DE1817938A1 - Preparation of hypocalcaemic peptides - Google Patents

Abstract

(I) H- -val-leu-ser-ala-tyr-try-arg-Q1-leu-Q1-Q1-phe-his-arg-phe-ser-gly- -Q2-gly-phe-gly-pro-Q3-thr-pro-OH. where Q1= asn and/or asp. Q2= met, val, norval, leu, ile, or norlen and Q3= glu or gln. (II) Acid addn. salts of (I) (III) Derivs. of (I), e.g. esters, amides, S-oxides etc. (IV) Complexes of (I) e.g. with metal ions, organic polymers. Hypocalcaemics and anti-inflammatories.

Description

New hypocalcemic peptides and methods of making them.

The invention relates to the dotriacontapeptide of the formula I Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH and derivatives of this compound, namely the C-terminal amide of the compound of the formula I (thyrocalcitonin) and its Met sulfoxide, also Nα-acyl derivatives of thyrocalcitonin and its sulfoxides, and acid addition salts and complexes of the compounds mentioned and processes for their preparation.

Obtaining a hypocalcemic called thyrocalcitonin Porcine thyroid peptides have been reported by Putter et al., Journ. Amer. Chem. Soc. 89, 5301 (1967). However, the authors do not give a structural formula, and various properties, e.g. the lack of N-dansylation, indicate that it is not the aforementioned dotriacontapeptide amide. Had before Copp et al., Endorinology 70, 638 (1962) by perfusion of the thyroid and parathyroid glands found in dogs with calcium-rich blood that the parathyroid gland has a forms hypocaicemic factor called "calcitonin". The only in a lot low level factor was not isolated. Hirsch et al., Endocrinology 73, 244 (1963) were able to show that crude acid extracts from thyroid tissue cause a have a hypocalcemic effect> and achieved, as Foster et al.

Nature 202, 1303 (1964) based on perfusion experiments on goats, to the view that the Copp et al. discovered hypocalcemic factor in the thyroid gland and not the parathyroid gland. The hormone was not isolated in pure form. Also in other publications such as Friedman et al., Science 150: 1465 (1965), Milhaud et al., C.r. hebd. Seances Acad. Sci. 261, 813 (1965), Aliopoulios et al., Science 151, 330 (1966) and Robinson et al., J. Endocrinol. 39, 71 (1967) was only the acquisition or examination of the effect of a more or less purified crude extract described; for lack of a pure product It was not possible to specify a gross formula or determine the structure. the Amino acid composition of thyrocalcitonin was also presented at the "Symposium on Thyrocalcitonin and the CCells: t London 17.-20. July 1967 (William Heinemann Medical Books Ltd. London, 1968) by the various research groups either not or not correctly specified.

As acid addition salts of the compounds according to the invention are especially salts of therapeutically applicable acids such as hydrochloric acid, acetic acid, $ Sulfuric acid, phosphoric acids, sulfonic acids to name a acyl groups for acylation the N @ are the residues of carboxylic acids, such as aliphatic, aromatic, araliphatic, heterocyclic and heterocycloylaliphatic carboxylic acids, especially of lower ones Alkanoic acids such as formic acid, acetic acid / propionic acid, butyric acids, of monocyclic acids aromatic carboxylic acids such as unsubstituted and substituted benzoic acid, of unsubstituted and aryl-substituted aryl-nieeralkylcarboxylic acids such as phenylacetic acid, of 5- to 6-membered heterocyclic acids with nitrogen, sulfur and / or Oxygen as heteroatoms, such as pyridinecarboxylic acids, thiophenecarboxylic acids, or of heterocyclyl-lower alkanoic acids such as pyridyl acetic acid, imidazole acetic acid, in which the substituents of the rings e.g. halogen atoms, nitro groups, Lower alkyl or lower alkoxy groups or lower carbalkoxy groups. Next are acyl residues of amino acids, especially a-amino acids such as the pyroglutamyl residue, also acyl residues, which differ from carbonic acid or thiocarbonic acid or their esters or derive amides, e.g.

Lower alkyloxycarbonyl groups such as ethoxycarbonyl or tert-butyloxycarbonyl, Benzyloxyearbonyl groups, carbamoyl and thiocarbamoyl groups such as N-substituted ones Carbamoyl and thiocarbamoyl groups, e.g. N-lower alkylcarbamoyl, N-phenylcarbamoyl- or N-phenyl-thiocarbamoyl groups in which the aryl radicals are substituted as indicated above could be.

The structure of the complexes has not yet been clarified Compounds to understand the addition of certain inorganic or organic Substances to long-chain peptides are created and give them a prolonged effect. Such substances are described, for example, for ACTH and other adrenocorticotropic substances effective peptides. Mention should be made, for example, of inorganic compounds that are different from metals such as calcium, magnesium, aluminum, cobalt and especially zinc especially poorly soluble salts such as phosphates, pyrophosphates and polyphosphates as well Hydroxides of these metals, also alkali metal polyphosphates such as wCalgon N ", "Calgon 322", "Calogon 188" or "Polyron B 12". Organic substances that are an extension cause the effect are, for example, non-antigenic gelatine, e.g. polyoxygelatine, Polyvinyl pyrrolidone and carboxymethyl cellulose, also sulfonic acid or phosphoric acid esters of alginic acid, dextran, polyphenols and polyalcohols, especially polyphlorestin phosphate and phytic acid, as well as polymers and copolymers of amino acids, e.g. protamine or polyglutamic acid.

The new compounds, especially the amide of the compound of Formula I, have a hypocalcemic effect. They lower the plasma calcium and -phosphate content of the blood of mammals, as determined by experiments on Wistar rats has been proven. erner-they have anti-inflammatory properties, as in the example of kaolin edema in the rat.

The compounds are also effective on humans.

With parenteral administration from 0.01 to 1 mg in 0.1 to 1 m. Acetate buffer from pH 4.6 dissolved C-terminal amide of the compound of formula 1 is the serum calcium and serum phosphate decreased. This effect is not only evident in hypercalcemia patientsJ - but also normal calcemic people who suffer from osteoporosis, for example.

The new compounds can therefore be used to treat> hypercalcemia and used by bone diseases such as oesteoporosis.

The process according to the invention for the preparation of the new compounds is characterized in that a) from the compound of the formula I or the said derivatives of this compound, in which at least the a-amino group and the terminal and side-chain carboxyl groups are protected, in a manner known per se splitting off the protective groups or b) compounds of the formula II H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-A la -Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe -His-Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH II or derivatives oxidized to disulfide or c) compounds of the formula II or derivatives in which the mercapto groups are replaced by the Trityl group are protected, oxidized directly to disulfides or d) compounds of the formula III or IV wherein A 1 to 21 of the amino acid residues following cysteine and R is hydrogen or an acylated amino group, condensed with the remaining C-terminal sequence of the peptide in a known manner, with the proviso that the azide method, the anhydride method or the activated ester method are used when the C-terminal sequence has a free carboxyl group and, if desired, cleaves off the α-acyl group and; if desired, the compounds obtained are converted into their acid addition salts or complexes.

In the preparation of the starting materials for the 1st variant of the invention Process as well as all intermediate products required in the 4 process variants Especially those from the synthesis of long-chain peptides come as protective groups known as well as some new protecting groups, which are easily split off can e.g.

by hydrolysis, reduction, aminolysis or hydrazinolysis.

For example, acyl or acyl groups are used as protective groups for amino groups Aralkyl groups such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, p-toluenesulfonyl, o-Nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl groups (these sulfenyl groups can also be caused by the action of nucleophilic reagents, e.g. sulfites, thiosulfates, see English patent 1 104 271 are split off) optionally substituted, such as substituted by lower alkoxy groups, especially o- or p-methoxy groups Benzyl, or diphenyl or triphenylmethyl groups or from carbonic acid itself deriving groups such as, if appropriate, e.g. by halogen atoms such as chlorine or bromine, Benzyl or benzhydryloxycarbony groups substituted by nitro groups or lower alkoxy groups, e.g. carbobenzoxy, p-bromo- or p-chloroarbobenzoxy, p-nitrocarbobenzoxy or p-methoxycarbobenzoxy, colored benzyloxycarbonyl groups such as p-phenylazo -benzyloxycarbonyl and p- (p1-methoxy-phenylazo) -benzyloxycarbonyl, aliphatic oxycarbonyl groups such as adamantyloxycarbonyl, cyclopentyloxycarbonyl, Trichloroethyloxycarbonyl, tert. Amyloxycarbonyl or especially tert-butyloxycarbonyl, aromatic-aliphatic oxycarbonyl groups such as 2-phenyl-isopropyloxycarbonyl, 2-tolyl-isopropyloxycarbonyl and in particular 2-p-diphenylisopropyloxycarbonyl (see application G.Nr. 1073/67 (Case 6106).

The amino groups can also be obtained by forming enamines by reaction of the amino group with 1,3-diketones e.g. Benzoylaeeton, Acetylacetone or dimedone.

Carboxyl groups are formed, for example, by amide or hydrazide formation or protected by esterification. The amide and hydrazide groups can optionally be substituted, the amide group e.g. by the 3,4-dimethoxybenzyl or bis (p-methoxyphenyl) methyl group, the hydrazide group e.g. through the carbobenzoxy group> the trichloroethyloxycarbonyl group, the trifluoroacetyl group, the trityl group, the tert-butyloxycarb.onyl group or the 2-p-diphenylso-propyloxycarbonyl group. Suitable for the esterification are e.g.

lower optionally substituted alkanols such as methanol, ethanol, Cyanomethyl alcohol, benzoylmethyl alcohol or, in particular, tert-butanol, also Aralkanols such as aryl-lower alkanols, e.g. optionally through lower alkyl or lower alkoxy groups or halogen atoms substituted benzyl or benzhydryl alcohols such as p-nitrobenzyl alcohol, p-Methoxybenzyl alcohol or 2,4,6-trimethylbenzyl alcohol, optionally by electron-withdrawing Substituent substituted phenols and thiophenols such as thiophenol, thiocresol, p-nitrothiophenol, 2,4,5- and 2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenolJ p-cyanophenol, or p-methanesulfonylphenol, further e.g. N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxypiperidine> 8-hydroxyquinoline.

The hydroxy groups of the serine, threonine and tyrosine residues can e.g. protected by esterification or etherification. As acyl residues in the Esterification are e.g.

Lower alkanoyl radicals such as acetyl, aroyl radicals such as benzoyl and above all residues derived from the carbonic acid such as benzyl oxycarbonyl or ethyloxycarbonyl suitable. Groups suitable for etherification are e.g. benzyl, tetrahydropyranyl or tert. Butyl residues. Also suitable for protecting the hydroxyl groups are those in Ber.-100 (1967), 3838-3849 described 2,2,2-trifluoro-1-tert-butyloxyearbonylamino- or -l-benzyloxycarbonylaminoethyl groups (Weygand). The hydroxyl groups need but not necessary to be protected.

The mercapto groups of the cysteine residues are e.g.

protected by acylation or alkylation. Suitable for acylation is e.g. the acetyl or benzoyl radical, the ethylcarbamoyl radical or the optionally substituted carbobenzoxy radical. Suitable for alkylation are, for example, the tert-butyl or benzylthiomethyl radical or optionally substituted arylmethyl groups such as Benzyl, p-nitrobenzyl, diphenylmethyl, dimethoxybenzhydryl or trityl, also phenylcyclohexyl, Thienylt2) -cyclohexyl et al., See Ber. 1968, 101: 681.

To protect the amino group in the guanidino group of arginine The nitro group and the tosyl group or the carbobenzoxy radical are particularly important Use, but the guanidino group does not need to be protected will.

Likewise, the imino group of histidine does not necessarily need to be protected but it can be advantageous to protect them, e.g. by benzyl, Trityl, carbobenzoxy, adamantyloxycarbonyl, or the Weygand's mentioned above Groups.

If the new peptides after Merrifield's solid support synthesis can be built up, the side chains of serine, threonine and tyrosine e.g. Benzyl, that of cysteine through benzyl, or p-methoxybenzyl, that of histidine l-Benzyloxyearbonylamino-2,2,2-trifluoroethyl, that of arginine through the nitro group and those of glutamic acid are protected by benzyloxy. As a-amino protecting group for example, tert-butyloxycarbonyl is used.

The cleavage of the protected peptide from the resin and the protecting groups happens e.g. with anhydrous hydrogen fluoride.

Preferably used in the 1st variant of the invention Process for protecting the amino groups the tert-butyloxyearbonyl group, for protection the carboxyl group of the side chain and optionally the terminal carboxyl group the tert-butyl ester group, to protect the hydroxyl groups of the serine, threonine and tyrosine residues, if they are protected at all, the tert-butyl ether group and, if desired, to protect the imino group of the histidine, the 2,2,2-trifluoro-1-tert.-butyloxyearbonylaminoethyl group. All these protective groups can, if desired, in one stage by acid hydrolysis, e.g. by means of trifluoroacetic acid. When building the in the first variant of the process using protected dot triacontapeptide used as starting material of protective groups which can be split off with trifluoroacetic acid are the mercapto groups preferably protected by benzyl or trityl. The S-trityl groups can be made from the protected peptide in organic solution selectively (while maintaining the with Groups that can be split off from trifluoroacetic acid) with mercuric acetate and hydrogen sulfide be split off. The S-benzyl groups can be selected from the protected peptide be split off with sodium in liquid ammonia. In both cases one obtains the protected peptide with free mercapto groups. This can lead to the protected disulfide, e.g. with iodine in glacial acetic acid, with diiodoethane in organic solvents, with atmospheric oxygen be oxidized in liquid ammonia. It is particularly advantageous to use the mercapto groups by protecting trityl groups and removing them from the protected peptide at the same time To remove the formation of the disulfide bridge with iodine in methanol, see Swiss application Ges.Nr. 6999/68 (Case 6461). This formation of the disulfide ring can take place on the stage a partial sequence containing the two cysteine residues, e.g. of the nonapeptide 1-9, or at the stage of the dot triacontapeptide.

In the 2nd variant of the method according to the invention Procedure the free open-chain peptide of the formula II used as starting material preferably also prepared with the protective groups mentioned for variant 1) will. The S-trityl groups are useful as are all other protecting groups removed with trifluoroacetic acid.

The free open-chain peptide is replaced by potassium ferricyanide in a known manner oxidized in aqueous solution or by iodine or with air in liquid ammonia.

In the 5th variant of the method, the trityl groups are as described above mentioned process with iodine and methanol removed with simultaneous disulfide formation.

In the fourth variant of the method according to the invention, directly active derivatives of the peptides shown in formula I or their analogues behave, namely peptides whose α-amino group is either acylated or absent.

A special α-amino protective group is not necessary in this case. The compounds can, however, if desired, subsequently in a manner known per se Way, e.g. by hydrolysis or hydrogenolysis, into corresponding peptides with free a-amino group are converted.

The free peptides obtained can subsequently be added in a manner known per se be converted into their acid addition salts, derivatives or complexes.

So you can use the peptide for the production of Na acyl derivatives free a-amino group in usual Wise N-acylate e.g. by reaction with a mixed anhydride or acid azide containing the acyl radical in question or above all an activated ester such as phenyl or substituted phenyl ester.

The sulfoxide is advantageously obtained by oxidation with dilute hydrogen superoxide solution produced in a weakly acidic medium.

Complexes with inorganic substances such as sparingly soluble metal, e.g. Aluminum or zinc compounds are known in the same way as for ACTH, e.g. by reaction with a soluble salt of the metal in question, e.g.

Zinc chloride or zinc sulfate, and precipitation with an alkali metal phosphate and / or hydroxide produced. Complexes with organic compounds such as polyoxygelatine, Carboxymethyl cellulose, polyvinylpyrrolidone, polyphioretin phosphate, polyglutamic acid etc. are made by mixing these substances with the peptide in aqueous solution obtain. In the same way, insoluble compounds with alkali metal polyphosphates can also be used getting produced.

The peptides used as starting materials are behaved by the amino acids, if necessary or desired, using easily cleavable ones Protective groups, individually in the order mentioned or after formation linked smaller peptide units, optionally at a suitable level of synthesis the disulfide bridge is formed. One works appropriately according to those for the production of long-chain peptides, taking into account the disulfide bridge, suitable linking methods, as they are known from the literature.

The amino acid and / or peptide units are linked therefore, for example, in such a way that an amino acid or a peptide with a protected a-amino group and activated terminal carboxyl group with an amino acid or a peptide with free α-amino group and free or protected, e.g. esterified or amidated terminal carboxyl group or that one amino acid or a peptide with activated α-amino group and protected terminal carboxyl group with an amino acid or a peptide with a free terminal carboxyl group and protected a-amino group. The carboxyl group can, for example, by Conversion into an acid azide, anhydride, imidazolide or an activated ester, such as cyanomethyl ester, thiophenyl ester, p-nitrothiophenyl ester, thiocresyl ester, p-methanesulfonylphenyl ester> p-nitrophenyl ester, 2> 4-dinitrophenyl ester, 2 4,5- or 2,4,6-trichlorophenyl ester, Pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 8-hydroxyquinoline ester, N-hydroxypiperidine ester, or by reaction using a carbodiimide (optionally with the addition of N-hydroxysuccinimide) or -N, N'-carbonyldiimidazoles, or Isoxazolium salt, e.g. Woodward reagent, the amino group e.g. by reaction be activated with a phosphite. The most common methods are to be mentioned the carbodiimide method, the azide method, the activated ester method and the Anhydride method, also the Merrifield method and the N-carboxyanhydride method or N-thiocarboxy anhydrides.

In addition to the production of the end products, there is also production the starting materials, especially the peptide fragment containing the disulfide bridge and its linkage with the remaining part of the peptide is a particular subject of the invention It has been found that it is advantageous to start from a sequence which comprises the first 7-12 N-terminal amino acids (1-7, 1-8, 1-9, 1-10, 1-11 or 1-12) and with this N-terminus the entire remaining sequence, i.e. 8-32, 9-32, 10-32, 11-32, 12-32 or 13-32 to condense. Instead you can also use the mentioned N-terminal sequence of the first 7-12 amino acids with the fragment up to the 24th amino acid (Glycine) and link the tetracosapeptide with the octapeptide of amino acids 25-32 condense. The condensation of said N-terminal fragment with the sequence up to the 32 or up to the 24th amino acid is expediently based on the azide method from the azide or hydrazide, or the Wünsch's method (carbodiimide in the presence of N-hydroxysuccinimide), starting from the peptide with a free terminal Carboxyl group carried out.

The following describes the manufacture of the N-terminal fragment the synthesis of the nonapeptide (1-9) explained in more detail. In a very corresponding way can the heptapeptide (1-7)> the octapeptide (1-8), the decapeptide (1-10), the Undecapeptide (1-11) and the dodecapeptide (1-12) can be produced.

The nonapeptide sequence is, for example, from the sequences 1-4 and 5-9 or 1-6 and 7-9 constructed, as can be seen from FIGS. 1-9; one can however also use other fragments to build up the sequence 1-9. As a protecting group for the α-amino group on the cysteine is preferably the tert-butyloxycarbonyl group or an equivalent group which can be split off by acid hydrolysis is used, or if a Na-acylated Dotriacontapeptide is to be produced, the corresponding one Acyl- 'e.g. acetyl group. In addition, it is expedient to use it as mercapto protecting groups those which are selective towards the gpp N amino protecting group which can be split off by acid hydrolysis .B. tert-butyloxycarbonyl group) can be split off, e.g. the benzyl or Trityl group.

The terminal carboxyl group of the nonapeptide is not necessary to be protected, e.g. not when condensation using the azide or anhydride method be carried out (cf.

e.g. Fig. 1, columns 5 and 8). But you can also go through this group Protect esterification as indicated above, e.g.

by esterification with methanol (cleavage of the ester group with dilute Sodium hydroxide solution or conversion into the hydrazide) or with benzyl alcohol or analogs (Cleavage of the ester group e.g. by hydrogenolysis). Protection of the amino groups the intermediate products are made by means of the usual protective groups, e.g. carbobenzoxy, Trityl, tert-butyloxycarbonyl and especially 2-para-diphenyl-isopropyloxycarbonyl. The carboxyl groups of the intermediates are, if necessary, in the usual way Esterified way. The hydroxyl groups of the serine residues and the threonine residue can protected by etherification, e.g. with tert-butanol or equivalents.

In the following figures and examples: 1) means the azide method 2) the mixed anhydride method 3) the activated ester method, especially p-nitrophenyl ester (ONP) or hydroxysuccinimide ester (OSU) 4) the carbodiimide method 5) the method according to Wünsch BOC tert-butyloxycarbonyl, DPC p-diphenyl-isopropyloxycarbonyl, Z carbobenzoxy TRI trityl Bzl Benzyl NPS o-Nitrophenylsulfenyl OtBu tert-butyl ester OBzl benzyl ester ONB p-nitrobenzyl ester ONP p-nitrophenyl ester OMe methyl ester tBu tert-butyl ether Ac Acetyl TFA trifluoroacetic acid.

The carbobenzoxy, p-nitrobenzyl ester and benzyl ester groups are split off by hydrogenolysis in the presence of palladium carbon, the N-trityl group with aqueous acetic acid, the tert-butyloxycarbonyl group with trifluoroacetic acid, the o-nitrophenylsulfenyl group with hydrogen chloride in organic solvents or e.g. with hydrogen cyanide or sulphurous acid as in English patent 1 104 271 described; the diphenylisopropyloxycarbonyl group e.g.

with a mixture of glacial acetic acid, formic acid (82.8% in) and water (7: 1: 2) as in the Swiss application G.No. 1073 / 6Y (Case 6106). Of the p-Nitrobenzyl or methyl ester is converted into the hydrazide with hydrazine hydrate. With diluted Sodium hydroxide solution, the methyl ester group is hydrolyzed. The tert-butyl ester is cleaved with trifluoroacetic acid, as is the tert-butyl ether. The S-trityl groups are removed with mercuric acetate and hydrogen sulfide, which S-Benzyl group with sodium in liquid ammonia, with any existing p-nitrobenzyl ester groups are split off at the same time. The ring closure to the disulfide takes place e.g.

by oxidation with 1,2-diiodoethane.

The C-terminal sequence to be joined to the N-terminal sequence comprising the 8th to 13th to 32nd amino acid is preferably selected from the C-terminal Fragment 25 32 and the sequence up to the 24th amino acid, i.e. 8 - 24, 9 - 24, 10 - 24, 11 - 24, 12 - 24 or 13 - 24 assembled.

In the schematic representation of FIGS. 11 and 12, the synthesis of the C-terminal octapeptide amide (25-32). Preferably that will Tripeptide amide 30 or the tetrapeptide amide 29-32 using the carbodiimide method linked to a fragment from the preceding amino acids. The γ-carboxyl group the glutamic acid is expediently protected by the tert-utyl ester group. The a-amino protecting group. DPC is treated with glacial formic acid (82.8 % ig) water (7: 1: 2) is split off.

The synthesis of the fragment with the amino acid sequence from the 8th - The 13th to the 24th amino acid can be carried out in completely different ways. Preferably will the peptide up to the amino acid in position 13 - (Tryptophan), i.e. 8-13, 9-13, 10-13 etc. and this either condensed with the undecapeptide 14-24, or initially with a fraction of this Undecapeptide sequence, e.g. 14-16, 14-17, 14-18, 14-19, and then with the remainder Fragments linked up to amino acid 24. Figures 13 and 14 illustrate the synthesis of the tetrapeptide of the 10q - 13th amino acid in the form of the hydrazide, the flat of the azide method with the following amino acids, e.g. the said undecapeptide or a fragment can be condensed from it.

The undecapeptide with the sequence 14-24 is preferred according to the invention made up of the sequences 14-19 and 20-24 or 14-20 and 21-24. The hexapeptide 14-19 or the heptapeptide 14-20 are expediently using the azide method the remaining sequence linked up to the 24th amino acid. Figures 15, 16 and 17 illustrate the synthesis of the hexapeptide hydrazide 14-19 and the heptapeptide hydrazide 14 20th

The amino acid derivative H-His-N2H2-BOC shown in FIG. 17A is obtained by condensation of Z-His-OH with BOC-NH-NH2 using dicyclohexylcarbodiimide in ethyl acetate and hydrogenoyltische cleavage of the carbobenzoxy group. In analog Thus, the protected phenylalanine hydrazide is prepared in Figures 15, A and 16A. The BOC group is formed from the hydrazine residue with trifluoroacetic acid, the carbobenzoxy group split off by hydrogenolysis. Instead of the BOC and carbobenzoxy groups, equivalents can be used Protecting groups are used. Figures 18-20 illustrate the Synthesis of the sequences 20-24 and 21-24, which correspond to the sequence 14-19 (in Fig. 16 stage J, after hydrogenolytic cleavage of the Z group from the hydrazine residue) or 14-20 can be linked to the undecapeptide 14-24.

However, the sequence 14-19 or 14-20 of FIG. 16K can also be used or of FIGS. 15 J or IT L after the α-amino protective group has been split off by hydrogenolysis respectively.

with trifluoroacetic acid with the sequence 10 - 13 according to the azide method link to the sequence 10-19 or 10-20, then from the protected hydrazide die Split off the BOC or carbobenzoxy protective group as above and the sequence 10 - 19 or 10-20 according to the azide method with the sequence 20-24 or 21-24 to the pentadecapeptide Connect 10 - 24.

The sequence of the pentadecapeptide 10-24, as e.g. Combining the fragments shown in FIGS. 13-20 results can for example according to the Wünsch method with the octapeptide amide 25-32, such as 11 or 12 is condensed to the protected tricosapeptide amide 10-32 will.

But you can also use the pentadecapeptide 10-24 initially with the N-terminal Condense sequence 1-9, expediently in the same way as below for the tricosapeptidainide 10-32 indicated, and the tetracosapeptide, for example, with the octapeptide amide, if desired Link 25 - 32.

Another possibility according to the invention, the protected tricosapeptide amide build up of amino acids 10 - 32, consists in having the Sequence 10-16, preferably swetSq according to the azide method, linked to the sequence 17-32. Sequence 10-16 can be obtained, for example, by condensation of those shown in Figs Derivatives of the sequence 10-13 with the tripeptide 14-16 (H-Art-Asn-Leu-OH, e.g. from Z-Arg-Asn-Leu-OHzl obtained by hydrogenolysis) by the azide method. The sequence 17-32 is preferably composed of the sequences 17-24 and 25-32 (octapeptide amide), for example by condensation according to the Wünsch method. One can use the octapeptide derivative Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-OH with protected α-amino group and optionally protected hydroxyl group of the serine residue, for example by condensation of Z-Asn-Asn-Phe-His-N2H3 (from Fig. 17 F with trifluoroacetic acid) with H-Arg-Phe-Ser (tBu) -Gly-OH (Fig. 20 H) obtained by the azide method.

The tricosapeptide amide 10-32 with a protected α-amino group becomes this protective group removed in a suitable manner (BOC with trifluoroacetic acid, with which also a tert-butyl ester group on the W-carboxyl group of glutamic acid30 and any tert-butyl ether groups present are hydrolyzed; DPC with glacial acetic acid-formic acid-water, where tert. Butyl ester and ether groups are not attacked) The linkage the sequence 1-9, which contains the disulfide bridge and in which at least the a-amino group is protected, with the sequence 10-32, takes place, as already mentioned, preferably by the azide method.

In this way, the Dotriaeontapeptidamid with a protected α-amino group is obtained and optionally protected side chain hydroxyl and / or carboxyl groups. These protecting groups are split off as already mentioned.

The Dotriacontapeptide to be used for the method according to variant 2) with free SH groups can be protected in an analogous manner to that described above Dotriacontapeptide can be produced, with the difference that the "'protected SH groups are retained until the end of the synthesis. Only after all the rest Protective groups have been removed from the protected Dotriacontapeptide, cleaves the SH protecting groups are removed as mentioned above.

The synthesis according to variant 4) of the process is particularly suitable for the production of end products of the formula I with acylated or modified ones Amino group, especially the acetylated compound. The fragment of formula III, See, e.g., Fig. 9K, can follow the procedures illustrated above for Sequences 1-9 getting produced; However, you can also use the α-amino protective group from the start Select acyl group to be retained. The synthesis methods correspond to those described above.

Depending on the mode of operation, the new compounds are obtained in shape of bases or their salts. From the salts can in a manner known per se Bases are obtained. The latter, in turn, can be reacted with acids, which are suitable for the formation of therapeutically usable salts, win salts, such as those with inorganic acids, such as hydrohalic acids, betspiel- way Hydrochloric acid or hydrobromic acid, ferchloric acid, nitric acid or thiocyanic acid, Sulfuric or phosphoric acids, or organic acids such as formic acid, vinegar Acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, Succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, Ascorbic acid, hydroxymaleic acid, dihydroxymaleic acid, benzoic acid, phenylacetic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, anthranilic acid, cinnamic acid, mandelic acid, Salioylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, methanesulfonic acid, Ethanesulfonic acid, hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, Naphthalenesulfonic acid or sulfanilic acid0 The peptides obtained according to the process can be used in the form of pharmaceutical preparations.

These contain the peptides in a mixture with one for the enteral or parenteral administration suitable pharmaceutical, organic or inorganic Carrier material For the same, such substances come into question: those with the polypeptides do not react, like .z.3. Gelatin; Lactose, glucose, table salt, starch, magnesium stearate, Talc, vegetable oils, benzyl alcohol, rubber, polyalkylene glycols, petrolatum, cholesterol or other known excipients. The pharmaceutical preparations can z, B. as a lyophilisate or in liquid form as solutions, suspensions or emulsions previous. If necessary, they are sterilized and / or

or contain auxiliaries such as preservatives, stabilizers, wetting agents or emulsifiers. You can also use other therapeutically valuable substances contain.

The invention is described in the following examples, The temperatures are given in degrees Celsius.

The following systems are used in thin layer chromatography.

System 43A-: tert-amyl alcohol-isopropanol-water (100: 40: 10) System 43C: tert-amyl alcohol-isopropanol-water (51:21:28) System 45: sec.butanol-3% aqueous ammonia ( 70:30) System 52: n-butanol-glacial acetic acid-water (75: 7.5: 21) System 52A z n-butanol-glacial acetic acid-water (67:10:23) System 53: n-butanol-formic acid-water (60: 0.75: 39) system 54 t sec, butanol-isopropanol 9% monochloroacetic acid. (58: 8: 34) system 55: etha, nol-8 plus aqueous sodium chloride (75:25) system 59: methyl ethyl ketone Pyridine-water (60:15:25) system 70: ethyl acetate-pyridine-water (40:20:40) system 89: ethyl acetate-acetone-water (72: 24: 4) system .96: sec. Eutanol-glacial acetic acid Water (67:10:23) system 100: ethyl acetate-pyridine-glacial acetic acid-water (62: 21: 6: 11) system 101: n-butanol-pyridine-glacial acetic acid-water (38: 24: 8: 30) system 10IA : n-butanol-pyridine-glacial acetic acid-water - (42: 24: 4: 30) system 101B: n-butanol-pyridine-glacial acetic acid-water (40: 24: 6: 30) system 102E: ethyl acetate-methyl ethyl ketone-glacial acetic acid- Water (50: 30: 10: 10) System 104: chloroform-methanol-17% aqueous ammonia (41: 4k: 18) system 111A: n-butanol-pyridine-ammonia (26%) -water (42: 24: 4: 30) system 121: isopropanol- Ammonia (26%) -water (70:10:20) System 121A: isopropanol-ammonia (26%) -water (85: 5: 10) System 87: isopropanol-glacial acetic acid-water (77: 4: 19) System 110: ethyl acetate-n-butanol-pyridine-glacial acetic acid-water (42: 21: 21: 6: 10) System 1: benzene-ethanol (80:20) 2: benzene-ethanol (90:10) "3: benzene -Aethanol (95: 5) "4: n-amyl alcohol-formic acid-water (70:20:10) 5: n-butanol-acetic acid-water (6636: 16ß7: 16.7) 6: n-butanol-pyridine- Acetic acid-water (66.6: 12.5: 4.2: 16.7) "7: n-amyl alcohol-pyridine-water (50:30:20)" 8: choroform-methanol-glacial acetic acid (87.4: 9.7: 2.9).

Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 10 11 12 13 Ser Ala Tyr Try A BOC # OH Z # OH BOC # OH H # OMe B BOC 2) -5) OMe CH OMe DZ OMe EH OMe F BOC OMe G BOC N2H3 10 11 12 13 Fig. 12th Fig. 13: - - 14 15 16 17 18 19 Arg Asn Leu Asn Asn Phe AZ # OH Z # ONP Z # ONP Z # ONP Z # ONP H # N2H2-BOC OSU OSU OSU BZ 3) N2H2-BOC CH N2H2 -BOC DZ 3) N2H2-BOC EH N2H2-BOC FZ 3) N2H2-BOC GH N2H2-BOC HZ 3) N2H2-BOC IH N2H2-BOC JZ 4) 5) N2H2-BOC KZ N2H3 14 15 16 17 18 19 Fig. 14 14 15 16 17 18 19 Arg Asn Leu Asn Asn Phe A BOC # OH BOC # OH BOC # OH BOC # OH BOC # OH H # N2H2-Z B BOC N2H2-Z CH N2H2-Z D BOC N2H2-Z EH N2H2 -Z F BOC N2H2-Z GH N2H2-Z H BOC N2H2-Z IH N2H2-Z j BOC N2H2-Z KH N2H2-Z 14 15 16 17 18 19 Fig. 15 14 15 16 17 18 19 20 Arg Asn Leu Asn Asn Phe His AZ # OH Z # ONP Z # OH Z # ONP Z # ONP Z # OH H # NH-NH-BOC BZ 2) -5) NH-NH-BOC CH NH-NH-BOC DZ 3) NH-NH -BOC EH NH-NH-BOC FZ 3) NH-NH-BOC GH NH-NH-BOC HZ 2) -5) NH-NH-BOC IH NH-NH-BOC JZ 3) NH-NH-BOC KH NH- NH-BOC LZ 4) 5) NH-NH-BOC MZ NH-NH2 14 15 16 17 18 19 20 Fig. 16 .: '-. r.

# Fig. 18 19 Example 1 Leu-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg (H2 +) - Asn-Leu-Asn-Phe-His-Arg (H2 +) - Phe-Ser (tBu) -Gly-Met-Gly-Phe -Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2 are dissolved in 20 ml of 90% trifluoroacetic acid with ice cooling and the solution is left at 0 ° under nitrogen for 30 minutes. It is then poured into 300 ml of ice-cold, peroxide-free ether. The resulting precipitate is filtered off with suction, washed several times with peroxide-free ether and vacuum dried over caustic soda. The powder is then again dissolved in 20 ml of ice-cold 90% strength trifluoroacetic acid, warmed to 25 °, nitrogen left at 250 for 1 hour. The solution is then again poured into 300 ml of ice-cold, peroxy-free ether, and the resulting precipitate is filtered off with suction, washed with ether and dried in vacuo at room temperature over caustic soda. The following operations are carried out under nitrogen and using solvents free of peroxide and oxygen .

To convert it into the acetic acid salt - the above obtained Trifluoroacetate of I in 20% acetic acid dissolved and over an acid (1.2 x 15 cm) of a weakly basic ion exchange resin (Merck No. II, acetate form) filtered. The column is washed with 20 acetic acid until the U.V. control of the eluate no longer shows the presence of 1. Thereupon the eluate evaporated with addition of n-octanol in vacuo at 409, bath temperature, the residue freed from octanol by washing with petroleum ether and dried. It will acetic acid salt d, it obtained Dotriacontapeptide as a pale yellowish resin. To the: Purification is the product of a countercurrent distribution in the system n-ßutanol CL liter) -l-n. Acetic acid (1 liter) -ammonacetate (500 mg) subjected. For this purpose one solves it in 30 ml each upper and lower phase of this system and fills the Lö solution into the the first three pipes of the distribution apparatus.

After 250 distribution steps, the individual fractions are by means of Thin layer chromatography (on alumina plate, system 104, Rf = 0.50) on their Checked purity. The fine dotriacontapeptide is in fractions 140 - 165 (maximum of the weight curve in fraction 156, K = 1.65). These factions will combined, evaporated in vacuo at 40 ° bath temperature and the residue for removal dried by ammonium acetate at 40 ° and 0.01 mm Hg. The dotriacontapeptide obtained in this way proves to be uniform in thin-layer chromatography on aluminum oxide (proof with Reindel-Hoppe reagent); it shows the following Rf values: Rf45 = 0.33; Rf52 = 0.59; Rf104 = 0.56.

The sulfoxide of I has the following Rf values: Rf45 = 0.30; Rf52 = 0.54; Rf104 = 0.50.

For electrophoresis on cellulose acetate foil ("Cellogel") in acetic acid-formic acid buffer, pH = 1.9, 90 minutes running time, 8 volts / cm, 1 and its sulfoxide show a running distance, 1.1 cm against the cathode.

The starting material can be prepared as follows: 1) H-Thr (tBu) -OMe 12.92 g (40 mmol) of Z-Thr (tBu) -OsHe are dissolved in 200 ml of glacial acetic acid and 3 g of Pd-charcoal (10 %) hydrogenated at room temperature. The intake of Wasserstorr is after an hour completed.

The solution is filtered off from the catalyst and in a water jet vacuum evaporated at 350. After drying in a high vacuum at 35 °, 7.3 g of one result Cels, which is uniform according to the thin-layer chromatogram and is used further directly will.

2) DPC-Ser (tBu) -Thr (tBu) -OMe 19.3 g (38.6 mmol) DPC-Ser (tBu) -OH, cyclohexylamine salt are taken up in 500 ml of chloroform and at 00 three times with 25 ml of l-n, citric acid and five times with 40 ml; half-saturated saline solution. shaken off '. After this Drying the solution over sodium sulfate is evaporated and the foam obtained taken up in 250 ml of ethyl acetate. 5.36 ml (38.6 mmol) of triethylamine are added, the solution cools to -10 ° and mixed with 5.13 ml (38.6 mmol) of isobutyl chlorocarbonate while stirring. It is stirred for 10 minutes at -10 ° and then the cooled to 120 Solution of 7.3 g g (38.6 mmol) of H-Thr (tBu) -OMe in 100 ml of ethyl acetate is added dropwise in such a way that that the reaction temperature -10 ° t 3 exceeds. After completing the entry Stirred for another hour at -10 and then stand overnight at room temperature calmly. The solution is filtered off from the precipitated triethylamine hydrochloride and at 0 ° three times with 20 ml l-n each time. Citric acid and five times with saturated saline washed, dried and evaporated. Crude product (oil): 22.07 g. For cleaning 1 g is chromatographed on a silica gel column (2.5 cm, 30 cm). With petroleum ether and ethyl acetate (1: 1), 787 mg of pure product are eluted after a forerun of 110 ml.

In the thin-layer chromatogram on silica gel in toluene-acetone (7: 3) is Rf = 0.51 ".

3) Z-Val-Leu-OMe Dissolve 19.9 g (100 mmol) of H-Leu-OMe, HCl in 120 ml of dimethylrormamide and 14.6 ml (105 mmol) of triethylamine are added at 0 °. The excreted Triethylamine hydrochloride is filtered off, the filtrate to a solution of 25.1 G (100 mmol) of Z-Val-OH in 200 ml of dimethylformamide (0 °) and then 22.6 g (110 mmol) dicyclohexylcarbodiimide was added dry.

After an hour of stirring at 00 and over power in the refrigerator will be filtered and the solution evaporated at 400 on a high vacuum. The oily residue is aurgepommen in 30 ml of ethyl acetate, the solution cooled to 0 ° and excreted vo Dicyclohexylurea freed. After addition of n-hexane, the product crystallizes out from the filtrate Night out, F.

102 -105 °; [α] D: -41 ° (c = 2.88 in methanol).

- In the thin layer chromatogram on silica gel in chloroform-methanol (98: 2) is Rf: 0.15 and in toluene-acetone (7: 3) is Rf = 0.6o.

4) DPC-Ser (tBu) -Thr (tBu) -NH-NH2 4.253 g (7.4 mmol) DPC-Ser (tBu) -Thr (tBu) -OMe 5.55 ml (approx. 110 mmol) of hydazine hydrate are added to 18 ml of methanol and 10 Left at room temperature for 2 hours and at 400 for 2 hours. The reaction solution is taken up in 450 ml of ethyl acetate and four times with half-saturated saline solution washed. After the solution has been dried over sodium sulfate, it is concentrated to approx. 15 ml and about 5 ml of petroleum ether are added. 3.17 g of the hydrazide crystallize overnight from F. 132 - 1340.

In the thin-layer chromatogram on silica gel in toluene-acetone (7: 3) is Rf = 0.40 5) H-Val-Leu-OMe, HCl 5.66 g (15 mmol) of Z-Val-Leu-OMe are dissolved in 75 ml of methanol in the presence of 15 mmol hydrochloric acid and 859 mg Pd-charcoal (10%) at room temperature hydrogenated. The uptake of hydrogen has ended after 3 hours. It gets off the catalyst filtered off and the solution concentrated to about 15 ml. Crystallized on addition of ether the product. F. 136-139 °.

In the thin layer chromatogram on silica gel in chloroform-methanol (9: 1) is Rf = 0.45; in toluene-acetone (1: 1) Rf is 0.43.

6) TRI-Cys (TRI) -Vai-Leu-OMe 7.13 g (10 mmol) TRI-Cys (TRI) -OH, diethylamine salt are in chloroform at 0o with l-n. Citric acid and half-saturated saline solution washed, the solution dried, evaporated and the resulting Schawn in 50 ml Ethyl acetate added. A 1.4 ml (10 mmol) of triethylamine is added to this Solution of 3.22 g (10 mmol) of H-Val-Leu-OMe, hy-, drocnlorid in 40 ml of ethyl acetate of 00, from which the precipitated triethylamine4dr.ochoride has been filtered off. Then moved one at = 0 ° with 2.26 g (1 mmol) of dry dicyclohexylcarbodiimide, stirred 2 1/2 Hours at 0 ° and leave to stand in the refrigerator overnight. The excreted dicyclohexylurea is filtered off and the solution at 0 ° with 1-n citric acid, 1-n. Sodium bicarbonate and half-saturated sodium chloride solution and dried over sodium sulfate. The solution is concentrated to approx. 20 ml, cooled to 0 ° and further dicyclohexylurea filtered off. Evaporation to dryness gives 853 g of ester. This is used for cleaning Crude product chromatographed on a silica gel column (5 cm; 55 cm).

After a forerun of 400 ml, the main product is mixed with 300 ml of petroleum ether / ethyl acetate (1: 1) eluted in pure form.

In the thin-layer chromatogram on silica gel in chloroform-methanol (99: 1) is Rr = 0.43.

7) H-Cys (TRI) -Val-Leu-OMe 11.08 g (13.3 mmol) TRI-Gys (TRI) -Val-Leu-OMe are dissolved in 75 ml of acetic acid and 12.3 ml of water are added dropwise, so that there is always a clear solution. After stirring for one hour at room temperature 64 ml of water are added to the clear solution, the mixture is filtered, the precipitate is removed and washed with cold 50% acetic acid. The filtrate becomes a high vacuum at 40 ° Evaporated oil, this taken up in 250 ml of ethyl acetate and at 0 ° with 1-n. Sodium bicarbonate and saturated saline solution. After drying over sodium sulfate is elngedampft, 7.07 g of a white product: tes are obtained. The thin layer chromatogram on silica gel in the chloroform-methanol (98: 2) system shows when sprayed with Reindel-Hoppe reagent a spot of Rf 0.30.

8) DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OMe. To 13.6 g (23.8 mmol) DPC-Ser (tBu) -Thr (tBu) -NH-NH2 in 220 ml of dimethylformamide. At -12 ° 32.26 ml Hydrochloric acid in ethyl acetate (1.84-n) 59.25 mmol) and 3.26 ml (28.55 mmol) tert-butyl nitrite given. After 15 minutes at -10 ° .. you then let the solution, cooled to -10 °, of 14.03 g (23.8 mmol) of H-Cys- (TRI) -Val-Leu-OMe. and 8.315 ml (59.25 mmol) of triethylamine in 100 ml of dimethylformamide so that the reaction temperature never -9 ° exceeds. It will be another hour stirred at -100 and then left overnight at room temperature. It is made from the excreted triethylamine hydrochloride filtered off, the filtrate in a high vacuum at 400 a.

evaporated, the oily residue taken up in 500 ml of ethyl acetate, with l-n. Citric acid, l-n. Washed sodium bicarbonate and saturated saline, dried, concentrated to approx. 40 ml and mixed with approx. 10 ml of petroleum ether. Above At night the protected pentapeptide ester crystallizes from F. 177-1780.

In the thin layer chromatogram on silica gel in chloroform-methanol (98: 2) is Rf = 0.45; in toluene-acetone (7: 3), Rf = 0.57.

9) DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OH 2.830 g (2 mmol) DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu -OMe are dissolved in 60 ml of 75 figem dioxane and 3 ml of 2-n.

Sodium hydroxide solution (6 mmol) added, after 1 1/2 hours at room temperature the dioxane is evaporated in a water jet vacuum at 400, ethyl acetate and water added and at, 0 ° with 1-n. Citric acid adjusted to pH 3. The ethyl acetate phase is washed with brine, dried and evaporated.

The result is a product which, according to the thin-layer chromatogram, is obtained Silica gel is uniform. ar in chloroform-methanol t7i3) = 0.40; Rf45 = 0.55.

10) H-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OH 2.22 g (2 mmol) DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val -Leu-OH are dissolved in 20 ml of methylene chloride and 12 ml of chloroacetic acid in water (from 75 g of chloroacetic acid and 25 ml of water) were added. The clear solution is 15 minutes stirred at room temperature, cooled to 0 ° and then 70 ml of water are added, The pH is adjusted to 6.5 with concentrated ammonia, whereupon the product precipitates. The aqueous solution is decanted off and the residue is washed three times with water triturated and then lyophilized. Rub the product three times with ether supplies a white powder which, according to thin-layer chromatography, is uniform. It will continue to be used in this form.

In the thin-layer chromatogram on silica gel, R'f45 = 0.48; Rf101 = 0.65; Rf52 = 0.75.

11) Z-Asn-Leu-OMe 16.7 g of H-Leu-OMe and 46.0 g of Z-Asn-ONP are used in 100 ml of freshly distilled dimethylformamide dissolved. The solution will be 19 hours left to stand at 25 °. Then add 1.2 liters of water and suck the crystalline one Precipitation from; The dipeptide derivative is vacuum dried at 400 and then twice recrystallized from methanol-water. 180-181 °; [α] D20 = + 9 ° (c = 2.05 in chlorine form).

12) H-Asn-Leu-OMe 15.0 g of Z-Asn-Leu-OMe are dissolved in 400 ml of t-butanol-water (9: 1) dissolved and hydrogenated in the presence of 2 g palladium-carbon (10% Pd). After completion the hydrogenation is filtered off with suction from the catalyst and evaporated at 400. The residue is used immediately.

13) Z-Ser (tBu) -Asn-Leu-OMe 8.90 g H-Asn-Leu-OMe and 11.0 g Z-Ser (tBu) -OH are dissolved in 100 ml of freshly distilled dimethylformamide. The solution will be on Cooled to 0 ° and with 3.90 g of N-hydroxysuccinimide and then with 8.70 g of dicyclohexylcarbodiimide offset. After standing at 0 ° for 30 minutes and at 25 ° for 18 hours, the body is excreted Dicyclohexylurea suction filtered and the filtrate poured into 800 ml of ice water. The resulting crystalline precipitate is dried in vacuo at 400 and then recrystallized from methanol-water. That in analytically pure form Z-Ser (tBu) -Asn-Leu-OMe obtained melts at 202 ° -205 °; [α] D20 = -18 ° (c = 1.05 in glacial acetic acid).

14) H-Ser (tBu) -Asn-Leu-OMe 6.3 g of Z-Ser (tBu) -Asn-Leu-OMe are used in 600 ml of methanol dissolved and hydrogenated in the presence of 1.2 g of palladium-carbon (10% Pd). After 1.25 hours, the catalyst is filtered off with suction and in vacuo at a bath temperature of 30 ° evaporated to dryness.

The crystalline tripeptide derivative obtained in this way shows on thin-layer chromatography on silica gel plates in the system chloroform-methanol (90:10) Rf = 0.11.

15) BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-OMe 5.0 g H-Ser (tBu) -Asn-Leu-OMe and 6.7 g of BOC-Cys (TRI) -ONP are in 30 ml of freshly distilled dimethylformamide solved.

The yellow solution is left to stand at 260 for 42 hours.

This is followed by the addition of 500 ml of ice water and the precipitated Product sucked off. It is dissolved in ethyl acetate and, the solution with 5% citric acid solution, and then washed with water. This is followed by cooling with ice to remove p-Nitrophenol 5 x with a mixture of 1 part by volume of 5% potassium carbonate and 5% potassium bicarbonate solution and then washed with water. The ethyl acetate solution leaves a resinous product after drying and evaporation. This will be four times reprecipitated from acetone and petroleum ether, the tetrapeptide derivative obtained as a solid powder has a temperature of 181-182 °; [α] D20 = -11 ° (c = 2.09 in methanol).

By thin layer chromatography on silica gel plates, Rf43A is 0.57; Rf in ethyl acetate = 0.10; Rf in toluene-acetone (7: 3) = 0.05; Rf in chloroform-methanol (9: 1) = 0.45.

16) BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-NH-NH2 7.0 g BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-OMe are dissolved in 70 ml of methanol and the solution, cooled to 0, with 7 ml of hydrazine hydrate offset. After 16 hours at 20, an ice-cold solution of 600 ml of 2-n. acetic acid admitted, the gelatinous precipitate grated well, sucked off and with a lot of water washed neutral. The powder obtained is dried at 40 ° in a vacuum and then suspended in 100 ml of acetonitrile. After rubbing, it is sutured and with 400 dried in vacuo. The obtained BOC-Cys (TRI) -Ser (tBu) -Asn-Leu hydrazide shows F. 216-2180.

In thin layer chromatography on silica gel plates it shows.

the following Rf values: Rf = p.55 in dioxane-water (98: 2); Rf - 0.52 in chloroform-methanol (8: 2); Rf102E = 0.70.

17) BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val -Leu-OH At -15 °, 1.075 g (1.26 mmol) of BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-NH-NHS in 15 ml of dimethylformamide 1.71 ml of hydrochloric acid in ethyl acetate (1.84-n; 3.15 mmol) and 0.174 ml (1.51 mmol) of tert-butyl nitrite (l, 74 ml from a solution of 1 ml of tert-butyl nitrite made up to 10 ml with dimethylformamide).

The solution is stirred for 15 minutes at -10 ° and then the to -12 ° cooled solution of 1.104 g (1.26 mmol) of H-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OH and 0.6.1 ml (4.41 mmol) of triethylamine in 15 ml of dimethylformamide were added dropwise. That Reaction mixture is one hour at 100 and four hours at room temperature touched. After evaporation in a high vacuum to a small volume and addition of water a powdery product is obtained, which is rubbed twice with water and then is dried in a high vacuum over potassium hydroxide solution., This crude product is dissolved by order purified from methanol. Separates from a methanolic solution saturated at 500 over, night at 00 a white powder, which is in the Thin-layer chromatogram proves to be uniform.

In the thin-layer chromatogram on silica gel, Rf43C = 0.54; Rf121A = 0.65; Rf45 = 0.50; Rf70 = 0.75; Rf in chloroform-methanol (8: 2) = 0.40.

846 mg (0.5 mmol) BOC-Cys (Tri) -Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Val-Leu-OH are dissolved in 8 ml of dimethylformamide and 350 mg (1.1 mmol) of mercury (II) acetate added in 4 ml of methanol, from the clear solution begins after about 5 minutes A gelatinous product is to be excreted, the reaction mixture is 'stirred' for 60 minutes at room temperature, diluted with 59 ml of dimethylformamide and then passed for 20 minutes Hydrogen sulfide and nitrogen for 15 minutes. The black precipitate is filtered off through "Celite" and washed with dimethylformamide. The filtrate concentrated, it is concentrated to 40 ml and nitrogen is passed through for 15 minutes. This solution is' simultaneously with a solution of 170 mg (0.6 mmol) of diiodoethane in 40 ml of methanol at room temperature to 20 ml of dimethylformamide and 20 ml of methanol within a Added dropwise with stirring for an hour. After 10 hours at room temperature the solvent becomes evaporated in a high vacuum at 40 ° and the oily residue first three times with petroleum ether, then rubbed twice with water and lyophilized. The purification of the brownish The crude product is achieved through countercurrent advantages in the System: methanol buffer-chloroform-carbon tetrachloride (10: 3: 5: 4). The buffer is made from 28.6 ml of glacial acetic acid and 19.25 g of ammonium acetate Water made up to 1 liter, received.

After 135 distribution steps, the product is in the elements 49-63 (rmax = 53; K = 0.65). The contents of these elements are evaporated to dryness and freed from ammonium acetate overnight in a high vacuum at 400. It result 254 mg of a chromatographically uniform product.

In the thin-layer chromatogram on silica gel, Rf45 = 0.42; Rf121A = 0.70; Rf70 = 0.75; Rf53 = 0.43; Rf43A = 0.22.

To a stirred solution of 3.73 g (14.78 mmol) of iodine in 500 ml of methanol 2.50 g (14.78 mmol) of BOC-Cys (TRI) -Ser- (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OH are added at room temperature added dropwise in 500 ml of methanol within 45 minutes. After the end of the entry, stir continue for 1 hour and then decolorize the solution at 00 with 1.0-n. aqueous thiosulfate solution (26.05 ml l-n.

Thiosulfate). The clear solution is in a water jet vacuum at 30 ° concentrated approx. 100 ml. Then 1.5 liters of water are added and the precipitated product is filtered off Product and wash it off with water.

After drying over potassium hydroxide, the crude product weighs 2.3 g. It is triturated twice with petroleum ether and designed to purify a countercurrent distribution in the methanol-buffer-chloroform-carbon tetrachloride system (10: 3: 5: 4). [Buffer: 28.6 ml of glacial acetic acid; 19.25 g ammonium acetate, made up to 1 liter with water]. After 135 steps, the main product is in elements 50-79 (rmax = 64; K = 0.96). The contents of these elements are evaporated to dryness in a high vacuum (40 °) and the ammonium acetate is sublimed off. The received Leu-OH (1.49 g = 83% of theory) turns out to be uniform in the thin-layer chromatogram (silica gel). It shows the same Rf values as the product described under a).

19) Z-Tyr (tBu) -Try-OMe Add to a solution of 29.8 g of Z-Tyr (tBu) -OH 11.1 ml of triethylamine and 20.5 g of H-Try-OMe chlorohydrate in 190 ml of chloroform are stirred 30 minutes at room temperature and then cool to 0 °. At this temperature it drips a solution of 29.9 g of dicyclohexylcarbodiimide in 50 ml of chloroform is added. One stirs 1 hour at 0 ° and all night at room temperature. After filtering off the dicyclohexylurea the solution is concentrated in vacuo, the residue is taken up in ethyl acetate and this solution at Oo with 5% citric acid solution and 2-n. Sodium carbonate shaken out. The oil obtained after evaporation of the ethyl acetate is chromatographed on 1 kg of silica gel. The crude product is placed on the column in 250 ml of toluene, with 9 liters of toluene, 6.5 liters of toluene-chloroform (9: 1) and 15 liters of toluene-chloroform (1: 1) are eluted.

The fractions which are pure according to the thin-layer chromatogram are combined and evaporated to dryness in vacuo. 27.9 g of Z-Tyr (tBu) -Try-OMe are obtained; Rf = 0.65 in chloroform-acetone (1: 1) on silica gel.

20) H-Tyr (tBu) -Try-OMe 26.2 g g of Z-Tyr (tBu) -Try-OMe are in 520 ml Dissolved methanol in the presence of 4 g of 10% palladium-carbon and 21, b ml of 2,1-n. Hydrochloric acid hydrogenated at room temperature. When the hydrogenation is complete, the catalyst is removed filtered off and the filtrate evaporated to dryness in vacuo at 300. You get 22.0 g of H-Tyr (tBu) -Try-OMe-chlorohydrate Rf = 0.45 in chloroform-acetone in 21) Z-Ala-Tyr (tBu) -Try-OMe 20.8 g of H-Tyr (tBu) -Try-OMe, HCl are dissolved in 40 ml of dimethylformamide with 16,, 7 g of Z-Ala-ONP and 6.1 ml of triethylamine were added and the mixture was stirred until the mixture crystallized. After standing overnight, 300 ml of ethyl acetate are added, the insoluble one Material is filtered off and the filtrate at 0 ° with dilute potassium carbonate solution Waxed nitrophenol-free, then shaken out with 0.1 m citric acid and water. After the solution has been dried and the ethyl acetate has evaporated, the residue is poured into 25 ml of chloroform dissolved, drawn up on a column of 200 g of silica gel and treated with chloroform eluted.

The product obtained is recrystallized from ethyl acetate-hexane. The Z-Ala-Tyr (tBu) -Try-OMe melts from 108 ° with slow decomposition. Rf = 0.5 in chloroform-acetone (1: 1).

22) H-Ala-Tyr (tBu) -Try-0Me 10.3 g carbobenzoxy compound of 21) are in 200 ml of methanol in the presence of. 0.5 g of 10% palladium-carbon as before shown hydrogenated. The decarbobenzoxylated product shows Rf = 0.3 in the chloroform-methanol system (9: 1).

23) DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-OMe 8.3 g DPC-Ser (tBu) -OH (from the Cyclohexylammonium salt released with citric acid) in 25 ml of ethyl acetate at -10 ° with 2.7 ml of triethylamine and 2.7 ml of isobutyl chloroformate. After 5 minutes at -10 °, the solution of 8.1 g of H-Ala-Tyr (»Bu) -Try- 'becomes the mixed anhydride OMe in 20 ml of dimethylformamide and 10 minutes at -10 °, 2 hours at 0 ° and stirred for 12 hours at room temperature.

The mixture is diluted with ethyl acetate, at 0 ° with 0.1 m citric acid and, shaken saturated potassium carbonate solution, then at room temperature with Washed water, dried and evaporated to dryness in vacuo. The residue -w; Lrd recrystallized from ethyl acetate-hexane. 10.5 g of DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-OMe are obtained with a temperature of 148 - 149 ° (decomposition). Rf = 0.7 in the system chloroform-methanol (9: 1) 24) DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-NH-NH2 1.34 g of the tetrapeptide derivative of 23) dissolved in 7.5 ml of methanol, treated with 0.75 ml of hydrazine hydrate and 24 hours at Left to stand at room temperature. By adding 7.5 ml of methanol and 15 ml of water the DPC-Ser crystallizes (tBu) -Ala-Tyr (tBu) -Try-NH-NH2 from. That Product is suction filtered, washed well with 50% methanol and water and im Dried under high vacuum over concentrated sulfuric acid.

The product melts from 136 ° with slow decomposition.

Rf = 0.35 in the chloroform-methanol (9: 1) system.

25) Z-Asn-Phe-NHNH-BOC 1.4 g H-Phe-NHNH, -BOC, 1.94 g Z-Asn-ONP and 3 ml of dimethylformamide are stirred at room temperature until the mixture solidifies. After leaving to stand overnight, the dipeptide derivative is filtered off with ether and washed with'-ether Ni trophenol-free. The product starts at 211 ° below Melt decomposition. Rf '= 0.55 in chloroform- "methanol (8: 2) on silica gel.

26) H-Asn-Phe-NH-NH-BOC 28.75 g of the carbobenzoxy compound of 25) are: dissolved in 1 liter of methanol, in the presence of 2.9 g of 10% palladium, um carbon hydrogenated at room temperature. After the decarbobenzoxylation is complete, the catalyst becomes filtered off and the filtrate evaporated to dryness. The product melts at 160 - 161 °. Rf = 0.15 in chloroform-methanol (8: 2).

27) Z-Asn-Asn-Phe -NH-NH-BOC 20.93 g of H-Asn-Phe-NH-NH-BOC are used in 45 ml of dimethylformamide dissolved with warming, at room temperature with 22.7 g of Z-Asn-ONP added and stirred until the mixture solidifies.

After standing overnight, the festo mass is poured into 160 ml Dimethylformamide dissolved with warming, precipitated by dropping into 1 liter of ether, the product filtered off and washed nitrophenol-free with acetone, Rf = 0.24 in chloroform-methanol (8: 2).

28) H-Asn-Asn-Phe-NHNH-BOC 35.8 g of the carbobenzoxy compound described under 27) are dissolved in 560 ml of dimethylformamide with heating. After cooling to room temperature 3.6 g of 10% palladium-carbon are added and the mixture is hydrogenated. After complete decarbobenzoxylation the catalyst is filtered off and the dimethylformamide solution is concentrated to approx. 100 ml ud the H-Asn-Asn-Phe-NHNH-BOC is precipitated by dropping it into 1 liter of asther. Rf100 = 0.35 on silica gel.

29) Z-Leu-Asn-Asn-Phe -NHE-BOC 2s, 5g of H-Asn-Asn-Phe-NHNH-BOC will be dissolved in 58 ml of warm dimethylformamide, at room temperature with a solution of 24.2 g of Z-Leu-ONP in 16 ml of dimethylformamide are added and the mixture is stirred until the mixture stiffens. After standing overnight it is triturated with ether, filtered off, in 190 ml of dimethylformamide are dissolved and the product is added dropwise to 1.5 liters of ether like again. It is filtered off and washed with ether nitrophenol-free Rf = 0.45 in the system chloroform-methanol (7: 3) on silica gel.

30) H-Leu-Asn-Asn-Phe-NHNH-BOC 36.0 g of the carbobenzoxy derivative obtained under 29) will in 800 ml of dimethylformamide as under 28). described hydrogenated and worked up. 27.9 g of H-Leu-Asn-Asn-Phe-NHNH-BOC are obtained. Rf100 = 0.35.

31) Z-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC 27.9 g of H-Leu-Asn-Asn-Phe -NH-NH-BOC are dissolved in, 145 ml of dimethylformamide 'warm, at room temperature with; one '; Solution of 21.6 g of Z-Asn-ONP in 20 ml of dimethylformamide and stirred until the mixture solidifies. After leaving to stand overnight, work-up is carried out as described under 29). 34.2 g = 88% of theory are obtained. Th. Of the pentapeptide derivative Rf100 = 0.5.

32) H-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC 34.2 g of the one described under 31) Pentapeptide derivatives are hydrogenated and worked up as described under 30).

The H-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC shows Rf100 = 0.2.

33) Z-Arg-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC hydrochloride 23.6 g of Z-Arg-OH are suspended in 87 ml of dimethylformamide and cooled with cold water with 21.2 ml 3,6-n. Hydrochloric acid in dioxane is added, whereby solution occurs. 28.2 g des pentapeptide derivative mentioned under 32) are dissolved warm in 440 ml dimethylformamide, cooled to room temperature and added to the solution of Z-Arg-OH, HCl, then another 17.4 g of dicyclohexylcarbodiimide. The mixture is stirred for 24 hours, and the crystallized out is filtered off Dicyclohexylurea and the filtrate is concentrated to about 250 ml. By dropping them the crude product is precipitated from this solution in 2 liters of ether. This will dissolved by adding while stirring in 280 ml of hot dimethylformamide. After cooling down the product is brought to room temperature with 870 ml of saturated sodium chloride solution and 1740 ml of water precipitated. The precipitated product is suction filtered and well with half-saturated Washed saline. The moist suction filter is dissolved in 1 liter of warm dimethylformamide dissolved and dehydrated azeotropically by concentrating the solution twice. The unusual one Common salt is filtered off, the 'filtrate is added dropwise to 2.4 liters of acetonitrile, the resulting precipitate was filtered off after a few hours with acetonitrile and ether washed and dried in vacuo at 40 °. The Z-Arg-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC hydrochloride has Rf100 = 0.25.

34) Z-Arg-Asn-Leu-Asn-Asn-Phe-NHNH2 hydrochloride 10.6 g of the under 33) described hexapeptide derivative who with stirring and ice cooling in 50 ml 90% trifluonacetic acid dissolved, warmed to room temperature and 20 minutes at leave this temperature. The solution is then poured into 500 ml of ether were added dropwise, the precipitate was filtered off and washed acid-free with ether. The product is introduced into 90 ml of warm dimethylformamide and stirred for some time and then precipitated in powder form with 1 liter of ether. This crude product will stirred with 170 ml of isopropanol for 1 hour at room temperature, filtered off, with isopropanol and ether and washed in a vacuum 400 dried. You get the Z-Arg-Asn-Leu-Asn-Asn-Phe-NHNH2 hydrochloride, the Rf96 = 0.38 on silica gel having.

35) Z-Phe-Ser (tBu) -OCH3 27.0 g (90 mmol) of Z-Phe-ON are in 250 ml dissolved in absolute tetrahydrofuran together with 12.5 ml (90 mmol) of triethylamine and chilled. 14.7 ml (112 mmol) of isobutyl chlorocarbonate are added dropwise at -18 ° and stirred for half an hour at <-10 °. To the white suspension (triethylamine hydrochloride) are now 18.1 g of H-Ser (tBu) -OCH3 hydrochloride (85.5 mmol) in finely powdered Form, diluted with 350 ml. Tetrahydrofuran and then leaves 15.0 b1 (108 mmol) of triethyl'-amine add dropwise in 100 ml of tetrahydrofuran. The mixture is stirred for a further 3 hours at about -10 ° and then overnight at room temperature. The suspension is then reduced to about 250 in vacuo ml concentrated, taken up in a lot of ethyl acetate and diluted with dilute citric acid solution (3 x 200 ml), dilute sodium carbonate solution (3 x 200 ml) and saturated saline solution (5 x 200 ml) shaken out. dried over sodium sulfate and evaporated. That The crude product is dissolved in 200 ml of ethyl acetate-hexane (1: 1). Out of the solution When cooling down, white drusen crystallize out, which are isolated and dried, The product melts at 92.5 ° -94 °.

[α] D20 = + 3 ° # 0.5 ° (c = 2.2% in methanol).

Thin layer chromatography on silica gel plates shows the product the following Rf values: In chloroform-acetone (1: 1) Rf = 0.70; in chloroform-methanol (95: 5) Rf = 0.70; Rf43A = 0.65; Rf89 = 0.86.

36) Z-Phe-Ser (tBu) -hydrazide 25.4 g Z-Phe-Ser (tBu) -OCH3 (55.7 mmol) was dissolved in 250 ml of methanol 'and with ice-cooling with 28 ml (575 mmol) of hydrazine hydrate offset. The solution is left in the refrigerator for 67 hours, with a thick layer Crystal cake forms.

The product is isolated and dried. It melts at 158.5 - 159.5 °. [α] D20 = + 16 ° # 0.5 ° (c = 2.3% in glacial acetic acid).

In the thin-layer chromatogram on silica gel layers, Rf = 0.27 in chloroform-methanol (95: 5); Rf43A = 0.57; Rf89 = 0.65.

37) Z-Phe-Ser (tBu) -Gly-OCH3 18.4 g of Z-Phe-Ser (tBu) hydrazide (40.4 mmol) are dissolved in 100 ml of dimethylformamide and at -20 ° with 41.6 ml of hydrogen chloride in ether (2.43 n., 101 mmol) were added. 7.15 ml of t-butyl nitrite are added to the clear solution (59 mmol) in ko ml of dimethylformamide and rinsed with 20.ml of dimethylformamide. After 10 14.0 ml (101 mmol) of triethylamine in 60 minutes at temperatures below -9 ° ml of dimethylformamide zu.und enters 8.85 g of H-Gly-OCH3 hydrochloride (70 mmol). In Three portions of 5.6 ml each of triethylamine are added at intervals of 10 minutes (total 121 mmol). The pH is then approx.

8.5. The cooling mixture is then removed and kept for three hours at 00, then stirred overnight at room temperature. ' After concentrating the reaction mixture in a vacuum to form a thick paste, this is taken up in a large amount of ethyl acetate and washes the solution successively with 3 servings of diluted citric acid solution, 3 servings of dilute sodium carbonate solution and 5 servings of saturated saline solution The: .. after drying and evaporation of the solution obtained crude product is in Dissolved ethyl acetate, filtered through a glass frit and with approx. 800 ml Aether displaced. The protected tripeptide ester crystallizes out very slowly, P. 140-1419.

[α] D20 = + 13 ° # 0.5 ° (c = 2.3% in glacial acetic acid).

'In the thin-layer chromatogram on silica gel layers is in chloroform-methanol (95: 5) Rf = 0.61; in chloroform-acetone (9: 1) Rf = 0.28; Rf102E = 0.82; Rf43c = 0.72.

38) H-Phe-Ser (tBu) -Gly-OCH3 7.4 g of Z-Phe-Ser (tBu) -Gly-OCH3 will be decarbobenzoxylated in methanol with hydrogen in the presence of 1.0 g of palladium carbon (10% Pd). After the uptake of hydrogen has ended, it is filtered, evaporated and the resulting product Raw product processed further directly. The product shows in the thin layer chromatogram the following Rf values on silica gel layers: in chloroform-metha, nol (95: 5): Rf = 0.26; Rf43A = 0.60; Rf52 = 0.51.

39) Z-Arg-Phe-Ser (tBu) -Gly-OCH3 hydrobromide 5.3 g Z-Arg-OH (17.3 mmol) are slurried in 15 ml of dimethylformamide and, after cooling with an ice-common salt mixture, with 5.9 ml of hydrobromic acid in methanol (3.06 n., 18 mmol) were added. After a short stir creates a clear, colorless one Solution. To do this, leave it at -18 ° a solution of 5.6 g of .H-Phe-Ser (tBu) -Gly-OCH3 (14.4 mmol) in 15 ml of dimethylformamide added dropwise and a further 7 ml of dimethylformamide are added. Then, one adds a solution of 3.85 g of dicyclohexylcarbodiimide in 5 ml of methylene chloride are added, replaced after 1 hour the cooling mixture through ice water, and leaves after another three hours overnight stir at room temperature. The deposited precipitate of dicyclohexylurea is then filtered off, the filtrate concentrated to half in vacuo and cooled to 0 ° and stirred into 600 ml of ether. The resulting precipitate is decanted off, the precipitate washed with ether, dissolved in about 50 ml of methanol and again with 600 ml of ether precipitated. After renewed dissolution in methanol, some.

steam t and dried in a high vacuum. The product shows in the thin layer chromatogram on silica gel layers: Rf52 ~ Rfioo. = 0.54; on Cellulose Selekta Rf45 40) H-Arg-Phe-Ser (tBu) -Gly-OCH3-Hydrochloride-Hydrobromide 6.3 g of Z-Arg-Phe-Ser (tBu) -Gly-OCH3-hydrobromide (8.5 mmol) are dissolved in 350 ml of methanol dissolved and treated with 2.9 ml of hydrogen chloride in dioxane (3.09-n., 8.9 mmol). It is hydrogenated in the presence of 1.2 g of 10% palladium-carbon at room temperature and atmospheric pressure. After the hydrogen uptake has ended, it is filtered off, evaporated, the residue obtained was dissolved in 30 ml of methanol and the solution in 700 ml of ether are stirred in. This creates a yellowish paste that can be used again in is solved and felled in the same way.

Finally the product is redissolved in methanol and filtered and evaporated. In the thin-layer chromatogram, the product shows a layer of silica gel the following values: Rf59 = 0.12; Rf96 = 0.38; Rf100 = 0.13.

41) Z-His-Arg-Phe-Ser (tBu) -Gly-OCH3 Slurry 7.6 grams of Z-His-NH-NH2 (25 mmol) in 60 ml of dimethylformamide, treated with 31 ml of hydrogen chloride at -16 ° in ether (2.43-n. ', 75.3 mmol) and mixes the clear, yellowish color thus obtained Solution at 180 with 3.2 ml of t-Bu-.

tyl nitrite (95%, 26.5 mmol) in 10 ml of dimethylformamide.

After 10 minutes, 10.4 ml of triethylamine (75 mmol) are pre-cooled in 25 ml Dimethylformamide and 8.5 g of H-Arg-Phe-Ser (tBu) -Gly-OCH3, 2HCl (14 mmol) in 50 ml added pre-cooled dimethylformamide. It is diluted with 40 ml of dimethylformamide and 4 portions of 1.0 ml triethylamine each (total 28.7 mmol) was added. It is left for three hours at 0 ° and for 20 hours at room temperature stand, the precipitated salts are then filtered off and the reaction solution is concentrated on the vacuum to about 3/4 'of the volume. By repeating. Stir in the product in a lot of ethyl acetate a crude product is obtained which is distributed in the countercurrent System methanol-buffer-chloroform-carbon tetrachloride (9: 3.5: 8: 2) over 130 steps is cleaned, (buffer as under 18); rmax = 54; K ~ 0.7). The pure fractions are combined, evaporated, freed from ammonium acetate at 40 ° in a high vacuum and after solution lyophilized in water.

In the thin-layer chromatogram of the silica gel layers, Rf52A = 0.34; Rf101A = 0.71; Rf121 = 0.76; Rf111A = 0.40.

42) H-His-Arg-Phe-Ser (tBu) -Gly-OCH3, 2 CH3-COOH 2.9g Z-His-Arg-Phe-Ser (tBu) -Gly-OCH3 (3.3 mmol) are dissolved in 300 ml of methanol-glacial acetic acid (9: 1) with the addition of 500 mg of palladium-carbon (10 % Pd) hydrogenated at room temperature and atmospheric pressure. After the hydrogen uptake has ceased is filtered and evaporated to dryness in vacuo, whereby one over the residue twice 50 ml each time, toluene evaporated. The product, a white powder, shows up in thin-layer chromatograms The following Rf values on silica gel plates: Rf101B = 0.51; Rf96 = 0.10; Rf111A = 0.28; on cellulose Selekta Rf45 = 0.55; Rf52A = 0.44.

43) H-His-Arg-Phe-Ser (tBu) -Gly-OH-CH3-COOH 2.84 g H-His-Arg-Phe-Ser (tBu) -Gly-OCH3, 2 CH3-COOH are dissolved in 40 ml of water with 15:, ml of 2N piperidine solution in water Stirred for 14 minutes at room temperature. The clear, slightly yellow solution will then with 15 ml 2-n. aqueous acetic acid neutralized, filtered and lyophilized. The lyophilist, a slightly yellow, deliquescent mass, becomes in a high vacuum Stripped off in a water bath of 60 ° piperidine acetate.

Now wird.in 10 ml of methanol and dissolved in 400 ml of ethyl acetate stirred in. The white, flaky precipitate is filtered and washed with ethyl acetate and dried. 2.0 g of pure product are obtained in the form of a white powder, in the Thin layer chromatogram on silica gel layers is Rf101B = 0.45; Rf121 = 0.47; on cellulose Selekta: Rf45 = 0.48; Rf55 = 0.38; Rf96 = 0.36.

In paper electrophoresis (paper S + S S 2043 6; 5 hours; 2000 Volt; pH 6.3) the substance migrates 6.5 cm to the cathode.

In electrophoresis on Selekta cellulose plates (pH 6.3; 3 hours; 1000 volts) the substance migrates 8.5 cm to the Ka method.

44) Z-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OHCH3-COOH 3.77 g of Z-Arg-Asn-Leu-Asn-Asn-Phe-NH-NH2 are slurried in 100 ml of dimethylformamide, and after cooling with an ice-salt bath with 3.0 ml of hydrogen chloride in dioxane (3.09-n., 9.27 mmol) were added. Part of the suspension goes into solution. Now you give in -10 ° 0.5 ml of t-butyl nitrite (95%, 4.14 mmol). After 5 minutes at -10 to 12 ° a further 0.08 ml of t-butyl nitrite are added. After a total of 11 minutes of reaction time 1.29 ml of triethylamine (9.26 mmol) are added and, immediately afterwards, the precooled solution of 2.40 g H-His-Arg-Phe-Ser (tBu) -Gly-OH, CH3-COOH (3.6 mmol) in 30 ml of dimethylformamide, then another 20 ml of cold dimethylformamide.

1.08 ml of triethylamine are then added in four portions over a period of 2.5 hours (7.8 mmol), the pH ranges between 7 and 8. You leave the approach Night at 0 ° and gives in the morning. the solution of the in the same way as described above made from 1.81 g of Z-Arg-Leu-Asn-Asn-Phe-NH-NH2 Azids too, then another 0.62 ml (4.47 mmol) of triethylamine, again the batch is left over Night at 00 ,. in the morning in a vacuum at a bath temperature of 450% approx.

15 ml volume and stirs the batch in 800 ml of ethyl acetate. That The crude product filtered off is washed with ethyl acetate and dried. It will be one countercurrent distribution in the n-butanol-Elsessig-Wasser (4: 1: 5) system over 300 stages subject, rmax = 94; K = 0.46. Those determined by thin layer chromatography pure fractions are combined, evaporated, dissolved in water, filtered and lyophilized.

The product shows in the thin-layer chromatogram on silica gel layers: Rf101B = 0.56; Rf104 = 0.07; on cellulose Selekta: Rf45 = 0.37.

45) H-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH, 2 CH3-COOH 1.48 g of Z-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH are dissolved in 350 ml of dimethylformamide-water (4: 1) with the addition of 700 mg of 10% palladium-carbon at room temperature and Hydrogenated at atmospheric pressure. After 15 hours of shaking under a hydrogen atmosphere is filtered, evaporated in vacuo, dissolved in water and lyophilized. That pure product shows the following Rf values in a thin layer chromatogram on silica gel plates: Rf101B = 0.42; Rf101A = 0.34; on cellulose Selekta: Rf45 = 0.27; on Alox-Camag: Rf52A = 0.51; Rf111A = 0.28; Rf121 = 0.27.

46) DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH 756; mg DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-NHNH2 in 15 ml of degassed dimethylformamide at 300 with 0.6 ml of 3,6-n, Chlo, 'r-.

hydrogen in dioxane and then with 0.124 ml of t-butyl nitrite offset. After 15 minutes at -15 °, 1.7 ml of 2-n.

Sodium carbonate solution was added dropwise (pH r - 8) and the azide with ice-cold Water precipitated. The precipitate is sucked off and washed with ice-cold water. 272 mg of H-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH, acetate are dissolved through gentle warming in 5.5 ml of dimethylformamide, gives 0.51 ml of 1-m at room temperature. Triethylamine in dimethylformamide is added and the above solution is added at 00.00 produced solid azide. The suction filter is filled with 4 ml of cold dimethylformamide and 2 ml of cold ethyl acetate washed out. The solution is now stirred at 0o, whereby after 20 minutes 0.5 ml water and after 2 hours 0.05 ml l-m. Triethylamine in dimethylformamide be admitted. After 4 hours the reaction mixture with a lot of ether? pleases. The product, which initially precipitated out as an oily product, was obtained after repeated trituration with ether and reprecipitation from methanol-ether as a flaky product. The received The crude product is obtained by Craig distribution over 150 stages in a mixture of methanol, buffer, chloroform and carbon tetrachloride (10: 3: 7: 4) (buffer as under 18)). The pure substance is dependent on the distribution elements 75-112.

These are combined and the organic solvents are removed in vacuo and the aqueous solution lyophilized, the protected pentadecapeptide is obtained, that by chromatography on silica gel in the system ethyl acetate-pyridine-water (49:24:27).

Rf = 0.4 shows.

47) Z-Gly-Phe-Gly-Pro-OtBu.

15.5 g of Z-Gly-Phe-Gly-BH (shown according to J.R. Vaughan & YES. Eichler, J. Am. Chem. Soc. 75, 5556 (1953)] in 100 ml of absolute tetrahydrofuran and 5.3 ml of absolute triethylamine 'and leaves to the solution cooled to -200 Stir 4.64 ml of isobutyl chlorocarbonate are added dropwise so that the internal temperature is -150 does not exceed. This is followed by a reaction at -10 ° for 10 minutes, then again cooled to -20 ° and a solution of 7.6 g of H-Pro-OtBu in 30 ml of dimethyl form.

amide was added dropwise with stirring in such a way that the internal temperature was 0 ° does not exceed. The mixture is left to stand at 0 ° for 18 hours, then triethylamine hydrochloride is sucked off and the filtrate evaporates at a bath temperature of 490. The residue is in 200 ml of ethyl acetate dissolved and the solution with sodium bicarbonate solution, water, 10% Washed tartaric acid solution and water, dried with sodium sulfate and evaporated. The residue is crystallized from ethyl acetate-petroleum ether, mp 66 ° -71 °; [α] D20 = -45 ° (c = 1% in chloroform).

48) Z-Gly-Phe-Gly-Pro-OH.

25.6 g of Z-Gly-Phe-Gly-Pro-OtBu are dissolved in 260 ml of 90% strength Trifluoroacetic acid and let the solution stand at 220 for 20 minutes. This is done on the rotary evaporator Concentrated to an oily consistency at a bath temperature of 250 ml, then 0 250 ml Add water and knead the viscous mass through at 0.

The aqueous phase is decanted off and the residue twice more washed with 100 ml of water in the same way.

It is then dried in a high vacuum at a bath temperature of 30 °. Of the the residue obtained is mixed with 600 ml of ether; petroleum ether (1: 1), triturated, sucked off and dried. The product is processed further directly.

49) Z-Thr (tBu) -Pro-NH2.

5.27 g of Z-Thr (tBu) -OH and 1.56 g of H-Pro-NH2 are dissolved in 60 ml of acetonitrile, the solution cools to 00 and 3.11 g of dicyclohexylcarbodiimide are added Hour at 0 ° and 20 hours at room temperature is sucked off from the dicyclohexylurea, the 'filtrate evaporated and the residue taken up in ethyl acetate. The ethyl acetate solution is washed with dilute citric acid solution, soda solution and water, with sodium sulfate dried and evaporated. The colorless residue shows on thin layer chromatography on silica gel plates Rf45 = 0.76; Rf430 = 0.70; Rf52 = 0.62.

50) H-Thr (tBu) -Pro-NH2 hydrochloride.

5.88 g of Z-Thr (tBu) -Pro-NH2 are dissolved in 200 ml of methanol and 13.7 ml of 1-n. HCl dissolved and hydrogenated in the presence of 66o mg palladium carbon (10% Pd) Completion of the hydrogen take-up is turned to dryness at a bath temperature of 350 steams and dried. The residue 'is crystallized from ethanol-ether; F0 170 - 173 °.

51) Z-Glu (OtBu) -Thr (tBu) -Pro-NH2.

3.0 g of H-Thr (tBu) -Pro-NH2-hydrochloride and 4.92 g of Z-Glu (OtBu) -ONP are suspended in a mixture of 10 ml of dimethylformamide and 1.64 ml of triethylamine. The reaction mixture is stirred for 20 hours at 30e, then diluted with 150 ml of ethyl acetate and the Ethyl acetate solution at 0 ° four times with 50 ml of saturated potassium carbonate solution each time, 50 ml of water, 2 x 50 ml of 5% citric acid solution and 2 x 50 ml of water, dried with sodium sulfate and evaporated. The evaporation residue is made from ethyl acetate-hexane crystallized, mp 159-161 ° 52) H-Glu (OtBu) -Thr (tBu) -Pro-NH2 4.4 g of Z-Glu (OtBu) -Thr (tBu) -Pro-NH2 are dissolved in 120 ml of methanol and in the presence of 1.2 g of palladium carbon (10 X Pd) hydrogenated. After the hydrogenation has ended, the catalyst is suctioned off and the filtrate is evaporated, the tripeptide derivative being obtained as a crystalline crust. In thin-layer chromatography on silica gel plates, it shows the Rf value = 0.50 in the system chloroform-methanol (9: 1) 53) Z-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2.

4.47 g of Z-Gly-Phe-Gly-Pro-OH and 3.00 g of H-Glu (OtBu) -Thr (tBu) -Pro-NH2 are dissolved in 40 ml of acetonitrile and the solution is added with 1.76 g of dicyclohexylcarbodiimide. After 12 hours at 25 °, the excreted dicyclohexylurea is suctioned off and the filtrate evaporated to dryness. The residue "is taken up in ethyl acetate and washed the solution with tartaric acid solution, water, sodium bicarbonate solution and water, dried with sodium sulfate and evaporated. The residue is used for purification twice reprecipitated from ethyl acetate solution by adding petroleum ether. With thin layer chromatography on silica gel plates, Rf43c = 0.52; Rf52 = 0.57.

54) H-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2.

3.95 g of Z-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-Nh2 are dissolved in 80 ml of methanol and hydrogenated the solution of hydrogen from '600 mg of palladium carbon (10% Pd). When the hydrogenation has ended, the catalyst is filtered off with suction, the filtrate evaporated to a volume of 10 ml, 20 product m: added, an additional 10 ml concentrated and the product extracted with 70 ml petroleum ether. The greasy precipitation is grated to a powder, suction filtered and dried; Yield 3.3g. With thin layer chromatography on silica gel plates, Rf43C = 0.27; Rf52 = 0.24; Rf in chloroform-methanol (8: 2) = 0.10.

55) DPC-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2 Suspend 3.72 g DPC methionine (G.Nr. 17108/67, Case 6106 / 1-3) and 5.21 g H-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro- NH2 in 30 ml of acetonitrile and then 1.98 g of dicyclohexyl carbodiimide are added. after the Air in the reaction vessel, Nitrogen is displaced, is stirred at 30 ° for 20 hours. It is then diluted with 45 ml of methanol, slightly heated and then sucked off the still undissolved dicyclohexylurea and the filtrate concentrated in vacuo. The residue is dissolved in ethyl acetate and at 0 ° as below 52), washed neutral. The vinegar solution leaves behind when it dries and evaporation 9.12 g of colorless product which crystallizes from ethyl acetate-hexane: F. 1820 q In thin-layer chromatography on silica gel plates, Rf43C = Rf, in chloroform-methanol (9: 1) = 0.30.

56) H-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2, 3.0 g of the under 55) obtained octapeptide derivatives are dissolved in 35 ml of methylene chloride and at Room temperature with 28 ml of a mixture of monochloroacetic acid and water (3: 1) offset. After 10 minutes at room temperature, in-170 ml ice-cold, 2-n. Soda solution poured. The octapeptide derivative which separates out is mixed with 209 ml of n-butanol / ethyl acetate (1: 1) extracted and the organic phase is then washed neutral with water Evaporated to dryness, the octapeptide derivative with free amino group as colorless residue is obtained, which is obtained by thin layer chromatography on silica gel plates Rf100 = 0.23 and Rf43C shows 0.51.

57) DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His Arg-Phe-Ser (tBu) -Gly-OH, Ditosyla 258 mg of the pentadecapeptide diacetate described under 46) at 400, dissolved in 4 ml of absolute pyridine and then at 0 ° with 1.025 ml of a 4 % solution of toluenesulfonic acid monohydrate (2 equivalents). The solution is evaporated to dryness in a high vacuum at a bath temperature of 35 ° and the residue rubbed with ether. This is followed by suction and drying (at 30?) With the Ditosylate, as a solid powder, is obtained. RflOlA = 0.58; Rf110 = 0.09; Rf87 = 0.48.

58) DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-Met-Gly-Phe-Gly -Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2 254 mg of the ditosylate obtained under 57), 138 mg H-Met Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2 and 22.3 mg of N-Hy droxysuccinimid are dissolved in 1 ml of dimethylformamide with heating dissolved to 60 ° and the solution cooled to 250 with 0.3 ml of a dimethylformaid solution added, which contains 30 mg of dicyclohexylcarbodiimide. The reaction mixture is under Leave nitrogen for 4 hours at 25 ° and 12 hours at 450, the deposited Dicyclohexylurea filtered off and the filtrate with a mixture of 15 ml of benzene and 40 ml of petroleum ether precipitated. The resulting precipitate is centrifuged off, dried and after dissolving in 2.5 ml of dimethylformamide again by adding 30 ml of benzene petroleum ether.

(1: 2) like. After decanting the solution, the precipitate becomes dried. Yield 390 mg of crude product. For cleaning, it is multiplicatively distributed over 200 levels in the methanol-buffer-chloroform-carbon tetrachloride system (10: 3: 6: 5; buffer such as under 18). The pure product 'is obtained from' the elements 103-132, the maximum is located in element 122 (K = 1.5). The yield is 213 mg; Rf110 = 0.36; Rf52a = 0.25; Rf = 0.42.

59) H-Ser (tBu) -Ala-Tyr (tBu) Try-Arg-Asn-Leu-As'n-Asn-Phe -His-Arg-Phe-Ser (tBu) GlyMet-Gly-Pbe-Gly-ProrGlu (OtBu) Thr (tBu) -Pro-NH2-tritosylate.

210 mg of the protected tricosapeptide amide obtained under 58) are dissolved in 10 ml of glacial acetic acid (83%) - water (7: 1: 2) and leaves the solution for 1 hour stand at 300. Then the product with free α-amino group is obtained by adding 100 ml of ether precipitated and centrifuged off. The supernatant is mixed with 25 ml of petroleum ether added, concentrated to a volume of 3 'ml and the resulting precipitate centrifuged off.

Both precipitates are combined and dissolved in 5 ml of methanol-0.1-M acetic acid (-2: 1) solved. The solution is passed through a column of Merok ion exchanger No. II filtered in the acetate form, (column 9 mm; 125 mm). The eluate is concentrated to 2 ml and then lyophilized. It is then dried in a high vacuum at 45 ° to constant weight and dissolve the residue in 10 ml of 90% pyridine. 27 mg of toluenesulfonic acid monohydrate are then added and the solution evaporates to dryness in a high vacuum a. The residue is dissolved in 2 ml of water, the solution is lyophilized and then in a high vacuum at 40 ° dried.

This gives 167 mg of tritosylate of the protected tricosapeptide- 'admis with a free α-amino group. Rf96 = 0.27; Rf101A = 0.58; Rf = 0.15.

Leu-OH, 1.30 g of the tritosylate of sequence 10 obtained under 59) - 32, 38 mg of N-methylmorpholine and 86 mg of N-hydroxysuccinimide are fresh in 7 ml distilled dimethylformamide dissolved with heating to 70 °. The solution is then cooled to 250 and gives 115 mg of dicyclohexylcarbodiimide. The reaction mixture will left to stand under nitrogen for 9 hours at 250 and 8 hours at 45 °. On that - Is filtered off from the dicyclohexylurea and the filtrate with 80 ml of benzene and 400 ml of petroleum ether are added., The crude protected Dotriacontapeptidamid which precipitates out is dissolved in 25 ml of methanol and by adding 100 ml of benzene and 250 ml of petroleum ether failed. The precipitate is then suction filtered and dried.

For purification, a multiplicative distribution in the system methanol-buffer (as under 18) - chloroform-carbon tetrachloride (10: 3: 6: 5) is subjected over 300 steps (phase volume: 25 ml). Thin-layer chromatographic control (systems as below) shows that fractions 101-130 contain the pure, protected sequence 1-32 (maximum element 116, K = 0.63). In the case of thin layer chromatography on silica gel, Rf52 = 0.20; Rf96 = 0>44; Rf100 = 0.23; Rf87 O, on aluminum oxide, Rf45 = 0.67.

Leu-OH in 0.7 ml of degassed dimethylformamide and 0.116 ml of an lm. Solution of N-methylmorpholine in dimethylformamide are at -15 ° with 0.116 ml of a lm. Solution of pivaloyl chloride in dimethylformamide are added. After 3 minutes at -10 ° to -15 a solution of 172 mg H-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-hrg-Phe- Ser (tBu) -Gly-OH, diacetate in 0.4 ml of dimethylformamide and 0.04 ml of a solution of N-methylmorpholine in dimethylformamide were added dropwise. After 30 minutes at -5 ° to -10 °, it is precipitated with ether and the solid product is isolated. This crude product is purified by a Craig distribution over 530 stages; System: methanol buffer-chloroform-Te: carbon trachloride (10: 3: 7: 4) (buffer as under 18). The pure fractions (partition coefficient: 0.9-0.95) are combined, the solvent is evaporated off in vacuo and the remaining aqueous solution is lyophilized. 155 mg of a white powder are obtained. Rf96 = 0.4; Rf43C = 0.18.

Leu-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH, 2 HC1 and 85 mg of the peptide of the sequence 25-32 obtained under 56) are under Passing nitrogen with 10 mg of N-hydroxysuccinimide, dissolved in 0.4 ml of dimethylformamide, added and stirred at 4 until everything is dissolved. After adding 12.5 mg of dicyclohexylcabrodiimide, dissolved in 0.1 ml of dimethylformamide, the mixture is stirred at 400 for a further 12 hours. With 8 ml of peroxide-free ether, the crude product is precipitated and centrifuged. To the Cleaning is distributed as described under 60). The product obtained is with completely identical to that described under 60).

Example 2: Tyr (tBu) -Try-Arg (H2 +) - Asn-Leu-Asn-Asn-Phe-His-Arg (H2 +) - Phe-Ser (tBu) -Gly-Met-Gly-Phe-Gly-Pro-Glu ( OtBu) -Thr- (tBu) -Pro-NH2 are hydrolyzed as described in Example 1 with trifluoroacetic acid to split off the protective groups, converted into the acetate and purified by countercurrent distribution.

The product proves itself after thin-layer chromatography and electrophoresis on cellulose acetate film than with the Dotriacontapeptiamid obtained in Example 1 identical.

The starting material can be produced as follows: 1. BOC-Val-Leu-OBzl 19.9 g (92 mmol) of BOC-Val-OH are dissolved in 200 ml of freshly distilled dimethylformamide Dissolved with heating, after cooling to .09 25.9 ml of absolute triethylamine were added and the solution, cooled to -10 °, is mixed with 8.64 ml of ethyl chlorocarbonate. After 10 minutes at -10 °, a solution of 23.2 g (90 mmol) of H-Leu-OBzl. HCL in 90 ml of dimethylformamide were added. -The mixture is 30 minutes at -10 °, 3 hours left at 0 ° and at 200 for 16 hours. Then the undissolved material is filtered off and the filtrate was concentrated to dryness in a high vacuum. The residue is dissolved in ethyl acetate dissolved and the solution with dilute citric acid solution, water, sodium bicarbonate solution and water, dried with sodium sulfate and evaporated. After adding the product crystallizes from petroleum ether overnight.

M.p. 88.5-89.5 °; [α] D21 -22 ° (c = 2 in dimethylformamide); Rf2 = 0> 73 2. H-Val-Leu-OBzl-HCl 22.8 g (54 mmol) of BOC-Val-Leu-OBzl are dissolved in 228 ml of methylene chloride. This solution takes 11/2 hours gaseous hydrogen chloride passed. After evaporation to dryness, the residue becomes rubbed three times with ether.

F. 192-195.

Rf2 = 0.27.

3. BOC-Cys (Bzl) -Val-Leu-OBzl 14.2 g (32.8 mmol) BOC-Cys (Bzl) -ONP and 11> 7 g (32.8 mmol) of H-Val-Leu-OBzl.HCl are dissolved in 137 ml of dimethylformamide and 4.7 ml of triethylamine and 0.5 ml of glacial acetic acid are added to the solution, which has been cooled to 00 admitted. The yellow suspension is 3 hours at 00 and overnight at room temperature touched.

Now the precipitated triethylamine hydrochloride is filtered off, and the filtrate was evaporated to dryness in a high vacuum. The residue is dissolved in ethyl acetate dissolved and shaken out, twice with water, twice with 0.1-n hydrochloric acid, twice with 10% saline solution, six times with 10% soda solution and twice with saturated saline solution.

After drying over sodium sulfate, it is evaporated to dryness. The protected tripeptide crystallizes from ethyl acetate / petroleum ether with a melting point of 113-115 °; [α] 20 D -33 ° (c = 1 in dimethylformamide); .Rf3 = 0.30.

4. H-Cys (Bzl) -Val-Leu-OBzl, TFA 10 g (16> 3 mmol) BOC-Cys (Bzl) -Val-Leu-OBzl are dissolved in 20 ml of 90% trifluoroacetic acid and 1 hour at room temperature left. Then 200 ml of peroxide-free ether is added and 2 hours below Stirred ice / table salt cooling.

The precipitate is suction filtered and twice with peroxide-free ether washed and dried in vacuo over caustic soda.

168-173 °; Rf4 = 0.77.

5. BOC-Ser-Thr-OBzl is added to a solution of 14.7 g (38> 2 mmol) BOC-Ser-OH, dicyclohexylammonium salt in 100 ml of methylene chloride 9> 4 g (38.2 mmol) H-Thr-OBzl, HCl and 6o ml of methylene chloride, stirred for 10 minutes at room temperature and then cools to 5 °. A solution of 8.0 g (38.6 g) is added dropwise at this temperature mmol) of dicyclohexylcarbodiimide in 18 ml of methylene chloride. The mixture is stirred for 3 hours 55 ° and all night at room temperature. After filtering off the dicyclohexylurea and dicyelohexylamine hydrochloride shake the solution three times with 0.1 N hydrochloric acid, twice with 20% saline solution, once with 10% sodium bicarbonate solution and dried twice with 20% saline solution and over sodium sulfate. The solution is concentrated to about 100 ml, cooled to 50 and more dicyclohexylcrns'toff filtered off. Evaporation to dryness gives 15.8 g of ester. This is used for cleaning Product crystallized from ethyl acetate-hexane.

110-111 °; [α] D20 -8.5 ° (c = 2 in dimethylformamide) Rf2 = 0.33 6. H-Ser-Thr-OBzl, TFA 9'3 g (23> 5 mmol) BOC-Ser-Thr-OBzl are dissolved in 13> 8 ml of 90% trifluoroacetic acid and the solution for 20 minutes left at 22 °. Then it is added dropwise with stirring to 138 ml of dry ether, Stirred for one hour, and left to stand at -10 ° for 2 days. The resulting precipitation is filtered off and washed three times with dry ether and in vacuo over caustic soda dried.

128-129 °; Rf2 = 0.65; Rf4 = 0.50.

7. BOC-Asn-Leu-N2H2 -Z 34> 8 g (150 mmol) of BOC-Asn-OH are in 300 ml of acetonitrile slurried and mixed with 38 g (150 mmol) of Woodward's reagent K.

21 ml (150 mmol) of triethylamine are then left with stirring and cooling Add dropwise so that the internal temperature does not exceed + 30 °. After completing the registration is stirred for 45 minutes at 250. 52.2 g are added to the almost clear solution (165 mmol) H-Leu-N2H2-Z, HCl and 23 ml (165 mmol) triethylamine in 185 ml acetonitrile, the reaction mixture, which soon solidifies, stirs overnight at room temperature, cools to -10 ° and stir for 2 hours at -10 °. Then you suck the crystalline lens Precipitate and wash the dipeptide derivative once with cold acetonitrile, once with ethyl acetate and eight times with water until chloride-free. The product obtained will dried at 400 in vacuum.

F. 194-196; [α] 20 D -39 ° (c = 2 in dimethylformamide); Rf1 = 0.45.

8. H-Asn-Leu-N2H2-Z, TFA 46.3 g (94 mmol) BOC-Asn-Leu-N2H21-Z become dissolved in 92.6 ml of 90% trifluoroacetic acid and left at 200 for 1 hour. Thereon the solution is added dropwise with stirring to 975 ml of ether and stirred for 15 minutes. The ether is decanted from the precipitation, another 210 ml of ether are added and the mixture is stirred 1 hour at 350 under reflux, cools down to 100, and refined the product a few hours. It is dried over caustic soda in a vacuum.

175-179 °; Rf4 = 0.46.

9. BOC -Ser-A sn-Leu-N2H2-Z 16 6 g (78 mmol) BOC-Ser-OH are in 200 ml of acetonitrile dissolved, the solution cooled to 0 ° and with 20 g (78 mmol) of Woodward's Reagent K and 11 ml of triethylamine are added. After 11/2 hours of stirring at 00 becomes To the clear solution 39.6 g (78 mmol) of H-Asn-Leu-N2H2-Z-TFA and 11 ml of triethylamine added in 200 ml of dimethylformamide. After stirring overnight at room temperature the solution is evaporated in a high vacuum at 500, the oily residue in 500 ml Acetate added, twice with O, 2-N hydrochloric acid, twice with 10% sodium chloride solution, twice with 10% sodium bicarbonate solution and twice with 10% saline solution extracted, 'dried over sodium sulfate, and concentrated to 100 ml. After adding crystallized from 20 ml of hexane the tripeptide derivative. Twice Crystallization from ethyl acetate-10% water gives uniform BOC-Ser-Asn-Leu-N2H2-Z.

20 F. 149-151; [c] D -18.50 (c = 2 in dimethylformamide); Rf1 = 0.33.

10. BOC-Ser-Asn-Leu-N2H 15.3 g (26.7 mmol) BOC-Ser-Asn-Leu-N2H2-Z are in 456 ml of dimethylformamide in the presence of 2.4 g of Pd-charcoal (10%) at room temperature hydrogenated. The reduction is complete after 5 hours. The solution becomes from the catalyst filtered off and concentrated in a high vacuum. Rubbed in three times with ether delivers a white powder.

20 m. 179-181 °; [a] D -14 ° (c = 2 in dimethylformamide) Rf6 = 0.73.

11. BOC-Ser-Asn-Leu-Ser-Thr-OBzl 8.4 4 g (18.7 mmol) BOC-Ser-Asn-Leu-N2H; in 49 ml of dimethylformamide are at 200 with 19.1 ml of hydrogen chloride in tetrahydrofuran (1.97-n; 37.6 mmol) and 2.53 ml of iso-amyl nitrite were added. After 8 minutes at 200 a pre-cooled solution of 8 g (18.7 mmol) of H-Ser-Thr-OBzl.1,13 TFA and 8.3 ml of triethylamine in 49 ml of dimethylformamide were added. The reaction mixture is 3 Leave days at 0 °. Then it will be retired Triethylamine hydrochloride filtered off, the filtrate concentrated in a high vacuum at 400 to 40 ml, with stirring 400 ml of ethyl acetate are added and leave at -10 overnight. The precipitation will sucked off and washed twice with ethyl acetate.

The still moist crude product is treated with a mixture of 60 ml of 0.03 N * hydrochloric acid and 40 ml. ethyl acetate stirred, and left at -100 overnight. The following morning is decanted from the precipitation, the residue dissolved in acetone-water, in a water jet vacuum the acetone evaporated, whereby the water-insoluble pentapeptide derivative precipitates. The precipitate is filtered off, washed with water, dried, and from dimethylformamide / ethyl acetate crystallized.

207-208 °; [α] D20 -19.5 ° (c = 2 in dimethylformamide) Rf6 = 0.88 12. BOC-Ser-Asn-Leu-Ser-Thr-OH 10 g (14.1 mmol) BOC-Ser-Åsn-Leu-Ser-Thr-OBzl are in 300 ml of dimethylformamide with the addition of 1.25 g of Pd-charcoal (10%) at room temperature and atmospheric pressure hydrogenated. The reduction is complete after 2.5 hours. the The solution is filtered off from the catalyst over asbestos and concentrated in a high vacuum.

Trituration of the residue with ether gives a loud Thin layer chromatogram of uniform pentapeptide.

141-144 °; 20-13 ° (c = 2 in dimethylformamide) Rf6 = 0.34.

13. H-Ser-Asn-Leu-Ser-Thr-OH, TFA 8.5 g (13.7 mmol) BOC-Ser-Asn-Leu-Ser-Thr-OH are dissolved in 85 ml of 90% strength trifluoroacetic acid and left at 200 for 1 hour.

The solution is then concentrated to approx. 25 ml, with stirring 20Q ml of ether are added and left to stand at 100 overnight. The resulting precipitation is filtered off and again stirred with 100 ml of ether for 5 hours, filtered off, washed twice with ether and dried in vacuo over caustic soda.

161-168 °; [α] D -11 ° (c = 2 in dimethylformamide) Rf: 0.09.

14. BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-Thr-OH 5> 3 g (13 mmol) BOC-Cys (Bzl) -OSU and 7.4 g (10 mmol) of H-Ser-Asn-Leu-Ser-Thr-OH, 1.93TFA are in 75 ml of dimethylformamide solved. From a solution of 150 mmol of triethylamine in 100 ml of dimethylformamide is 18.1 ml (27.2 mmol) were added dropwise with stirring until the reaction mixture was damp Indicator paper shows a pH of 6.4. The solution is 3 days at room temperature stirred, then evaporated to dryness in a high vacuum. The residue is twice Stirred with 250 ml of ethyl acetate, then three times with a mixture of 100 ml of ethyl acetate and 20 ml of 5N citric acid solution, filtered off and in vacuo at 400 dried.

F. 184-185; 20-13 ° (c = 2 in dimethylformamide); Rf6 = 0.45.

15. BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-Thr-Cys (Bzl) -Val-Leu-OH 3.90 g (6 mmol) H-Cys (Bzl) -Val-Leu-OH. 1.15TFA are dissolved in 50 ml of freshly distilled dimethylformamide dissolved, mixed with 4.48 g (5> 5 mmol) BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-Thr-OH and and stirred at room temperature until dissolved. The solution is then cooled to 00 from, gives 1.26 g (11 mmol) of N-hydroxysuccinimide and 0.98 ml of triethylamine in 45 ml Dimethylformamide, cools down further to -22 °, and gives 1.13 g (5.5 mmol) of dicyclohexylcarbodiimide in 10 ml of dimethylformamide. The mixture is stirred for 1 hour at 220 and then for 3 days Room temperature, filtered from the precipitated dicyclohexylurea and steamed in a high vacuum to dryness. The residue is stirred twice with a mixture of 200 ml of ethyl acetate and 20 ml of 5% citric acid solution, then once with a mixture of 200 ml of ethyl acetate and 20 ml of 5% sodium bicarbonate solution, the product obtained is filtered off and dried at 40 ° in a high vacuum.

Mp 218-221 °; [α] D20 -28.5 ° (c = 2 in dimethylformamide); Rf8 = 0> 43.

16. BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH 2.5 g (1.9 mmol) BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-Thr- Cys (Bzl) -Val-Leu-OH are suspended at -40 ° in 250 ml of dry liquid ammonia. While stirring at the boiling point of ammonia, sodium is added in such a way that the colour of the reaction mixture only becomes light blue. After 50 minutes the nonapeptide derivative has completely dissolved. The mixture is stirred for a further 10 minutes while being maintained the blue color, then add 1 g of ammonium chloride and evaporate in a high vacuum (approx. 1 mm) to dryness. The residue is, with 30 ml of 0.1-n. Mixed hydrochloric acid, the precipitate filtered off, washed six times with water and dried, yield 1.38 g. The aqueous filtrate is extracted three times with ethyl acetate. The ethyl acetate extract is washed twice with l0iger and once with saturated sodium chloride solution, over Dried sodium sulfate and evaporated to dryness. The product is in the thin layer chromatogram uniformly.

Rf5 = 0.86; Rf6 = 0.76; Rf7 = 0.81.

17th

234 mg (0.225 mmol) of BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH dissolved in 100 ml of degassed dimethylformamide and 1 ml of a triethylamine solution added in dimethylformamide (0.95 ml of triethylamine in 10 ml of dimethylformamide, i.e. 0.675 mmol). This solution is simultaneously with a solution of 69.8 mg (0.248 mmol) Diiodoethane in 100 ml of degassed methanol at room temperature to a degassed Mixture of 150 ml of dimethylformamide and 150 ml of methanol within 90 minutes Stir added dropwise. 1 ml of cyclohexane is added and the mixture is evaporated to dryness in a high vacuum a. The residue is twice mixed with a mixture of 10 ml of 2% sodium bicarbonate and 20 ml of chloroform. The pale yellow colored powder is dried in a high vacuum and turns out to be uniform in the thin-layer chromatogram.

Rf5 = 0.82; Rf6 = 0.75; Rf7, = 0.78.

18th

360 mg of the tritosylate obtained under 59) of the sequence 10-32, 15 mg of N-methylmorpholine, 50 mg of N-hydroxysuccinimide and 15 ml of dimethylformamide stirred under nitrogen at 450 for 2 hours. Then cool down to Oo, add 8 mg Add dicyclohexylcarbodiimide, stir for 2 hours at 0 ° under nitrogen, then add more 50 mg of dicyclohexylcarbodiimide are added and the mixture is stirred at 450 for 8 hours under nitrogen. Then it is poured into 100 ml of peroxide-free ether, the precipitate is filtered off with suction and washed it with ether and dry it in a vacuum at 35 °. The crude product is distributed by countercurrent as described under 60).

Example 3 Leu-OH are dissolved in 30 ml of freshly distilled dimethylformamide and 123 mg of N-methylmorpholine. The solution is then cooled to -15 °, and 150 mg of pivaloyl chloride are added with stirring; and the reaction mixture is kept for 5 minutes. Then a solution, cooled to 150, of 2.4 g (0.9 mmol) of H-Ser-Ala-Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe-His is added dropwise -Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH2-discetat in 50 ml of dimethylformamide, 100 mg of N-methylmorpholine, and water. (The best way to get the triacosapeptide solution is to heat the peptide in the mixture of dimethylformamide and N-methylmorpholine to 500 under nitrogen and add water dropwise while stirring until the peptide dissolves).

After the addition has ended, the mixture is left at 0 ° under nitrogen overnight and then in a high vacuum at a bath temperature of 40 ° to a volume of approx. 30 ml. The concentrate is poured into 500 ml of peroxide-free ether and the resultant is sucked off Precipitation from. The thus obtained roche Nα-acetyldotriacontapeptide is used for Purification countercurrent distribution over 100 'stages in the n-butanol-glacial acetic acid-water system (5: 1: 4) subjected Pure Rf52 is isolated from the distribution elements on concentration = 0.55 and Rf104 = 0.69.

The nonapeptide derivative used as the starting material can be prepared as follows: 50 ml, concentrated hydrochloric acid are felt to -10, then 3.0 g finely powdered entered with stirring, displaced the air by nitrogen, heated to 00 and left at 00 for 10 minutes; Then 250 ml of ice water and 20 ml of glacial acetic acid are added and the mixture is filtered through a column of Merok ion exchanger No. II, weakly basic, acetate form. The eluate is evaporated to dryness, the residue is dissolved in water and lyophilized. The lyophilizate is dissolved in 30 ml of water, the piI of the solution is adjusted to 6.8 by adding pyridine and three times the volume of dimethylformamide is added. A solution of 600 mg of p-nitrophenyl acetate in 5 ml of dimethylformamide is then added and the mixture is left to stand for 5 hours at room temperature. It is then evaporated to dryness at a bath temperature of 350 and 0.01 mm Hg and 100 ml of 1% strength solution of pyridine in ether are added. The precipitated nonpaptid derivative is grinded well, sucked off, washed with ether and dried in a vacuum desiccator over conc. Sulfuric acid. The product is cleaned in.

Butanol-water (3: 1) and dissolved over a in the same solvent prepared acid (3.8 cm; 95 cm) "Sephadex" LH-20 was chromatographed. You catch factions 10 ml each, checked for purity by thin-layer chromatography on silica gel plates (System 121A), combine the pure fractions, evaporate to dryness and dry at 35 bath temperature and 0.01 mm Hg.

The H-Ser-Ala-Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe used as the starting material Gly-Pro-Glu-Thr-Pro-NH2-diacetate is prepared as follows: 3.4 g DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe- His-Arg-Phe-Ser (tBu) Gly-Met-Gly-Phe-Gly-Pro-Glu (OtBu) Thr (tBu) -Pro-NH2 are dissolved in 60 ml of 80% acetic acid dissolved and the solution is left to stand at 35 ° for 6 hours. Then you steam dry them, grind the residue with peroxide-free ether, suck it up powder obtained, it dries and dissolves it in 50 ml of 95% trifluoroacetic acid.

The mixture is left to stand under nitrogen at 250 for 1.5 hours and then evaporated to a volume of about 5 ml and pour into 300 ml of peroxide-free ether. The precipitate is filtered off with suction, washed with ether and dried over sodium hydroxide in the desiccator.- Then it is dissolved in 10% acetic acid and filtered through a column from Merck ion exchangers No. II (weakly basic, acetate form). The column is washed with 10% acetic acid until the eluate is in the water shows no more absorption at 280 mμ, the eluate evaporates to dryness, dissolves the Residue in water and lyophilized.

Example 4 Asn-Leu-Asn-Asn-Phe-His, -Arg-Phe-Ser-Gly-Met (0) -Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH2 (thyrocalcitonin sulfoxide).

25 mg of thyrocalcitonin are in 5 ml of 0.05-n. Acetic acid dissolved. In addition are added 0.5 ml of 5% aqueous hydrogen peroxide solution and left for 90 minutes stand at room temperature.

Then add a trace of platinum black and stir until the development of oxygen stops. The platinum is centrifuged off and the supernatant solution is lyophilized. The thyrocalcitonin sulfoxide thus obtained shows in thin-layer chromatography on alumina- Rf52 = 0.51 and Rf104 0.48. During electrophoresis on cellulose acetate (pH = 1.9; 90 minutes; 9 volts / cm) it migrates 1.5 cm against the cathode on Avicel cellulose 2.9 cm.

Example 5 Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met (O) -Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH2 (Nα-acetyl-thyrocalcitonin sulfoxide )).

Dissolve 15 mg of thyrocalcitonin sulfoxide in 10 ml of water and make adjust the pH to 6.5 by adding pyridine. 4 ml of a fresh one are added to the solution produced'0.5% solution of p-nitrophenyl acetate in dimethylformamide-water (1: 3) and let stand for 3 hours at 45 °. Then add 1.2 ml of 5-n. acetic acid and extracted the p-nitrophenol formed and the excess p-nitrophenyl acetate with ethyl acetate. The aqueous phase is concentrated to dryness and dissolved in 3 ml of water -and lyophilized. The crude product thus obtained is in a Craig distribution in System n-butanol-acetic acid-water (5: 1: 4; vol.) With phase volumes of 3 ml each over 181 levels distributed. From distribution elements no. 108 - 127 (µmax = 117; K = 1.83) one isolates a total of 11.5 mg of pure Nα-acetyl-thyrocalcitoninsulfXoxide when evaporating to dryness. The substance shows the following in the thin-layer chromatogram for aluminum oxide ("Camag") Rf values on: Rf52 = 0.51 and Rf104 = 0.64.

On cellulose thin-layer plates ("Avicel"): Rf54 = 0.61.

In contrast to thyrocalcitonin sulfoxide, it is ninhydrin negative.

During the electrophoresis on cellulose acetate strips ("Cellogel") migrates it (at pH 1.9 and 9 volts / cm) in 90 minutes, 0.5 cm to the cathode.

Example 6: Leu-Ser-Ala-Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met (O) -Gly-Phe-Gly-Pro-Glu-Thr-Pro -NH2 (Nα-phenylthiocarbamyl-thyrocalcitonin sulfoxide).

10 mg of thyrocalcitonin sulfoxide are dissolved in 2 ml of pyridine water (1: 1) dissolved and with 15 ml of phenyl isothiocyanate under nitrogen for 1 1/2 hours at 20 ° let react. The mixture is extracted 4 times with 3 ml of benzene and brought the aqueous solution in a vacuum desiccator to dryness. The residue is dissolved in 3 ml each of the lower and upper phases of the n-butanol-glacial acetic acid-water system (5: 1: 4) and cleans it through multiplicative distribution in the system mentioned (3 ml each upper and lower phase) over '177 steps. Fractions of 10 elements each are combined, concentrated and lyophilized. He holds 6 mg of pure Nα-phenylthiocarbamyl-thyrocalcitonin sulfoxide that with chloro-tolidine reagent, Barton's reagent and Pauly's reagent positive, reacts negatively with ninhydron, in electrophoresis on crystalline Cellulose ("Avicel"), the product shows a cathodic migration path at pH 1> 9 2.7 cm (starting product 3.5 cm); Rf value on Alox in the n-butanol-glacial acetic acid-water system (75: 7.5: 21) 0.55 (starting material. (0.45)] on cellulose in the same system 0.29 (Starting material 0.20). Specific activity: 40 MRC units / mg.

Example 7: Asn-Phe-His-Arg-Phe'-Ser-Gly-Met (0) -Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH2 (Nα-pyroglutamyl-thyrocalcitonone sulfoxide).

Dissolve 15 mg of thyrocalcitonin sulfoxide in 1 ml of dimethylformamide, 1 ml of 0.1% triethylamine in dimethylformamide (pH 6.7) and 0.3 ml of water are added 0.2 ml show pyroglutamyl-2,4,5-trichlorophenyl ester and leave 72 hours standing at 20 °. A little water is then added and the mixture is evaporated under reduced pressure one and lyophilized. The backlog becomes a multiplicative. Distribution in the system n-butanol-glacial acetic acid-water (4: 1: 5; upper and lower phase 3 ml each) over 397 steps subject. Nα-Pyroglutamyl-Thyrocalcitoninsulfoxid is in the Levels 270-345 (maximum in element 310). These factions are united that Solution concentrated and lyophilized.

The product obtained has the following in a thin layer chromatogram Rf values based on thyrocalcitonin sulfoxide = 1.0 on: made of aluminum oxide (Alox) : Rf52 = 1.0: Rf104 = 1.0; on cellulose: Rf101 = 1.05: Rf54 = 1.01.

In electrophoresis, it migrates based on thyrocalcitonin sulfoxide = 1.0 against the cathode: on Cellogel, pH 1.9, 140 volts: 0.39, on Cellulose Selecta, pH 1.9, 140 volts: 0.81.

Example 8 Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg Phe-Ser (tBu) -Gly-Met-Gly-Phe-Gly-Pro-Glu ( OtBu) -Thr (tBu) -Pro-OtBu are left in 2 ml of 95% trifluoroacetic acid for 1 hour at room temperature. The mixture is then poured into 20 ml of frozen, peroxide-free ether, filtered and washed with a little ether and the solution of the filtration residue in a little water is allowed to run over a small column of Amberl-ite CG-45 in acetate form. The eluate is monitored by means of a UV cell and the fractions with the substance are lyophilized.

Thin-layer chromatogram on aluminum oxide: Rf45 = 0.36, Rf52 = 0.39, Rf79 = 0.42 Electrophoresis: on selector plates (pH = 1.9, 1.5 hours 280 V) migration distance against the cathode 6.3 cm on Cellogel (pH = 1.9, 1.5 hours 280 V) migration distance against the cathode: 4.3 cm.

The starting material can be prepared as follows: 1. Z-Thr (tBu) -Pro-OtBu 6> 3 g of Z-Thr (tBu) -OH and 2.34 ml of N-methylmorpholine are dissolved in 100 ml of tetrahydrofuran dissolved and 'added at -170 with 3.0 ml of isobutyl chloroformate and after Stirring for 20 minutes at the same temperature with 6> 7 g of H-Pro-OtBu in 100 ml of tetrahydrofuran implemented; After standing at room temperature overnight, it is filtered and the filtrate evaporated to dryness in vacuo in ethyl acetate several times with dilute aqueous citric acid solution, dilute aqueous soda solution and saturated Saline solution shaken out. The oily compound obtained is a thin-layer chromatogram on silica gel: Rf43C = 0.89. Rf45 = 0.75, Rf52 = 0.64 [α] D20 = -44 ° # 1 ° (c = 1.0 in ethanol).

2. H-Thr (tBu) -Pro-OtBu, HCl 2.7 g of Z-Thr (tBu) -Pro-OtBu are in 200 ml of methanol with the addition of 2.9 ml of aqueous 2-n. Hydrochloric acid and 500 mg palladium ~ on-charcoal catalyst (10%) with hydrogen in the shaking duck at atmospheric pressure and room temperature decarbobenzoxylated.

After the hydrogen uptake has ended, filtration and evaporation the product is crystallized from hexane to dryness.

142-145 ° (decomposition).

Thin-layer chromatogram on silica gel: Rf43C = 0.45, Rf45 = 0.70, Rf52 = 0.55; [α] D20 = 66 ° # 0.5 ° [2.2% in ethanol] 3. Z-Glu (OtBu) -Thr (tBu) -Pro-OtBu 7.79 g of H-Thr (tBu) -Pro-OtBu, HCl and 9.79 g of Z-Glu (OtBu) -ONP are used together in 15 ml of dimethylformamide dissolved, mixed with 3.2 ml of triethylamine and overnight at Stirred at 27-30 °.

The solution is evaporated to dryness in vacuo, the residue, taken up in ethyl acetate and chloroform and mixed with citric acid, soda solution and saline solution as with 1.

extracted and the crude product obtained is chromatographed on silica gel. The substance can be eluted with hexane / ether 1: 1 and ether and crystallized then from ether / hexane.

135.137 °.

Thin-layer chromatogram on silica gel: RfCHCl3-MeOH (95: 5) = 0.53, RfCHCl3-acetone 1: 1 = 0.72, Rf45 = 0.82 [α] D20 = -44 ° # 0.5 ° (c = 2 in ethanol) 4th H-Glu (OtBu) -Thr (tBu) -Pro-OtBu 777 mg of the compound described under 3 hydrogenated in 250 ml of methanol with 300 mg of 10% palladium catalyst on charcoal. Man receives a thick oil that can be used for the next stage without further purification will.

Thin-layer chromatogram on silica gel: RfCHCl3-MeOH (9: 1) = 0.48, Rf43A = 0.50.

5. Z-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu 667 mg Z-Gly-Phe-Gly-Pro-OH with the 608 mg of H-Glu (OtBú) -Thr (tBu) -Pro (OtBu) obtained under 4. in 20 ml Acetonitrile dissolved and with the addition of 222 mg of N-hydroxysuccinimide with 269 mg of dicyclohexylcarbodiimide Stirred for 16 hours at room temperature. The precipitated dicyclohexylurea is filtered off off, evaporated in vacuo, the residue dissolved in a lot of ethyl acetate and shaken the Solution as described under 1. After drying and evaporation in a vacuum the crude product was purified by column chromatography on silica gel. It can be elute with ethyl acetate and ethyl acetate / ethanol (4: 1). Then becomes ether fell over.

F. 96990.

Thin-layer chromatogram on silica gel: RfCHCl - MeOH (9: 1) = 3 0.40, Rf102A = 0.75, Rf52 = 0.80.

6. H-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu 858 mg Z-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu are dissolved in 150 ml of methanol in the presence of 200 mg of palladium catalyst (10% Pd on carbon) hydrogenated. The product that you get after trituration with ether-hexane (1: 1) obtained as an amorphous powder, shows in the thin-layer chromatogram on silica gel the following values: RfCHCl3-MeOH (9: 1) = 0.16, Rf102E = 0.17, Rf52 = 0.41.

7. DPC-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu 410 mg DPC-Met-OH are with 750 mg H-Gly-Phe-Gly-Pro-Glu (OtBu) Thr (tBu) -Pro-OtBu in 15 ml of acetonitrile under nitrogen and with the addition of 152 mg of N-hydroxysuccinimide with 271 mg of dicyclohexylcarbodiimide Stirred for 19 hours at room temperature.

After filtering off the dicyclohexylurea, the filtrate becomes evaporated in vacuo, the residue taken up in a lot of ethyl acetate and as in 1. shaken out and evaporated. The crude product is analyzed by column chromatography purified on silica gel. It can be eluted with ethyl acetate / ethanol (4: 1).

Thin layer chromatogram on silica gel RfCHCl-MeOH (9) 3 0.42, Rf102E = 0.39, Rf53 = 0.29.

8. H-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu 190 mg of the below 7. The DPC compound obtained is dissolved in 3 ml of methylene chloride under nitrogen and 2.9 ml of the mixture of monochloroacetic acid / water (3: 1) are added. After 12 Minute reaction at room temperature, the batch is poured into 20 ml of ice-cold 2N soda solution in water and extracted this with three portions of 30 ml n-butanol / ethyl acetate (1: 1).

After washing until neutral with saturated aqueous sodium chloride solution, it is dried and evaporated, and the residue is extracted several times with ether. This extraction residue, an amorphous powder, has the following Rf values in the thin-layer chromatogram on silica gel: Rf43C = 0.31, Rf52 = 0.30, Rf100 0.27.

Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH-ditosylate is with 81 mg of H-Met-Gly-Phe.-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu in 0.2 ml of dimethylfornamide while passing in nitrogen after adding 9 mg of N-hydroxysuccinimide at 400 touched. After everything has dissolved 7.3 mg of dicyclohexylcarbodiimide are added in 1 ml of dimethylformamide and stirred for 22 hours at 40 °. After cooling down, it falls the product with 8 ml of peroxide-free ether and centrifuged off. The raw product becomes in the system methanol / buffer / chloroform / carbon tetrachloride (10: 3: 6: 5) over 100 levels distributed multiplicatively (K = 0.4). (Buffer as in Example 1, under 18). The tubes containing the product are poured together, evaporated and am High vacuum at 400 the ammonium acetate is driven off.

Rf52A = 0.24, Rf100 = 0.259, Rf110 = 0> 65 on silica gel plates.

Example 9 A 5 ml dry ampoule is made from synthetic Thyrocalcitonin, 0.5 mg lyophilized mannitol 20 mg.

For the purpose of subcutaneous injection, the contents are in 0.1 m. Acetate buffer dissolved by pH 4.6.

The contents of the ampoule are used, for example, once a day. -

Claims (5)

Claims 1. and its derivatives in which the amino and / or carboxyl group and / or hydroxyl groups are slotted. 2. BOG-Cys (TRI) -Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OH. 3. Val-Leu-OH. 4th 5. BOC-Cys (TRI) -Ser-Asn-Leu-Ser-Thr-Cys (TRI) -Val-Leu-OH.