - e - benzyloxycarbonyl-amino-capronic. Melting point: 155-157 ° C Rt3 = 0.30 [oi] d = + 39.0 ° (c = 1, methanol). Step 4: Da Aminoxy-e acid amide -III lower-butyloxycarbonylamino-caproic acid (ND-OLys (BOC) -NH2) is prepared as the method described in step 4 and in Example III from the oxalate of the amino acid Da-III of the pre-butyloxycarbonyl-aminoxy-e-aminocaproic acid. Melting point 118-120 ° C Rt4 = 0.28 [a] D = + 40.4 ° (c = 1, methanol) Step 5: Tri-Lys (BOC) - Lys (BOC) - Lys (BOC) -D- -OLys- (BOC) -NH2 is obtained as described in step 6 of example III from 8.7 g (1.0 mmol) Tri Lys (BOC) -Lys (BOC) -Lys (EOC) -N2H3 and 3.0 g (11.5 mmol) HD-OLys (BOC) -NH2. After evaporation of the 4Q ethyl acetate phase, the remaining crude product was added together with 20 ml of a mixture of ethyl acetate: (pyridine: acetic acid: water = 20: 6: 11 = 99: 1 on a silica gel column and chromatographed. fractions of 15 ml of 45 and analyzed by thin-layer chromatography. The fractions 85-192 containing the pure product are collected and the solvent is distilled off. 3.70 g (34.5%) of the substituted tetrapeptide protected with amimoxycarbonyl are obtained. - 50 amide Melting point 132-136 ° CR * t = 0.23 Stage 6: H - Lys (BOC) - Lys (BOC) - Lys (BOC) - D- -OLys (BOC) -NH2. HCl 2 g (2.10 mmoles) Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) D-OLys (BOC) -NH2 is dissolved in 20 ml of 80% acetic acid. at room temperature for one minute, then evaporated and the residue was triturated with ether to give 1.68 g of a substituted aminoxycarboxylic acid amido acetate tetrapeptide (mp 94-95 ° C) which was dissolved in 17 ml of methanol, then the pyridine hydrochloride solution obtained by adding 1.35 ml of pyridine and 1.35 ml of concentrated hydrochloric acid is added and the methanol is distilled off. 65 18 The residue is rubbed with 50 ml of water. After filtration, washing with water and drying, 1.2 g (72%) of H-Lys (BOC) -Lys (BOC) -Lys (BOC) -D -OLys (BOC) -NH 2 are obtained. HCl. Melting point 122-125 ° C Rt3 = 0.70 Step 7: BOC-D-OSer-Tyr-Ser-Met-Glu (OBut) - -His-Phe-Arg-Trp-Gly - Lys (BOC) - Pro-Val-Gly- -Lys (BOC) -Lys (BOC) - Lys (BOC) - D - OLys (BOC) - -NH2. 0.995 g (0.5 mmol) BOC - D-OSer-Tyr-Ser-Met -Glu (OBut) -His-Phe-Arg-Trp-Gly - Lys (BOC) -Pro- -Val-Gly-OH. HCl, 0.491 g (0.5 mmol) of H-Lys (BOC) -Lys (BOC) Lys (BOC) -D-OLys (BOC) -NH2 HCl and 0.800 g (3.0 mmol) of PCPOH are dissolved and the yields first 0.055 ml (0.5 mmol (N-methyl morpholine then 0.212 g (1.0 mmol) DCC. The reaction mixture is allowed to stand at room temperature for 1 day, then filtered off with DCV and the filtrate is diluted with 100 ml of ether. The separated product is filtered off, washed with ether and dried. 1.40 g (97%) of the protected, substituted amino oxycarboxylic acid octacipeptidamide is obtained, mp 170-178 ° C Rt6 = 0.52. Example V. H - D -OSer-Tyr-Ser-Met-Glu-His- -Fen-Arg-Trp '(- Gly-Lys-Pro-Val- Gly - Lys) Lys- -Lys-Lys-OGly-NH2. 0.400 g (0.134 mmol ) BOC-D-OSer-Tyr-Ser-Met- -Glu (OBut) -His-Fen-Arg-Trp-Gly - Lys (BOC) -Pro- -Val-Gly-Lys (BOC) -Lys (BOC) - Lysi (BOC) - OGly-NH2. HCl is dissolved in a mixture of 3.2 ml of trifluoroacetic acid and 0.4 ml of water. The solution is left to stand for 1 hour, then diluted with 80 ml of ether and the separated precipitate is filtered off. The nonadecapeptide-amide-trifluoroacetate was dissolved in 5 ml of water and filled with carboxymethyl cellulose (Whatman CM-52) (3.5 × 56 m) on the ion exchanger column and chromatographed with a linear gradient buffer prepared from 1.5 1 0.5 m (pH = 6.7) and 1.5 1 with 0.6 m (pH = 6.7). Fractions 78-86 containing product are collected and lyophilized. 0.166 g (41.5%) of the aminooxycarboxylic acid substituted nonadecapeptidamide Rt 5 = 0.22 is obtained. Peptide content 76%; content of the mixture - sulfoxide 2.3%. Amino acid analysis: Lys 4.5 (5), His 1.0 (1), Arg 0.9 (1), Ser 0.85 (1), Glu 1.0 (1) ), Pro 1.0 (1), Gly 1.9 (2), Val 0.85 (1), Met 0.75 (1), Tyr 0.95 (1), Fen 1.0 (1) Used as a starting product protected by substitution with amino oxycarboxylic acid, nonadecapeptidamide hydrochloride is obtained as follows: Step 1: Z - Lys (BOC) - Lys (BOC) - Lys (BOC) -Lys (BOO-OCH3 51 g (60 mmoles) ) Z - Lys (BOC) - Lys (BOC) -Lys (BOC) -OCH3 is dissolved in 100 ml of methanol and in the presence of 10 g of 10% palladium catalyst on activated carbon it is hydrogenated. After the reaction is complete, the catalyst is filtered off and the solution evaporated in vacuo. The residue was dissolved in 240 ml of ethyl acetate, 28.5 g (60 mmol) of Z-Lys (BOC) -OSu were added and the mixture was left to stand at room temperature. 1000 ml of ethyl acetate and 200 ml of water are added to the reaction mixture, after shaking, the aqueous phase is separated and the ethyl acetate phase is washed two times. 200 ml of 1N hydrochloric acid twice, 200 ml of an 8% sodium carbonate acid solution of 8% and evaporated after drying. The residue is triturated with ether, filtered off and washed with ether. The crude product is recrystallized from ethyl acetate. 52.05 g (81%) of Z-Lys (BOC) -Lys. (BOC) -Lys (BOC) -Lys (BOC) -OCH3 are obtained. Melting point 122-123 ° C. Stage 2: H - Lys (BOC) - Lys (BOC) - Lys (BOC) -Lys (BOO-OCH3. 35 g (32.4 mmol) Z-Lys (BOC ) -Lys (BOC) -Lys (BOC) -Lys (BOC) -OCH3 is dissolved in 700 ml of methanol and hydrated in the presence of 7 g of 15% palladium catalyst on activated carbon. After the reaction is complete, the catalyst is filtered off, the solution is filtered. evaporate and the residue is stabilized by an ether-diisopropyl ether mixture. After filtration and drying, the crude product is crystallized from an ether-diisopropyl ether mixture. 24.8 g (81%) H-Lys (BOC) - are obtained. Lys (BOC) -Lys (BOC) -Lys (60C) -OCH3. Melting point 101-103 ° C. Stage 3: Tri-Lys (BOC) - Lys (BOC) -Lys (BOC) -Lys 25 ( BOC) -OCH3 24 g (25.4 mmol) of H-Lys (BOC) -Lys (BOC) - Lys (BOC) - Lys (BOC) - OCH3 and 4.7 ml (33.6 mmol) of triethylamine are dissolved in 180 ml of dichloromethane, 8.5 g (30.5 mmol) of trityl chloride are added and the mixture is left at room temperature. After 24 hours, the dichloromethane is distilled off and the residue is added with 5 00 ml of ethyl acetate and 100 ml of water. After shaking, the aqueous phase is separated off and the ethyl acetate phase is shaken three times with 100 ml of 5% citric acid and three times with 100 ml of 8% sodium carbonate acid solution, dried and then evaporated. The residue is triturated with ether, filtered off and washed with diisopropyl ether and petroleum ether. 23.6 g (78.5%) Trilys (BOC) - Lys 40 (BOC) - Lys (BOC) - Lys are obtained (BOC) -OCH3. Melting point 166-168 ° C. Stage 4: Tri-Lys (BOC) - Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -OH3 is dissolved in 45 230 ml methanol. 5.7 ml (117 mmol) of hydrazine hydrate are added and left to stand at room temperature. After four days, the methanol is distilled off, the residue is triturated with ether, filtered off and dried under a vacuum. 23.0 g (98%) of 50 Tri-Lys (BOC) - Lys (BOC) - Lys (BOC) - Lys (BOC) - -N2H3 are obtained. Melting point 217-219 ° C. Stage 5: Tri-Lys (BOC) - Lys (BOC) -Lys (BOC) -Lys (BOC) - OGly - NH * 2.37 g (2.0 mmol) Tri-Lys (BOC) - Lys (BOC) - Lys (BOC) - Lys (BOC) - N2H3 is dissolved in 12 ml of dimethylformamide. The solution is cooled to -20 ° C and, with stirring, first 2 ml (8.0 mmol) of a hydrochloric acid solution in 4 N ethyl acetate and then 0.28 ml (2.35 mmol) of a third of butyl nitrite are added dropwise. The reaction mixture is kept for 5 minutes at -10 ° C and then cooled to -20 ° C and 0.216 g (2.4 mmol) of H-OGly-NH 2 [Riedl. Frank.Nonatshefle 92 730 (1961) and 1.03 ml (6.0 mmol) of diisopropylethylamine in 8.0 ml of dimethylformamide. The reaction mixture is left for 2 hours. at —5—0 ° C, then at 0 ° C until the next day. The solution is then evaporated. The residue is triturated with water, then drained and washed with water. 2.35 g (94%) of Tri-Lys (BOC) - Lys (BOC) - LyB (BOC) - Lys (BOC) OGiy-NH2 are obtained. Rt4 = 0.72. Step 6: H-Lys (BOC) - Lys (BOC) - Lys (BOC) - Lys (BOC) -OGly-NH2. HCl. [1.75 g (1.4 * mmol) Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -OGly - NH2 is dissolved in 35 ml of 80% aqueous acetic acid solution . After an hour the solution is evaporated and the residue is triturated with ether. After filtering, washing with ether and drying the crude product with a mixture of ethyl acetate - (pyridine: acetic acid: water = 20.6: 11) in a ratio of 4: 1 is introduced on a silica gel column (3.2 x 52 cm) and chromatographed. The eluate is collected in 15 ml fractions and their composition is determined by thin-layer chromatography. The fractions 1C5-112 containing pure product are collected, the solvent is evaporated, the residue is triturated with ether, filtered and, after drying, dissolved in 20 ml of methanol. . The solution of pyridine hydrochloride obtained by adding 0.22 ml of pyridine to 0.22 ml of concentrated hydrochloric acid is added to the solution. Then the methanol is distilled off, the residue is triturated with ether, filtered off and dried under a vacuum. Yield 0.95 g (65%) of H-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) - OGly NK2. HCl. Rt2 = 0.18 Step 7: BOC-D-Eight-Tyr-Ser - Met - Glu (OBu *) - -His-Fen-Arg-Trp-Gly-Lys (BOC) - Pro - Val - Gly- - Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) - OGly- -itc ^. hci. 0.398 g (0.20 mmol) of BOC-D-OSer-Tyr-Ser-Met -Glu (OBut) -His-Phe-Arg-Trp-Gly-ON. HCl, 0.208 g (0.20 mmol) H-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -OGly-NH2. HCl and 0.165 g (0.90 mmol) of pentafluorophenol are dissolved in 4 ml of dimethylformamide and first 0.22 ml (0.20 mmol) of N-methylmorpholine, then 0.063 g (0.30 mmol) of DCC are added. The reaction mixture is substituted at room temperature for 16 hours, then the DCV is filtered off and the filtrate is diluted with ether. The separated product is filtered off, washed with ether and dried in a vacuum. 0.540 g (91%) of protected nonadecapeptidamide hydrochloride is obtained, Rt6 = 0.63. Example VI. H-D-OSer-Tyr-Ser-Met-Glu- -His-Fen-Arg-Trp-Glu-Lys- Pse - Val - Gly-Lys- -Lys-Lys-Lys-OGly-Bzl. 0.400 g (0.130 mmol) BOC-D-OSer-Tyr-Ser-Met- -GkKOButJ-His-Fen-Arg-Trp-Gly - Lys (BOC) -Pro- -Val-Gly - Lys (BOC) - Lys ( BOC) - Lys (BOC) - Lys (BOC) -OGly-OBzl. The HCl is dissolved in a mixture of 3.2 ml. Of trifluoroacetic acid, 0.4 ml. Of angel and 0.4 ml. Of water. After one hour, 80 ml of ether are added and the separated precipitate is filtered off. The crude nonadecapeptidamide trifluoroacetate is dissolved in 5 ml of water and loaded with carboxymethylceiulose (Whatman CM-52) (3.5 × 52 cm) on the ion exchanger column and chromatographed with a linear gradient buffer prepared with 1.5 liters and 0.2 meters. (pH = 6.7) and 1.5 1 0.5 m (pH = 6.7) .21 The fractions 80-93 containing the product are collected and lyophilized. 0.151 g (30%) of the aminooxycarboxylic acid-substituted nondapeptidamide was obtained. Rt5 = 0.28. Peptide content: 80%; content of methienine sulfoxide 1.9%; Tyr-Trp ratio 1.05 Amino acid analysis: Lys 4.5 (5), His 0.95 (1), Arg 1.0 (1), Ser 0.8 (1), Glu 1.0 ( 1), Pro 1.0 (1), Gly 1.95 (2), Val 0.95 (1), Met 0.8 (1), Tyr 0.95 (1), Fen 1.0 (1) When used as a starting product protected by the substitution of amino oxycarboxylic acid with nonadecapeptidamide hydrogen chloride, it was obtained as follows: Step 1: BOC-OGly-OBzl 3.8 g (20 mmoles) BOC-OGly-OH (Hungarian Patent Specification No. 165 117 ) is dissolved in 5 ml of dimethylformamide and 4.44 ml (40 mmol) of triethylamine and 7.8 ml (66 mmol) of benzyl bromide are added. The mixture is heated in a water bath for 8 hours, then left to stand for the next Then it is diluted with 100 ml of ethyl acetate and washed with 50 ml of water and three times with 20 ml of 8% sodium acid carbonate solution. After drying, ethyl acetate is distilled off and the residue is recrystallized from ether. 4.5 g (80%) of BOC-OGly-OBzl are obtained. Melting point 79-80 ° C. Stage 2: H-OGly-OBzl. HCl. 11.2 g (40 mmol) of BOC-OGly-OBzl are dissolved in 22 ml of ethyl acetate and 22 ml of a 4N solution of hydrochloric acid in ethyl acetate are added. After 30 minutes, the reaction mixture is evaporated and the residue is triturated with ether. After filtering and drying, the crude product is recrystallized from ethyl acetate. 5.7 g (66%) of OGly-OBzl are obtained. HCl. Melting point 85-87 ° C. Stage 3: Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -OGly-OBzl. 7.11 g (6.0 mmol) of Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -N2H3 are dissolved in 36 ml of dimethylformamide. To cool the solution to -20 ° C., first 6 ml (24 mmol) of a 4N solution of hydrochloric acid in ethyl acetate and then 0.84 ml (7.1 mmol) of tert-butyl are added dropwise while stirring. The reaction mixture is kept for 5 minutes at -10 ° C, then cooled to -20 ° C and a mixture of 1.45 g (6.6 mmol) of H-OGly-OBzl is added. HCl and 4.24 ml (24.6 mmol) of diisopropylethylamine in 24 ml of dimethylformamide. The reaction mixture is stirred for 2 hours at -5 to 0 ° C. and allowed to stand at 0 ° C. overnight. The solution is then evaporated, the residue is triturated with water, filtered off and washed with water. The crude product is recrystallized from a methanol-water mixture (1: 1). 6.95 g (87%) of Tri-Lys (BOC) -Lys (BOC) -Lys (BOC) -OGly-OBzl are obtained. Rt4 = = 0.85 Step 4: H-Lys (BOC) - Lys (BOC) - Lys (BOC) - Lys (BOC) -OGly-OBzl. HCl. 1.87 g (1.4 mmol) of Tri-Lys (BOC) -Lys (BOC) -Lys (30C) -Lys (BOC) -OGly-0Bzl are dissolved in 37 ml of 80% aqueous acetic acid. After an hour, the solution was evaporated and the remaining rtp 22 was rubbed with ether. After filtering, washing with ether and drying, the crude product is loaded onto a column containing silica gel (3.2 × 49 cm) and chromatographed with a mixture of ethyl acetate - (pyridine: acetic acid: water = 20: 6: 11) in a ratio of 4. : 1. The eluate is collected in 15 ml fractions, the composition of which is determined by thin-layer chromatography. The fractions 83-97 containing the pure product are collected, the solvent is distilled off and the residue is triturated with ether. After filtration and drying, it is dissolved in 20 ml of methanol and a solution of pyridine hydrochloride is added. This solution is prepared previously with 0.22 ml of pyridine and 0.22 ml of concentrated hydrochloric acid. The methanol is then filtered off, the residue is triturated with ether, filtered and dried under vacuum. 0.905 g (57%) of H-Lys (BOC) -Lys (BOC) - -LyS {BOC) - Lys (BOC) is obtained. -OGly-OBzl. HCl. Rt2 = 0.23. Step 5: BOC - D - OSer-Tyr-Ser-Met-GlutOBu *) - -His-Fen-Arg-Trp-Gly-Lys (BOC) -Pro- Val - Gly- -Lys ( BOC) - Lys (BOC) - Lys (BOC) - Lys (BOC) - -OGly-OBzl. HCl. 0.398 g (0.20 mmol) of BOC-D-OSer-Tyr-Ser-Met- -GluCOBu → His-Phe-Arg-Trp-Gly-OB. HCl, 0.22 g (0.20 mmol) H - Lys (BOC) -Lys (BOC) - Lys (BOC) - -Lys (BOC) -OGly-OBzl. HCl and 0.165 g (0.90 mmol) of pentifluorophenol were dissolved in 4 ml of dimethylformamide and first 0.022 ml (0.20 mmol) of N-methylmorpholine was added then 0.063 g (0.30 mmol) of DCC. The reaction mixture is allowed to stand at room temperature, then the DCU is filtered off and the filtrate is diluted with ether. The separated product is filtered off, washed with ether and dried in a vacuum. 0.502 g (82%) of nonadecapeptidamide hydrochloride are obtained. Rt6 = 0.68. PL

Claims (7)

Claims 1. Method for producing peptides with ACTH activity of the general formula HO-OSer-Tyr-Ser-Met- -Glu-His-Fen-Arg-Trp-Gly-Lys-Pro - Val - Gly- - (Lys) nX wherein n is an integer of 3 or 4, and X is the group -OLys-OH, -O-OLys-OH or -OGly-OH as well as their acid addition salts, amides and benzyl ester, characterized by that in the peptide of general formula QD- -OSer-Tyr-Ser-Met-Glu- (OR) - His - Fen - Arg- -Trp-Gly-Lys ^ -Pro-yal-Gly-tLys ^ ln - ^, in where n = 3 or 4, x 'represents the groups -OLys (Q) -Y, 50 -D-OLys (Q) -Y or -OGly-Y, where Y represents the groups -OR or NH 2, where R is an ordinary carboxyl group protected, preferably a third butyl or benzyl group, Q is an amino group protected, preferably a benzyloxy carbonyl or formyl group, whereby the Q or R groups present in the peptide may be the same or different, the groups are removed protecting by one or more steps and optionally converting to an addition salt e with acids, amides or benzyl ester. 2. The method according to claim The starting product of claim 1, wherein the starting product is a protected peptide containing D-α-aminooxy-β-hydroxypropionic acid (D-OSer) in place of the first amino acid of the ACTH sequence. 3. The method according to p. The starting product of claim 1, wherein the starting product is a protected peptide containing L or D-amino-oxy-E-aminocaproic acid (OLis) at the 17th or 18th amino acid site of the ACTH sequence. 4. The method according to p. The process of claim 1, wherein the starting product is a protected lysine-containing peptide at the Arg17 site of the ACTH sequence. 5. The method according to p. A method according to claim 1, characterized in that the starting product is a peptide whose N-terminal oxyamino group and the amino groups are protected with III tert-butoxycarbonyl moieties and whose carboxyl groups are protected with III tert-butoxycarbonyl moieties. 6. The method according to p. The process of claim 1, wherein all protecting groups are removed by acid hydrolysis. 7. The method according to p. The process of claim 1, wherein all protecting groups are removed by the action of trifluoroacetic acid. LZG Zakl. No. 3 in Pab., Residing 523-77, nakl. 110 + 20 copies Ccns PLN 10 PL