CH517074A - Hypocalcaemic peptides - with anti-inflammatory activity - Google Patents

Abstract

Peptides H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala-Tyr-Try-Arg-Asn-Leu- -Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met (or Met (O))-Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH, or analogous cpds. in which one or more of asparagine gps. are replaced by the aspartic acid gp. and/or the methionine gp. in position 25 is replaced by the valine, norvaline, leucine, isoleucine or norleucine gp. or their C-terminal amines or acid addn. salts are useful in treatment of hypercalcaemia and bone diseases, e.g. osteoprosis. They are prepd. by condensation of corresponding aminoacids, where proline can be present as amine, or their activated derivs. with intermediate masking of reactive functional gps. to be excluded from reaction and under formation of disulphide bridge between Cys1 and Cys7 by oxidation. Pref. COOH gps. and pref. OH gps. are masked by tert.-butyl, pref. NH2- on Cys' is masked by tert.-butyloxycarbonyl.

Description

  

  
 



  Process for the preparation of new hypocalcemic peptides
The invention relates to a process for the preparation of the new hypocalcemically active peptide of the formula I H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala Tyr-Try-Arg-Asn-Leu-Asn -Asn-Phe-His-Arg-Phe-Ser-Gly-Met [or Met (O)] -Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH and corresponding compounds in which one or more of the asparagine residues in positions 3, 15, 17 and 18 by the aspartic acid residue and / or the glutamic acid residue, in position 30 by the glutamine residue and / or the methionine residue, in position 25 by the valine, norvaline, leucine, isoleucine or norleucine residue are, their C-terminal amides and acid addition salts and complexes of the compounds mentioned.



   As acid addition salts, particular mention should be made of salts of therapeutically applicable acids such as hydrochloric acid and acetic acid, but above all sparingly soluble salts such as sulfates, phosphates or sulfonates.



   Complexes are to be understood as meaning the compounds, which have not yet been clarified in their structure, which arise when certain inorganic or organic substances are added to long-chain peptides and which give them a prolonged effect. Such substances are described, for example, for ACTH and other adrenocorticotropic peptides. To be mentioned are z. B. inorganic compounds derived from metals such as calcium, magnesium, aluminum, cobalt and especially zinc, especially sparingly soluble salts such as phosphates and pyrophosphates and hydroxides of these metals, and also alkali metal polyphosphates such. B. Calgon N, Calgon 322, Calgon 188 or Polyron B 12. Organic substances that cause the effect to be prolonged are, for example, non-antigenic gelatin, e.g. B.

  Polyoxygelatine, polyvinylpyrrolidone and carboxymethyl cellulose, also sulfonic acid or phosphoric acid esters of alginic acid, dextran, polyphenols and polyalcohols, especially polyphloretin phosphate and phytic acid, as well as polymers and copolymers of amino acids, e.g.



  Protamine or polyglutamic acid.



   The new compounds, in particular the amide of the compound of the formula I, have a hypocalcemic effect. They lower the plasma, calcium and phosphate levels in the blood of mammals, as has been demonstrated by experiments on Wistar rats. They also have anti-inflammatory properties, as was shown in the example of kaolin edema in rats.



   The compounds are also effective on humans. With parenteral administration of 0.01 to 1 mg in 0.1-m.



  Acetate buffer with a pH 4.6 dissolved C-terminal amide of the compound of the formula I, the serum calcium and serum phosphate are lowered.



   This effect is evident not only in hypercalcemia patients, but in normal calcemic people who, for. B. suffer from osteoporosis.



   The new compounds can therefore be used for the treatment of hypercalcaemia and bone diseases such as osteoporosis.



   The inventive method for the preparation of the new compounds of the formula I and corresponding compounds in which one or more of the asparagine residues are replaced by the aspartic acid residue and / or the glutamic acid residue by the glutamine residue and / or the methionine residue by one of the above-mentioned residues, their C terminal amides and the acid addition salts of these compounds is characterized in that the corresponding amino acids, whereby the proline can also be present as an amide, or their activated derivatives, with intermediate protection of reactive functional groups to be excluded from the reaction and with the formation of the disulfide bridge between Cys1 and Cys7 condensed by oxidation.



   The compounds obtained can be converted into their acid addition salts or complexes.



   In the synthesis of the end products as well as all the necessary intermediates, the protective groups used are particularly those known from the synthesis of long-chain peptides, as well as some new protective groups that can be easily split off, e.g. by hydrolysis, reduction, aminolysis or hydrazinolysis.



   So one uses e.g. as protective groups for amino groups, acyl or aralkyl groups such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, p-toluenesulfonyl, o-nitrophenylsulfenyl, 2,4-dinitrophenylsulfenyl groups (these sulfenyl groups can also be removed by the action of nucleophilic reagents, e.g. sulfites, , see English Patent 1 104 271, are split off) optionally substituted, such as



  benzyl, or diphenyl or triphenylmethyl groups substituted by lower alkoxy groups, especially o- or p-methoxy groups, or groups derived from carbonic acid, such as e.g. benzyl or benzhydryloxycarbonyl groups substituted by halogen atoms such as chlorine or bromine, nitro groups or lower alkoxy groups, e.g. Carbobenzoxy, p-bromo- or p-chlorocarbobenzoxy, p-nitrocarbobenzoxy or p-methoxycarbobenzoxy, colored benzyloxycarbonyl groups such as p-phenylazo-benzyloxycarbonyl and p- (p'-methoxy-phenylazo) -benzyloxoxy, aliphatic oxyloxycarbonyl, aliphatic oxyloxy carbonyl, aliphatic oxyloxy carbonyl, aliphatic oxyloxy carbonyl, adamyl carbonyl, aliphatic oxyloxy carbonyl, adamicloxycarbonyl, adamicloxycarbonyl, adamoxycarbonyl, carbonyl, carbonyl, oxyloxy carbonyl groups, such as tert-amyloxycarbonyl or especially tert-butyloxycarbonyl, aromatic-aliphatic oxycarbonyl groups such as 2-phenyl-isopropyloxycarbonyl,

   2-tolyl-isopropyloxycarbonyl and in particular 2-p-diphenylisopropyloxycarbonyl (cf. Swiss patent 509 266). The amino groups can also be obtained by the formation of enamines by reaction of the amino group with 1,3 diketones, e.g. B. benzoylacetone, acetylacetone or dimedone are protected.



   Carboxyl groups are protected, for example, by amide or hydrazide formation or 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 z. B. by the carbobenzoxy group, the trichloroethyloxycarbonyl group, the trifluoroacetyl group, the trityl group, the tert-butyloxycarbonyl group or the 2-p-diphenylisopropyloxycarbonyl group. Suitable for esterification are, for. B. lower optionally substituted alkanols such as methanol, ethanol, cyanomethyl alcohol, benzene methyl alcohol or especially tert-butanol, also aralkanols such as aryl lower alkanols, eg.

  B. benzyl or benzhydryl alcohols optionally substituted by lower alkyl or lower alkoxy groups or halogen atoms, such as p-nitrobenzyl alcohol, p-methoxybenzyl alcohol or 2,4,6-trimethylbenzyl alcohol, phenols and thiophenols such as thiophenol, thiocresol, p-nitroth which may be substituted by electron-attracting substituents , 4,5- and 2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p-cyanophenol or p-methanesulfonylphenol, further e.g. B. N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxypiperidine, 8-hydroxyquinoline.



   The hydroxyl groups of the serine, threonine and tyrosine residues can e.g. B. protected by esterification or etherification. As acyl residues in the esterification e.g. Lower alkanoyl radicals such as acetyl, aroyl radicals such as benzoyl and, above all, radicals derived from the carbon acid such as benzyloxycarbonyl or ethyloxycarbonyl are suitable. Groups suitable for etherification are e.g. B. benzyl, tetrahydropyranyl or tert-butyl radicals. Furthermore, the protection of the hydroxyl groups in Ber. 100 (1967), 3838 to 3849 described 2,2,2-trifluoro-1-tert-butyloxycarbonylamino or -l-benzyloxycarbonylaminoethyl groups (Weygand). However, the hydroxyl groups do not necessarily need to be protected.



   The mercapto groups of the cysteine residues are z. B.



  protected by acylation or alkylation. Suitable for acylation is, for. B. the acetyl or benzoyl radical, the ethylcarbamoyl radical or the optionally substituted carbobenzoxy radical. Suitable for the alkylation are e.g. the tert-butyl or benzylthiomethyl radical or optionally substituted arylmethyl groups such as benzyl, p-nitrobenzyl, diphenylmethyl, dimethoxybenzhydryl or trityl, also phenylcyclohexyl, thienyl (2) -cyclohexyl and the like. a., cf. 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 primarily used, but the guanidino group does not need to be protected.



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



   If the new peptides are built according to Merrifield's solid support synthesis, the side chains of serine, threonine and tyrosine can e.g.



  by benzyl, that of cysteine by benzyl or p-methoxybenzyl, that of histidine by 1-benzyloxy carbonylamino-2,2,2-trifluoroethyl, that of arginine by the nitro group and that of glutamic acid by benzyloxy. Tert-butyloxycarbonyl, for example, is used as the α-amino protecting group.



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



   Preference is given to using the tert-butyloxycarbonyl group to protect the amino groups and the tert-butyloxycarbonyl group to protect the carboxyl group of the side chain and, if appropriate, the terminal carboxyl group. Butyl ester group, to protect the hydroxyl groups of the serine, threonine and tyrosine residues, if these are protected at all, the tert-eutyl ether group and, if desired, to protect the imino group of the histidine, the 2,2,2-trifluoro-1-tert. -butyloxycarbonylaminoethyl group. All of these protecting groups can, if desired, be added in one step by acid hydrolysis, e.g. by means of trifluoroacetic acid.

 

  When the protected dotriacontapeptide is built up using protective groups which can be split off with trifluoroacetic acid, the mercapto groups are preferably protected by benzyl or trityl. The S-trityl groups can be split off selectively from the protected peptide in organic solution (while retaining the groups which can be split off with trifluoroacetic acid) using mercuric acetate and hydrogen sulfide.



  The S-benzyl groups can be split off selectively from the protected peptide with sodium in liquid ammonia. In both cases, the protected peptide with free mercapto groups is obtained. This can lead to the protected disulfide, e.g. be oxidized with iodine in glacial acetic acid, with diiodoethane in organic solvents, with atmospheric oxygen in liquid ammonia. It is particularly advantageous to protect the mercapto groups by trityl groups and to remove them from the protected peptide with simultaneous formation of the disulfide bridge with iodine in methanol, cf. Swiss patent 498 805. This formation of the disulfide ring can be carried out at the stage of a partial sequence containing the two cysteine residues, e.g. B. of the nonapeptide 1-9, or at the stage of the Dotriacontapeptide.



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



   The sulfoxide can be prepared from peptides which contain methionine in the 25 position, advantageously by oxidation with dilute hydrogen peroxide solution in a weakly acidic medium.



   Complexes with inorganic substances such as sparingly soluble metal, e.g. Aluminum or zinc compounds are made in a manner analogous to that known for ACTH, e.g. By reaction in aqueous solution with a soluble salt of the metal in question, e.g.



  Zinc chloride or zinc sulfate, and precipitation with an alkali metal phosphate, pyrophate and / or hydroxide at a pH of about 7-9. Complexes with organic compounds such as polyoxygelatin, carboxymethyl cellulose, polyvinylpyrrolidone, polyphloretin phosphate, polyglutamic acid, etc. are obtained by mixing these substances with the peptide in an aqueous solution. Insoluble compounds with alkali metal polyphosphates can also be prepared in the same way.



   According to the invention, the amino acids are linked, if necessary or desired, using easily cleavable protective groups, individually in the order mentioned or after prior formation of smaller peptide units, the disulfide bridge being formed at a suitable stage of the synthesis if necessary. It is expedient to work according to the linking methods suitable for the production of long-chain peptides, taking into account the disulfide bridge, as are known from the literature.



   The linkage of the amino acid and / or Pep tide units is therefore z. B. in such a way that one amino acid or a peptide with a protected a amino group and activated terminal carboxyl group with an amino acid or a peptide with a free a-amino group and free or protected, z. B. esterified or amidated terminal carboxyl group or that one reacts an amino acid or a peptide with activated a-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, be converted 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'-carbonyldiimidazole, or isoxazolium salt, e.g. Woodward reagent, the amino group can be activated for example by reaction with a phosphite.

  The most common methods are the carbodiimide method, the azide method, the activated ester method and the anhydride method, also the Merrifield method and the N-carboxyanhydride or N-thiocarboxyanhydride method.



   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 to condense the entire remaining sequence, i.e. 8-32, 9-32, 10-32, 11-32, 12-32 or 13-32. Instead, one can also link the mentioned N-terminal sequence of the first 7-12 amino acids with the fragment up to the 24th amino acid (glycine) and condense the tetracosapeptide with the octapeptide of amino acids 25 to 32. The condensation of said N-terminal fragment with the sequence up to the 32nd or up to the 24th amino acid is expediently carried out by the azide method, starting from the azide or hydrazide, or the Wünsch method (carbodiimide in the presence of N-hydroxysuccinimide) carried out starting from the peptide with a free terminal carboxyl group.



   The production of the N-terminal fragment in the synthesis of the nonapeptide (1-9) is explained in more detail below. The heptapeptide (1-7), the octapeptide (1-8), the decapeptide (1-10), the undecapeptide (1-11) and the dodecapeptide (1-12) can be produced in a very similar manner.



   The nonapeptide sequence is built up, for example, from sequences 14 and 5-9 or 1-6 and 7-9, as can be seen from FIGS. 1-9; however, other fragments can be used to build the sequence 1-9. The tert-butyloxycarbonyl group or an equivalent group which can be split off by acid hydrolysis is preferably used as the protective group for the a-amino group on the cysteini, or if an Na-acylated Dotriacontapeptide is to be produced, the corresponding acyl, z. B. acetyl group. In addition, it is expedient to use as mercapto protective groups those which can be split off selectively with respect to the Na-amino protective group which can be split off by acid hydrolysis (e.g. 13th tert. Butyloxycarbonyl group), e.g. the benzyl or trityl group.

  The terminal carboxyl group of the nonapeptide need not necessarily be protected, e.g. B. not if condensations are carried out by the azide or anhydride method (see. For example, Fig. 1, columns 5 and 8). However, this group can also be protected by esterification, as indicated above, e.g. B. 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). The amino groups of the intermediates are protected by means of the usual protecting groups, e.g. Carbobenzoxy, trityl, tert-butyloxycarbonyl and especially 2-para-diphenyl-isopropyloxycarbonyl. If necessary, the carboxyl groups of the intermediates are esterified in the usual manner.

  The hydroxyl groups of the serine residues and the threonine residue can be etherified by ether, e.g. be protected with tert-butanol or equivalents. In the following figures and in the examples: 1) the azide method 2) the mixed anhydride method 3) the activated ester method, particularly p-nitrophenyl ester (ONP) or hydroxysucci nimide ester (OSU) 4) the carbodiimide method 5) the method as desired 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 acid Tert.-methyl-ethyl ester
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 z. B. with hydrocyanic acid or sulfurous acid as described in English patent 1,104,271; the diphenylisopropyloxycarbonyl group e.g.



  with a mixture of glacial acetic acid; Formic acid (82.8%) and water (7: 1: 2) as described in Swiss patent 509 266. The p-nitrobenzyl or methyl ester is converted into the hydrazide with hydrazine hydrate. The methyl ester group is hydrolyzed with dilute sodium hydroxide solution. 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, the S-benzyl group with sodium in liquid ammonia, with any p-nitrobenzyl ester groups that may be removed at the same time. The ring closure to the disulfide takes place z. B. by oxidation with 1,2-diiodoethane.



   The C-terminal sequence to be connected to the N-terminal sequence, comprising the 8th to 13th to 32nd amino acid, is preferably composed of the C-terminal fragment 25-32 and the sequence up to the 24th.



  Amino acid, composed as 8-24, 9-24, 10-24, 11-24, 12-24 or 13-24.



   In the schematic representation of Figures 11 and 12 the synthesis of the C-terminal octapeptide amide (25-32) is illustrated. The tripeptide amide 30-32 or the tetrapeptide amide 29-32 is preferably linked by the carbodiimide method with a fragment from the preceding amino acids. The r-carboxyl group of glutamic acid30 is expediently protected by the tert-butyl ester group. The a amino protecting group DPC is split off as indicated above with glacial acetic acid formic acid (82.8%) - water (7: 1: 2).



   The synthesis of the fragment with the amino acid sequence from the 8th-13th up to the 24th amino acid can be carried out in very different ways. The peptide is preferably used up to the amino acid in position 13 (tryptophan), i.e. 8-13, 9-13, 10-13 etc.



  produced and this either condensed with the undecapeptide 1P24, or first with a fraction of this undecapeptide sequence, e.g. 14-16, 1P17, 1plus, 1619, and then linked to the remaining fragment up to amino acid 24. Figures 13 and 14 illustrate the synthesis of the 10-13 tetrapeptide.



  Amino acid in the form of the hydrazide which is synthesized by the azide method with the following amino acids, e.g.



  the said undecapeptide or a fragment thereof can be condensed.



   According to the invention, the undecapeptide with the sequence 1624 is preferably constructed from the sequences 14 to 19 and 20-24 or 14-20 and 21-24.



  The hexapeptide 1P19 or the heptapeptide 14-20 are expediently linked by the azide method with the residual sequence up to the 24th amino acid. Fig.



  15, 16 and 17 illustrate the synthesis of the hexapeptide hydrazide 14-19 and the heptapeptide hydrazide 14-20.



  The amino acid derivative H-His-N-H-BOC mentioned in FIG. 17A is obtained by condensation of Z-His-OH with BOC-NH-NH2 using dicyclohexylcarbodiimide in ethyl acetate and hydrogenolytic cleavage of the carbobenzoxy group. The protected phenylalanine hydrazide is prepared in an analogous manner in FIGS. 15A and 16A.



  The BOC group is split off from the hydrazine residue with trifluoroacetic acid, the carbobenzoxy group by hydrogenolysis. Equivalent protecting groups can be used instead of the BOC and carbobenzoxy groups. 18-20 illustrate the synthesis of the sequences 20-24 and 21-24, which are linked with the sequence 1P19 (in FIG. 16 stage J, after hydrogenolytic cleavage of the Z group from the hydrazine residue) and 1P20 to form the undecapeptide 1P24 can.



   But you can also use the sequence 1P19 or



  14-20 of Fig. 16K or Fig. 15J or 17L after cleavage of the a-amino protective group by hydrogenolysis or with trifluoroacetic acid with the sequence 10-13 according to the azide method to the sequence 10-19 or



  Link 10-20, then split off the BOC or carbobenzoxy protective group from the protected hydrazide as above and the sequence 10-19 or 10-20 by the azide method with the sequence 20-24 or 21-24 to the pentadecapeptide 10- 24 connect.



   The sequence of the pentadecapeptide 10-24 as shown e.g. B. by joining the fragments shown in FIGS. 13-20, can be condensed for example by the Wünsch method with the octapeptide amide 25-32, as shown in FIG. 11 or 12, to form the protected tricosapeptide amide 10-32.



   But you can also first condense the pentadecapeptide 10-24 with the N-terminal sequence 1-9, expediently in the same way as indicated below for the tricosapeptide amide 10-32, and link the tetracosapeptide, for example as desired, with the octapeptide amide 25-32.



   Another possibility according to the invention of building up the protected tricosapeptide amide of amino acids 10-32 is that the sequence 10 to 16 is linked to the sequence 17-32, preferably by the azide method. The sequence 10-16 can be used e.g. B. by condensation of the derivatives of the sequence 10-13 given in Fig. 13 or 14 with the tripeptide 14-16 (H-Arg-Asn-Leu-OH, e.g. obtained from Z-Arg-Asn Leu-OBzl by hydrogenolysis) , using the azide method. The sequence 17-32 is preferably built up from the sequences 17-24 and 25-32 (octapeptide amide), for example by condensation according to the Wünsch method.

  The octapeptide derivative Asn-Asn-Phe-His-Arg-Phe-Ser-Gly OH with a protected α-amino group and optionally protected hydroxyl group of the serine residue can be obtained, for example, by condensation of Z-Asn-Asn-Phe His-N2Hs (from FIG. 171 with trifluoroacetic acid) with H-ArgPhe-Ser (tBu) -Gly-OH (FIG. 20 H) by the azide method.



   This protective group is removed in a suitable manner from the tricosapeptide amide 10-32 with a protected α-amino group (BOC with trifluoroacetic acid, which also hydrolyzes a tert-butyl ester group on the γ-carboxyl group of glutamic acid30 and any tert-butyl ether groups present; DPC with glacial acetic acid -Formic acid-water, whereby tert-butyl ester and ether groups are not attacked).



   The linking of the sequence 1-9, which contains the disulfide bridge and in which at least the α-amino group is protected, with the sequence 10-32, is carried out, as already mentioned, preferably by the azide method.



   This gives the dotriacontapeptide amide with a protected α-amino group and optionally protected side chain hydroxyl and / or carboxyl groups.



  As already mentioned, these protective groups are split off.



   Depending on the procedure, the new compounds are obtained in the form of bases or their salts. The bases can be obtained from the salts in a manner known per se. From the latter, in turn, salts can be obtained by reaction with acids which are suitable for the formation of therapeutically useful salts, e.g.

   those with inorganic acids, such as hydrohalic acids, for example hydrochloric acid or hydrobromic acid, perchloric acid, nitric acid or thiocyanic acid, sulfuric or phosphoric acids, or organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, oxalic acid, malonic acid, succinic acid, , Malic acid, tartaric acid, citric acid, acorbic acid, hydroxymaleic acid, dihydroxymaleic acid, benzoic acid, phenylacetic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, anthranilic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, methoxy sulfanoic acid, methoxy sulfanoic acid, 2-phenoxybenzoic acid, ethoxy sulfanoic acid, methoxy sulfanoic acid, 2-phenoxy-sulfanoic acid, methoxy sulfanoic acid, hydroxy-sulfanoic acid, 2-phenoxy sulfanoic acid, acetic acid, dihydroxymaleic acid, benzoic acid , Benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid or sulfanilic acid.



   The peptides obtained according to the method can be used in the form of pharmaceutical preparations. These contain the peptides in a mixture with a pharmaceutical, organic or inorganic carrier material suitable for enteral or parenteral administration.



   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: n-butanol-glacial acetic acid-water (67: 10: 23) System 53: n-butanol-formic acid-water (60 : 0.75: 39) System 54: sec. Butanol-isopropanol 9% monochloroacetic acid (58: 8: 34) System 55: Ethanol-8% 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.

  Butanol-glacial acetic acid-water (67: 10: 23) System 100: ethyl acetate-pyridine-lasacetic-water (62: 21: 6: 11) System 101: n-butanol-pyridine-glacial acetic acid-water (38: 24: 8: 30) System 101 A: 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 121 A: Isopropanol-ammonia (26%) -water (85: 5:10) System 87: isopropanol-glacial acetic acid-water (77: 4: 19) System 110: ethyl acetate-n-butamol-pyridine-glacial acetic acid
Water (42: 21: 21: 6:10) System 1: Benzene-Ethanol (80:20) System 2: Benzene-Ethanol (90:10) System 3:

  Benzene-ethanol (95: 5) system 4: n-amyl alcohol-formic acid-water (70:20:10) system 5: n-butanol-acetic acid-water (66.6: 16.7 16.7) system 6: n-butanol-pyridine-acetic acid-water (66.6: 12.5: 4.2: 16.7) system 7: n-amyl alcohol-pyridine-water (50: 30:20) system 8: chloroform-methanol Glacial acetic acid (87.4: 9.7:

   2.9).
EMI6.1


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 <tb>

    <SEP> tBu <SEP> tBu
 <tb> G <SEP> II <SEP> | <SEP> IOMe
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> H <SEP> Z <SEP> l <SEP> 2), 3) .5) <SEP> 1
 <tb> <SEP> OMe
 <tb> <SEP> t3u <SEP> tBu
 <tb> I. <SEP> H <SEP> I <SEP> I <SEP> OMe
 <tb> <SEP> OMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> J <SEP> BOC <SEP> l <SEP> w-LOMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> K <SEP> Boa <SEP> I <SEP> I <SEP> I <SEP> I <SEP> NH-NH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> L <SEP> BOC <SEP> I <SEP> 1) <SEP> OMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> M <SEP> 300 <SEP> 1 <SEP> Nile-Nile 2
 <tb> <SEP> SH <SEP> tBu <SEP> tBu <SEP> tBu <SEP> SH
 <tb> N <SEP> BOC <SEP> l <SEP> I <SEP> I <SEP> I <SEP> I <SEP> NH-NH2
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> O <SEP> 300 <SEP>, <SEP> l <SEP> l <SEP> | <SEP> / <SEP>.

   <SEP> NH-NH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb>



  Fig. 1
EMI7.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Tar <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> A <SEP> BOC --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> H --- N2H2-Z <SEP> BOC --- OH <SEP> N --- ONB <SEP> BOC --- OH <SEP> BOC --- OH <SEP> H --- OBzl
 <tb> <SEP> 3) <SEP> 1) -5) <SEP> 1) -5)
 <tb> B <SEP> BOC -------------- N2H2-Z <SEP> BOC ------------ ONB <SEP> BOC ------------- OBzl
 <tb> C <SEP> H -------------- N2H2-Z <SEP> H ------------ ONB <SEP> H ------------- OBzl
 <tb> <SEP> Bzl
 <tb> <SEP> 1) -5) <SEP> 1) -5)
 <tb> D <SEP> BOC ------------------------- N2H2-Z <SEP> BOC ------------------------- OBzl
 <tb> <SEP> Bzl
 <tb> E <SEP> BOC ------------------------- N2H3 <SEP> H ------------------------- OBzl
 <tb> <SEP> 1)
 <tb> F <SEP> BOC ---------------------------------------------- -ONB
 <tb> G <SEP>

   H ----------------------------------------------- ONB
 <tb> <SEP> Bzl
 <tb> <SEP> 1) -5)
 <tb> H <SEP> BOC ---------------------------------------------- ------------- ONB
 <tb> <SEP> Bzl
 <tb> I. <SEP> BOC ---------------------------------------------- ------------- N2H3
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> <SEP> 1)
 <tb> J <SEP> BOC ---------------------------------------------- ---------------------------------------------- OBzl
 <tb> <SEP> SE <SEP> SE
 <tb> K <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> L <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> Fig. 2 Fig.

   3
EMI8.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Tar <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> A <SEP> BOC --- OH <SEP> Z --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> H ---- OBzl <SEP> BOC --- OH <SEP> BOC --- OH <SEP> H --- OBzl
 <tb> <SEP> 1) -5) <SEP> 1) -5)
 <tb> B <SEP> BOC ---------------- OBzl <SEP> BOC -------------- OBzl
 <tb> C <SEP> H ------------------ OBzl <SEP> H -------------- OBzl
 <tb> <SEP> Bzl
 <tb> <SEP> 1) -5)
 <tb> D <SEP> BOC -------------------------- OBzl <SEP> BOC ------------------------ OBzl
 <tb> <SEP> Bzl
 <tb> E <SEP> H -------------------------- OBzl <SEP> H ------------------------ OBzl
 <tb> <SEP> 5)
 <tb> F <SEP> BOC ------------------------------------- OBzl
 <tb> G <SEP> H ------------------------------------- OBzl
 <tb> H <SEP>

   Z ------------------------------------------------ OBzl
 <tb> I. <SEP> H ---------------------------------------------- --OH
 <tb> <SEP> Bzl
 <tb> J <SEP> BOC ---------------------------------------------- --------------OH
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> K <SEP> BOC ---------------------------------------------- ---------------------------------------------- OBzl
 <tb> <SEP> SE <SEP> SE
 <tb> L <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> M <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb>
EMI9.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Tar <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> TRI <SEP>

   TRI
 <tb> A <SEP> BOC --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> H --- N2H2-Z <SEP> BOC --- OH <SEP> H --- ONB <SEP> TRI --- OH <SEP> BOC --- OH <SEP> H --- ONB
 <tb> <SEP> NPS
 <tb> <SEP> 1) -5)
 <tb> B <SEP> BOC ------------- ONB
 <tb> <SEP> like <SEP> in <SEP> fig. <SEP> 2 <SEP> G
 <tb> C <SEP> H ---------------------------------------------- -ONB <SEP> H ------------- ONB
 <tb> <SEP> TRI <SEP> TRI
 <tb> <SEP> 5)
 <tb> D <SEP> BOC ---------------------------------------------- ------------- ONB <SEP> TRI ------------------------ ONB
 <tb> <SEP> NPS
 <tb> <SEP> TRI <SEP> TRI
 <tb> E <SEP> BOC ---------------------------------------------- ------------- N2H3 <SEP> H ------------------------ ONB
 <tb> <SEP> TRI <SEP> TRI
 <tb> <SEP> 1)
 <tb> F <SEP> BOC ---------------------------------------------- --------------------------------------------- ONB
 <tb> <SEP> TRI <SEP> TRI
 <tb> G <SEP>

   BOC ------------------------------------------------- ------------------------------------------ N2H3
 <tb> <SEP> SH <SEP> SH
 <tb> H <SEP> BOC ---------------------------------------------- --------------------------------------------- N2H3
 <tb> I. <SEP> BOC ---------------------------------------------- --------------------------------------------- N2H3
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> Fig.

   4th
EMI10.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Tar <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> TRI <SEP> TRI
 <tb> A <SEP> BOC --- OH <SEP> Z --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> BOC --- OBzl <SEP> TRI --- OH <SEP> BOC --- OH <SEP> H --- ONB
 <tb> B <SEP> BOC -------------- ONB
 <tb> C <SEP> H -------------- ONB
 <tb> <SEP> TRI
 <tb> <SEP> like <SEP> in <SEP> fig.

   <SEP> 3 <SEP> I
 <tb> D <SEP> H ---------------------------------------------- --OH <SEP> TRI ------------------------ ONB
 <tb> <SEP> TRI <SEP> TRI
 <tb> E <SEP> BOC ---------------------------------------------- ---------------OH <SEP> H ------------------------ ONB
 <tb> <SEP> TRI <SEP> TRI
 <tb> <SEP> 5)
 <tb> F <SEP> BOC ---------------------------------------------- ----------------------------------------------- ONB
 <tb> <SEP> TRI <SEP> TRI
 <tb> G <SEP> BOC ---------------------------------------------- ----------------------------------------------- N2H3
 <tb> <SEP> SH <SEP> SH
 <tb> H <SEP> BOC ---------------------------------------------- ----------------------------------------------- N2H3
 <tb> I. <SEP> BOC ---------------------------------------------- ----------------------------------------------- N2H3
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <RTI

    ID = 10.1> Fig. 5
EMI11.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Tar <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> BOC --- ONP <SEP> Z --- OH <SEP> Z --- ONP <SEP> H --- OMe <SEP> DPC --- OH <SEP> H --- OMe <SEP> TRI --- OH <SEP> Z --- OH <SEP> H --- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> <SEP> 3) <SEP> 1) -5)
 <tb> B <SEP> Z -------------- OMe <SEP> DPC ------------- OMe <SEP> Z -------------- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> C <SEP> H -------------- OMe <SEP> DPC ------------- N2H3 <SEP> H -------------- OMe
 <tb> <SEP> tBu <SEP> TRI
 <tb> <SEP> 1) -5) <SEP> -5)
 <tb> D <SEP> Z -------------------------- OMe <SEP> TRI ------------------------- OMe
 <tb> <SEP> tBu <SEP> TRI
 <tb> E <SEP> H -------------------------- OMe <SEP> H ------------------------- OH
 <tb>

    <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> <SEP> 3) <SEP> 1)
 <tb> F <SEP> BOC ------------------------------------- OMe <SEP> DPC ---------------------------------------------- OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> G <SEP> BOC ------------------------------------- N2H3 <SEP> H ---------------------------------------------- OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> <SEP> 1)
 <tb> H <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> SH <SEP> tBu <SEP> tBu <SEP> tBu <SEP> SH
 <tb> I. <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> J <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5

    <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> Fit. 6th
EMI12.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Tar <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> BOC --- ONP <SEP> Z --- OH <SEP> Z --- OH <SEP> H --- OMe <SEP> Z --- OH <SEP> H --- OMe <SEP> TRI --- OH <SEP> Z --- OH <SEP> H --- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> <SEP> 1) -5)
 <tb> B <SEP> Z -------------- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> C <SEP> H -------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> <SEP> like <SEP> Fig.

   <SEP> 6 <SEP> D
 <tb> D <SEP> TRI -------------- OMe <SEP> TRI ------------------------ OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> E <SEP> TRI -------------- OH <SEP> H ------------------------ OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> F <SEP> TRI ---------------------------------------------- -OMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> <SEP> like <SEP> Fig.

   <SEP> 6 <SEP> G
 <tb> G <SEP> BOC ------------------------------------- N2H3 <SEP> H ---------------------------------------------- -OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> <SEP> 1)
 <tb> H <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> SH <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> I. <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> J <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> Fig.

   7th
EMI13.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> TRI --- OH <SEP> Z --- OH <SEP> Z --- ONP <SEP> H --- OMe <SEP> DPC --- OH <SEP> H --- OMe <SEP> TRI --- OH <SEP> Z --- OH <SEP> H --- OMe
 <tb> <SEP> 3)
 <tb> B <SEP> Z -------------- OMe
 <tb> C <SEP> H -------------- OMe
 <tb> <SEP> tBu
 <tb> D <SEP> Z ------------------------- OMe
 <tb> <SEP> tBu
 <tb> E <SEP> H ------------------------- OMe
 <tb> <SEP> TRI <SEP> tBu
 <tb> F <SEP> TRI ------------------------------------- OMe
 <tb> <SEP> TRI <SEP> tBu
 <tb> G <SEP> H ------------------------------------- OMe
 <tb> <SEP> TRI
 <tb> H <SEP> AC ------------------------------------- OMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> <SEP> like <SEP> Fig.

   <SEP> 6 <SEP> G
 <tb> I. <SEP> AC ------------------------------------- N2H3 <SEP> H ---------------------------------------------- -OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> J <SEP> AC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> K <SEP> AC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> Fig.

   8th
EMI14.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> <SEP> Cys <SEP> Ser <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> Val <SEP> Leu
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> TRI --- OH <SEP> Z --- OH <SEP> Z --- ONP <SEP> H --- OMe <SEP> DPC --- OH <SEP> H --- OMe <SEP> TRI --- OH <SEP> Z --- OH <SEP> H --- OMe
 <tb> <SEP> 3)
 <tb> B <SEP> Z -------------- OMe
 <tb> C <SEP> H -------------- OMe
 <tb> <SEP> tBu
 <tb> <SEP> 1) -5)
 <tb> D <SEP> Z ------------------------- OMe
 <tb> <SEP> tBu
 <tb> E <SEP> H ------------------------- OMe
 <tb> <SEP> tBu
 <tb> F <SEP> BOC ------------------------------------- OMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> <SEP> like <SEP> Fig.

   <SEP> 6 <SEP> G
 <tb> G <SEP> BOC ------------------------------------- N2H3 <SEP> H ---------------------------------------------- -OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> H <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu
 <tb> I. <SEP> BOC ---------------------------------------------- ----------------------------------------------OH
 <tb> J <SEP> H ---------------------------------------------- ----------------------------------------------OH
 <tb> K <SEP> AC ---------------------------------------------- ----------------------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
 <tb> Fig.

   9
EMI15.1

   <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28 <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> <SEP> Met <SEP> Gly <SEP> Pas <SEP> Gly <SEP> Pro <SEP> Glu <SEP> Thr <SEP> Pro
 <tb> <SEP> OtBu <SEP> tBu
 <tb> A <SEP> DPC --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> H --- OMe <SEP> Z --- OH <SEP> Z --- ONP <SEP> Z --- OH <SEP> H --- NH2
 <tb> <SEP> tBu
 <tb> <SEP> 2) -5) <SEP> 2) -5)
 <tb> B <SEP> Z ---------------- OMe <SEP> Z --------------- NH2
 <tb> <SEP> tBu
 <tb> C <SEP> H ---------------- OMe <SEP> H --------------- NH2
 <tb> <SEP> OtBu <SEP> tBu
 <tb> <SEP> 2) -5) <SEP> 3)
 <tb> D <SEP> Z ---------------------------- OMe <SEP> Z --------------------------- NH2
 <tb> <SEP> OtBu <SEP> tBu
 <tb> E <SEP> H ---------------------------- OMe <SEP> H --------------------------- NH2
 <tb> <SEP> OtBu <SEP> tBu
 <tb> <SEP> 2) -5) <SEP> 2) -5)
 <tb> F <SEP> DPC ---------------------------------------- OMe <SEP>

   Z --------------------------------------- NH2
 <tb> <SEP> OtBu <SEP> tBu
 <tb> G <SEP> DPC ---------------------------------------- OH <SEP> H --------------------------------------- NH2
 <tb> <SEP> OtBu <SEP> tBu
 <tb> <SEP> 2) -5)
 <tb> H <SEP> DPC ---------------------------------------------- ----------------------------------------- NH2
 <tb> <SEP> OtBu <SEP> tBu
 <tb> I. <SEP> H ---------------------------------------------- ----------------------------------------- NH2
 <tb> <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28 <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> Fig.

   10
EMI16.1

   <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28 <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> <SEP> Met <SEP> Gly <SEP> Pas <SEP> Gly <SEP> Pro <SEP> Glu <SEP> Thr <SEP> Pro
 <tb> <SEP> OtBu
 <tb> A <SEP> NPS --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> H-OtBu <SEP> Z --- OH <SEP> Z --- OH <SEP> H --- NH2
 <tb> <SEP> 2) -5) <SEP> 2) -5)
 <tb> B <SEP> Z ------------- OtBu <SEP> Z --------------- NH2
 <tb> C <SEP> H ------------- OtBu <SEP> H --------------- NH2
 <tb> <SEP> OtBu
 <tb> <SEP> 2) -5) <SEP> 2) -5)
 <tb> D <SEP> Z ------------------------- OtBu <SEP> Z --------------------------- NH2
 <tb> <SEP> OtBu
 <tb> E <SEP> H ------------------------- OtBu <SEP> H --------------------------- NH2
 <tb> <SEP> 2) -5)
 <tb> F <SEP> Z ------------------------------------- OtBu
 <tb> G <SEP> Z ------------------------------------- OH
 <tb> <SEP> OtBu
 <tb> H <SEP>

   Z ------------------------------------------------- -------------------------- NH2
 <tb> <SEP> OtBu
 <tb> I. <SEP> H ---------------------------------------------- ----------------------------- NH2
 <tb> <SEP> OtBu
 <tb> J <SEP> NPS ---------------------------------------------- ----------------------------------------- NH2
 <tb> <SEP> OtBu
 <tb> K <SEP> H ---------------------------------------------- ----------------------------------------- NH2
 <tb> <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28 <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> Fit. 11
10 11 12 13
Ser Ala Tyr Try A BOC --- OH Z --- OH BOC --- OH H --- OMe 2) -5) B BOC ------------------ OMe CH OMe D Z ----------------------------------- OMe EH OMe F BOC ----- --------------------------------------------- OMe G BOC-- ------------------------------------------------ N2H3
10 11 12 13
Fig.

   12
EMI18.1


 <tb> <SEP> 10 <SEP> 11 <SEP> 12 <SEP> 13
 <tb> <SEP> Ser <SEP> Ala <SEP> Tyr <SEP> Try
 <tb> <SEP> tBu <SEP> t3u <SEP>
 <tb> A <SEP> DPC --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> H-OMe
 <tb> <SEP> tBu
 <tb> B <SEP> Z ------ 2) -5) -------- OMe
 <tb> <SEP> tBu
 <tb> C <SEP> H --------------- OMe
 <tb> <SEP> tBu
 <tb> 2) -5)
 <tb> D <SEP> Z ------------------------- OMe
 <tb> <SEP> tBu
 <tb> E <SEP> H ------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> F <SEP> DPC -------------- 2) -5) ------------------------ OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> G <SEP> DPC --------------------------------------- N2H3
 <tb> <SEP> 10 <SEP> 11 <SEP> 12 <SEP> 13
 <tb> Fig.

   13
14 15 16 17 18 19
Arg Asn Leu Asn Asn Phe A Z --- OH Z --- ONP Z --- ONP Z ----- ONP Z --- ONP H --- N2H2 --- BOC
OSU OSU OSU
3) B Z ----------------- N2H2-BOC C H ----------------- N2H2-BOC 3) D Z --------------------------------- N2H2-BOC E H ------------ --------------------- N2H2-BOC 3) F Z ---------------------- ------------------------ N2H2-BOC G H --------------------- ------------------------- N2H2-BOC 3) H Z ------------------ ------------------------------------------ N2H2-BOC I H --- -------------------------------------------------- ------- N2H2-BOC
4) 5) J Z -------------------------------------------- ----------------------------- 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 C H --------------- N2H2-Z D BOC --------- --------------------- N2H2-Z E H ------------------------ ------ N2H2-Z F BOC --------------------------------------- ------ N2H2-Z G H --------------------------------------- ------ N2H2-Z H BOC --------------------------------------- -------------------- N2H2-Z I H ------------------------- ---------------------------------- N2H2-Z J BOC ----------- -------------------------------------------------- ------------ N2H2-Z K H --------------------------------- ---------------------------------------- N2H2-Z
14 15 16 17 18 19
Fig.

   15th
14 15 16 17 18 19 20
Arg Asn Leu Asn Asn Phe His A Z --- OH Z --- ONP Z --- OH Z --- ONP Z --- ONP Z --- OH H --- NH-NH-BOC
2) -5) B Z ---------------- NH-NH-BOC C H ---------------- NH-NH- BOC
3) D Z ----------------------------- NH-NH-BOC E H ---------- ------------------- NH-NH-BOC
3) F Z ------------------------------------------ NH-NH- BOC G H ------------------------------------------ NH-NH-BOC
2) -5) H Z ------------------------------------------- ------------ NH-NH-BOC I H ------------------------------- ------------------------ NH-NH-BOC
3) J Z ---------------------------------------------- ----------------------- NH-NH-BOC K H -------------------- ------------------------------------------------- NH -NH-BOC
4) 5) L.

   Z ------------------------------------------------- -------------------------------- NH-NH-BOC M Z ----------- -------------------------------------------------- -------------------- NH-NH2
14 15 16 17 18 19 20
Fig. 16
EMI22.1


 <tb> <SEP> 20 <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24
 <tb> <SEP> His <SEP> Arg <SEP> Phe <SEP> Ser <SEP> Gly
 <tb> <SEP> tBu
 <tb> A <SEP> Z --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> H-OMe
 <tb> <SEP> t3u <SEP>
 <tb> 2) -5)
 <tb> B <SEP> Z --------------- OMe
 <tb> <SEP> tBu
 <tb> <SEP> C <SEP> H --------------- OMe
 <tb> <SEP> 2) -5)
 <tb> <SEP> tBu
 <tb> Z -------------------------------- OMe
 <tb> <SEP> tBu
 <tb> E <SEP> H ----------------------------- OMe
 <tb> <SEP> tBu
 <tb> <SEP> 4) <SEP> 5)
 <tb> <SEP> F <SEP> Z ------------------------------------------ OMe
 <tb> <SEP> tBu
 <tb> <SEP> G <SEP>

   H ------------------------------------------ OMe
 <tb> <SEP> tBu
 <tb> 1), <SEP> 4), <SEP> 5)
 <tb> <SEP> H <SEP> Z ---------------------------------------------- --------- OMe
 <tb> <SEP> tBu
 <tb> I. <SEP> H ---------------------------------------------- --------- OMe
 <tb> <SEP> tBu
 <tb> J <SEP> H ---------------------------------------------- ---------OH
 <tb> <SEP> 20 <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24
 <tb> Fig.

   17th
EMI23.1


 <tb> <SEP> 20 <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24
 <tb> <SEP> His <SEP> Arg <SEP> Phe <SEP> Ser <SEP> Gly
 <tb> <SEP> N02
 <tb> A <SEP> Z --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> BOC --- OH <SEP> H --- OBzl
 <tb> 2) -5)
 <tb> B <SEP> BOC ---------------- OBzl
 <tb> C <SEP> H ---------------- OBzl
 <tb> 2) -5)
 <tb> D <SEP> BOC ------------------------------ OBzl
 <tb> E <SEP> H ------------------------------ OBzl
 <tb> <SEP> NO
 <tb> 2) -5)
 <tb> <SEP> F <SEP> BOC ---------------------------------------------- OBzl
 <tb> <SEP> NO2
 <tb> G <SEP> H ---------------------------------------------- OBzl
 <tb> <SEP> NO2
 <tb> <SEP> 1)
 <tb> <SEP> H <SEP> Z ---------------------------------------------- --------------- OBzl
 <tb> I. <SEP> H <SEP>.

   <SEP> OH
 <tb> <SEP> 20 <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24
 <tb> Fig. 18
EMI24.1


 <tb> <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24
 <tb> <SEP> Arg <SEP> Phe <SEP> Ser <SEP> Gly
 <tb> <SEP> NO2 <SEP> tBu
 <tb> A <SEP> Z --- OH <SEP> Z --- OH <SEP> Z --- OH <SEP> H --- OMe
 <tb> <SEP> 2) -5)
 <tb> <SEP> tBu
 <tb> B <SEP> Z ----------------- OMe
 <tb> <SEP> tBu
 <tb> C <SEP> H <SEP> @ <SEP> OMe
 <tb> <SEP> tBu <SEP>
 <tb> D <SEP> Z
 <tb> <SEP> tBu
 <tb> E <SEP> H <SEP> # <SEP> OMe
 <tb> <SEP> NO2 <SEP> tBu
 <tb> 2) -5)
 <tb> Z ------------------------------------------- OMe
 <tb> <SEP> tBu
 <tb> G <SEP> H ---------------------------------------------- OMe
 <tb> <SEP> tBu
 <tb> H <SEP> H ---------------------------------------------- OH
 <tb> <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24
 <tb> Fig.

   19th
EMI25.1


 <tb> <SEP> example <SEP> 1
 <tb> H-Cvs-Ser-Asn-Leu-Ser-Thr-Cvs-Val-Leu-Ser-Ala-Tvr- <SEP>
 <tb> Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH2 (I, thyrocalcitonin).
EMI25.2


 <tb>



    <SEP> 480 <SEP> mg <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu)
 <tb> Thr (tBu) -Cys-Val-Leu-Ser (tBu) -Ala-Tyr (tBu) -Try- <SEP>
 <tb> Arg (H2 +) -Asn-Leu-Asn-Asn-Phe-His-Arg (H +) -Phe Ser (tB u) -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 dried in vacuo over caustic soda. The powder is then again dissolved in 20 ml of ice-cold, 90% strength trifluoroacetic acid, heated to 250 and left at 250 for 1 hour under nitrogen.

  The solution is then again poured into 300 ml of ice-cold, peroxide-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 trifluoroacetate of I obtained above is dissolved in 20% acetic acid and filtered through an acid (1.2 × 15 cm) of a weakly basic ion exchange resin (Merck No. II, acetate form). The column is washed with 20% acetic acid until the U.V. control of the eluate no longer shows the presence of I. The eluate is then evaporated in vacuo at a bath temperature with the addition of n-octanol, the residue is freed from octanol by washing with petroleum ether and dried. The acetic acid salt of the Dotriacontapeptide is obtained as a pale yellowish resin. For purification, the product is subjected to countercurrent distribution in the n-butanol system (1 liter-1-n. Acetic acid (1 liter) -ammonacetate (500 mg)).



  For this purpose it is dissolved in 30 ml each of the upper and lower phases of this system and the solution is poured into the first three tubes of the distribution apparatus. After 250 distribution steps, the individual fractions are checked for purity by means of thin-layer chromatography (on aluminum oxide plate, system 104, Rf = 0.50). The pure Dotriacontapeptide is in fractions 140-165 (maximum of the weight curve in fraction 156, K = 1.65). These fractions are combined, evaporated in vacuo at a bath temperature of 400 and the residue is dried at 400 and 0.01 mm Hg to remove ammonium acetate. The dotriacontapeptide obtained in this way proves to be uniform on thin-layer chromatography on aluminum oxide (detection 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; Calls04 = 0.5O.



   In electrophoresis on cellulose acetate film (Cellogel) in acetic acid-formic acid buffer, pH = 1.9, 90 minutes running time, 8 volts / cm, I and its sulfoxide show a distance of 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) -OMe are hydrogenated in 200 ml of glacial acetic acid and 3 g of Pd-charcoal (10%) at room temperature. The uptake of hydrogen ceases after one hour. The solution is filtered off from the catalyst and evaporated at 350 in a water jet vacuum. After drying in a high vacuum at 350, 7.3 g of an oil result which, according to thin-layer chromatogram, is uniform and is used further directly.



  2. DPC-Ser (tBu) -Thr (tBu) -OMe
19.3 g (38.6 mmol) of DPC-Ser (tBu) -OH, cyclohexylamine salt are taken up in 500 ml of chloroform and extracted three times with 25 ml of citric acid and five times with 40 ml of semi-saturated saline solution at 0. After the solution has been dried over sodium sulfate, it is evaporated and the foam obtained is taken up in 250 ml of ethyl acetate. 5.36 ml (38.6 mmol) of triethylamine are added, the solution is cooled to -100 and mixed with 5.13 ml (38.6 mmol) of isobutyl chlorocarbonate while stirring. The mixture is stirred at -100 for 10 minutes and then the solution, cooled to -120, of 7.3 g (38.6 mmol) of H-Thr- (tBu) -OMe in 100 ml of ethyl acetate is added dropwise so that the reaction temperature never exceeds -100 .

  After the end of the entry, the mixture is stirred for a further hour at -100 and then left to stand overnight at room temperature. The solution is filtered off from the precipitated triethylamine hydrochloride and washed at 0 three times with 20 ml of 1N citric acid each time and five times with saturated sodium chloride solution, dried and evaporated. Crude product (oil): 2207 g. For purification, 1 g is chromatographed on a silica gel column (2.5 cm, 30 cm). With petroleum ether-ethyl acetate (1: 1), after a forerun of 110 ml, 787 mg of pure product are eluted.



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



  3. Z-Val-Leu-OMe
19.9 g (110 mmol) of H-Leu-OMe, HCl are dissolved in 120 ml of dimethylformamide and 14.6 ml (105 mmol) of triethylamine are added at 0. The precipitated triethylamine hydrochloride is filtered off, the filtrate is added to a solution of 25.1 g (100 mmol) of Z-Val-OH in 200 ml of dimethylformamide (00) and then 22.6 g (110 mmol) of dicyclohexylcarbodiimide are added dry. After stirring for one hour at 0 and overnight in the refrigerator, the mixture is filtered and the solution is evaporated at 400 in a high vacuum. The oily residue is taken up in 30 ml of ethyl acetate, the solution is cooled to 0 and freed from the dicyclohexylurea which has separated out. After adding n-hexane, the product crystallizes out of the filtrate overnight.

  F. 1021050; [a] D: -410 C (c = 2.88 in methanol).



   In the thin-layer chromatogram on silica gel in chloroform-methanol (98: 2), Rf = 0.55 and in toluene-acetone (7: 3), Rf = 0.60.



  4. DPC-Ser (tBu) -Thr (tBu) -NH-NHO
4.253 g (7.4 mmol) DPC-SerftBu) -Thr (tBu) -OMe in 18 ml methanol are mixed with 5.55 ml (about 110 mmol) hydrazine hydrate and left to stand for 10 hours at room temperature and 2 hours at 400. The reaction solution is taken up in 450 ml of ethyl acetate and washed four times with half-saturated sodium chloride solution. After drying the solution over sodium sulfate, it is concentrated to about 15 ml and mixed with about 5 ml of petroleum ether. 3.17 g of the hydrazide with a melting point of 1321340 crystallize out overnight.



   In the thin-layer chromatogram on silica gel in toluene-acetone (7: 3), Rf = 0.40.



  5. H-Val-Leu-OMe, HCl
5.66 g (15 mmol of Z-Val-Leu-OMe are hydrogenated in 75 ml of methanol in the presence of 15 mmol of hydrochloric acid and 850 mg of Pd-carbon (10%) at room temperature. The uptake of hydrogen ceases after 3 hours the catalyst is filtered off and the solution is concentrated to about 15 ml. Upon addition of ether, the product crystallizes out, mp 136-139.



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



  6. TRI-Cys (TRI) -Val-Leu-OMe
7.13 g (10 mmol) of TRI-Cys (TRI) -OH, diethylamine salt are washed in chloroform at 0 with citric acid and semisaturated sodium chloride solution, the solution is dried, evaporated and the foam obtained is taken up in 50 ml of ethyl acetate.



  A solution of 3.22 g (10 mmol) of H-Val Leu-OMe, hydrochloride in 40 ml of ethyl acetate from which the precipitated triethylamine hydrochloride has been filtered off is added to a 1.4 ml (10 mmol) triethylamine solution . 2.26 g (11 mmol) of dry dicyclohexylcarbodiimide are then added at 0, the mixture is stirred at 0 for 2 hours and left to stand in the refrigerator overnight. The precipitated dicyclohexylurea is filtered off and the solution is washed at 0 with in citric acid, in sodium bicarbonate and semi-saturated sodium chloride solution and dried over sodium sulfate. The solution is concentrated to about 20 ml, cooled to 0 and further dicyclohexylurea is filtered off.

  Evaporation to dryness gives 8.53 g of ester. For purification, the crude product is chromatographed on a silica gel column (5 cm; 55 cm). After a first run of 400 ml, the main product is eluted in pure form with 300 ml of petroleum ether / ethyl acetate (1: 1).



   In the thin-layer chromatogram on silica gel in chloroform-methanol (99 1), Rf = 0.43.



  7. H-Cys (TRI) -Val-Leu-OMe
11.08 g (13.3 mmol) TRI-Cys (TRI) -Val-Leu-OMe are dissolved in 75 ml of ethyl acetate and 12.3 ml of water are added dropwise so that a clear solution always remains. After stirring for one hour at room temperature, 64 ml of water are added to the clear solution, the precipitate is filtered off and washed with cold 50% acetic acid. The filtrate is evaporated to an oil in a high vacuum at 400, this is taken up in 250 ml of ethyl acetate and washed at 0 with sodium bicarbonate and saturated sodium chloride solution.



  After drying over sodium sulfate, it is evaporated to give 7.07 g of a white product. The thin-layer chromatogram on silica gel in the chloroform-methanol system (98: 2) shows a spot with an Rf value of 0.30 when sprayed with Reindel-Hoppe reagent.



  8. DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OMe
To 13.6 g (23.8 mmol) of DPC-Ser (tBu) -Thr (tBu) NH-NH., In 220 ml of dimethylformamide, 32.26 ml of hydrochloric acid in ethyl acetate (1.84-n .; 59.25 mmol) and 3.26 ml (28.55 mmol) of tert-butyl nitrite were added. After 15 minutes at -10, the solution, cooled to -100, 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 is then left Add dropwise ml of dimethylformamide so that the reaction temperature never exceeds -90. It is stirred for a further hour at -100 and then left to stand overnight at iLimmertempe- ratur.

  The precipitated triethylamine hydrochloride is filtered off, the filtrate is evaporated in a high vacuum at 400, the oily residue is taken up in 500 ml of ethyl acetate, washed with citric acid, in sodium bicarbonate and saturated sodium chloride solution, dried, concentrated to about 40 ml and with about 10 ml of petroleum ether offset. The protected pentapeptide ester from F. 177-1780 crystallizes out overnight.



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



  9. DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-OMe
2.830 g (2 mmol) of DPC-Ser (tBu) -Thr (tBu) Cys (TRI) -Val-Leu-OMe are dissolved in 60 ml of 75% dioxane, and 3 ml of 2N sodium hydroxide solution (6 mmol) are added. After 11/2 hours at room temperature, the dioxane is evaporated off in a water jet vacuum at 40, ethyl acetate and water are added and the pH is adjusted to 3 at 0 with citric acid. The ethyl acetate phase is washed with sodium chloride solution, dried and evaporated. The result is a product which, according to a thin-layer chromatogram on silica gel, is uniform.



   Rf in chloroform-methanol (7: 3) = 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) are added. The clear solution is stirred for 15 minutes 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 triturated three times with water and then lyophilized. Rubbing the product three times with ether yields a white powder which, according to the thin-layer chromatogram, is uniform. It is still used in this form.

 

   In the thin-layer chromatogram on silica gel, Rf45 = 0.48; Rf ioi = 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 dissolved in 100 ml of freshly distilled dimethylformamide. The solution is left at 250 for 19 hours. 1.2 liters of water are then added and the crystalline precipitate is filtered off with suction. The dipeptide derivative is dried in vacuo at 400 and then recrystallized twice from methanol-water.



   F. 180-181; [a] 20 = +90 (c = 2.05 in chloro 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) and hydrogenated in the presence of 2 g of palladium-carbon (10% Pd). After the hydrogenation has ended, the catalyst is suction filtered and evaporated at 400. The residue is used further directly.



   13. Z-Ser (tBu) -Asn-Leu-OMe
8.90 g of H-Asn-Leu-OMe and 11.0 g of Z-Ser (tBu) -OH are dissolved in 100 ml of freshly distilled dimethylformamide. The solution is cooled to 0 and 3.90 g of N-hydroxysuccinimide and then 8.70 g of dicyclohexylcarbodiimide are added. After standing at 0 for 30 minutes and at 250 for 18 hours, the precipitated dicyclohexylurea is suctioned off and the filtrate is poured into 800 ml of ice water. The resulting crystalline precipitate is dried in vacuo at 400 and then recrystallized from methanol-water. The Z-Ser (tBu) -Asn-Leu-OMe obtained in analytically pure form melts at a temperature of 202 to 2050; [n] 2D '-180 (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 dissolved in 600 ml of methanol 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 evaporated to dryness in vacuo at a bath temperature of 300. The crystalline tripeptide derivative obtained in this way shows in thin-layer chromatography on silica gel plates in the chloroform-methanol system (90:10) Rf = 0.11.



  15. BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-OMe
5.0 g of H-Ser (tBu) -Asn-Leu-OMe and 6.7 g of BOC Cys (TRI) -ONP are dissolved in 30 ml of freshly distilled dimethylformamide. The yellow solution is left to stand at 260 for 42 hours. It is then precipitated by adding 500 ml of ice water and the precipitated product is suction filtered. It is dissolved in ethyl acetate and the solution is washed with 5% citric acid solution and then with water. This is followed by washing 5 times with a mixture of 1 part by volume of 5% potassium carbonate and 5% potassium bicarbonate solution and then with water while cooling with ice to remove p-nitrophenol. The ethyl acetate solution leaves a resinous product after drying and evaporation. This is reprecipitated four times from acetone and petroleum ether.

  The tetrapeptide derivative obtained as a solid powder shows F. 181-1.820; [a] D = -110 (c = 2.09 in methanol).



   In thin layer chromatography on silica gel plates, Rf43A = 0.57; Rf in ethyl acetate = 1.10; 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 of BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-OMe are dissolved in 70 ml of methanol and 7 ml of hydrazine hydrate are added to the solution, which has been cooled to 0. After 16 hours at 20, an ice-cold solution of 600 ml of 2N acetic acid is added, the gelatinous precipitate is triturated well, suction filtered and washed neutral with plenty of water. The powder obtained is dried in vacuo at 400 and then suspended in 100 ml of acetonitrile.



  After trituration, it is suction filtered and dried at 400 in a vacuum. The BOC-Cys (TRI) Ser (tBu) -Asn-Leu hydrazide obtained shows F. 216-2180.



   When subjected to thin layer chromatography on silica gel plates, it has the following Rf values: Rf = 0.55 in dioxane-water (98: 2); Rf = 0.52 in chloroform methanol (8: 2); Rft02E = 0.70.



  17. BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-Ser (tBu) Thr (tBu) -Cys (TRI) -Val-Leu-OH
At -150 to 1.075 g (1.26 mmol) of BOC Cys (TRI) -Ser (tBu) -Asn-Leu-NH-NH2 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 (1.74 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 -100 and then the solution, cooled to -120, of 1.104 g (1.26 mmol) of H-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu OH and 0 , 61 ml (4.41 mmol) of triethylamine in 15 ml of dimethylformamide were added dropwise. The reaction mixture is stirred for one hour at -100 and four hours at room temperature. After evaporating to a small volume in a high vacuum and adding water, a powdery product is obtained which is triturated twice with water and then dried over potassium hydroxide solution in a high vacuum. This crude product is purified by dissolving it from methanol.

  From a methanolic solution saturated at 500, a white powder separates out overnight at 0, which turns out to be uniform in the thin-layer chromatogram
In the thin-layer chromatogram on silica gel, Rf48C = 0.54; Calls21 = 0.65; Rf45 = 0.50; Rf70 = 0.75; Rf in chloroform-methanol (8: 2) = 0.40.
EMI27.1


 <tb>



  18th <SEP> BOC-Cys-Ser- (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu)
 <tb> <SEP> Cys-Val-Leu-OII
 <tb>
846 mg (0.5 mmol) of 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 in 4 ml of methanol were added. A gelatinous product begins to separate from the clear solution after about 5 minutes. The reaction mixture is stirred for 60 minutes at room temperature, diluted with 50 ml of dimethylformamide, and then hydrogen sulfide is passed through it for 20 minutes and nitrogen for 15 minutes. The black precipitate is filtered off through Celite and washed with dimethylformamide. The filtrate is concentrated to 40 ml and nitrogen is passed through it for 15 minutes.

  This solution is simultaneously added dropwise 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 one hour while stirring. After 10 hours at room temperature, the solvent is evaporated off in a high vacuum at 400 and the oily residue is first triturated three times with petroleum ether and then twice with water and lyophilized. The brownish crude product is purified by countercurrent distribution in the system: methanol buffer chloroform-carbon tetrachloride (10: 3: 5: 4). The buffer is obtained from 28.6 ml of glacial acetic acid and 19.25 g of ammonium acetate, made up to 1 liter with water.

 

  After 135 distribution steps, the product is in 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. 254 mg of a chromatographically uniform product result.



   In the thin-layer chromatogram on silica gel, RfrÏ = 0.42; Rfl2lA = 0.70; RfTo = 0.75; Rf53 = 0.43; Rfnst = 0.22.



   b) 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- are added at room temperature Leu-Ser (tBu) -Thr (tBu) - Cys (TRI) -Val-Leu-OH in 500 ml of methanol was added dropwise within 45 minutes.



  When the addition is complete, stirring is continued for 1 hour and the solution is then decolorized at 0 with 1.0N aqueous thiosulphate solution (26.05 ml 1N thiosulphate). The clear solution is concentrated in a water jet vacuum at 300 to about 100 ml. 1.5 l of water are then added, and the precipitated product is filtered off and washed 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 of ammonium acetate, made up to 11 with water). After 135 steps, the main product is in elements 50 to 79 (rmax = 64; K = 0.96). The contents of these elements are evaporated to dryness in a high vacuum (400) and the ammonium acetate is sublimed off.



  The received
EMI28.1


 <tb> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu)
 <tb> <SEP> Cys-Val-Leu-OH
 <tb> (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
11.1 ml of triethylamine and 20.5 g of H-Try-OMe chlorohydrate are added to a solution of 29.8 g of Z-Tyr (tBu) OH in 190 ml of chloroform, the mixture is stirred for 30 minutes at room temperature and then cooled to 00. At this temperature, a solution of 29.9 g of dicyclohexylcarbodiimide in 50 ml of chloroform is added dropwise. The mixture is stirred for 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 is shaken out at 0 with 5% citric acid solution and 2N sodium carbonate. 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, eluted with 9 liters of toluene, 6.5 liters of toluene-chloroform (9: 1) and 15 liters of toluene-chloroform (1: 4). The fractions, which are pure chromate grams according to the thin layer, are combined and evaporated to dryness in a vacuum. 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 of Z-Tyr (tBu) -Try-OMe are dissolved in 520 ml of methanol in the presence of 4 g of 10% palladium carbon and 21.8 ml of 2.1N hydrochloric acid are hydrogenated at room temperature. When the hydrogenation is complete, the catalyst is filtered off and the filtrate is evaporated to dryness in vacuo at 30. 22.0 g of H-Tyr (tBu) -Try-OMe-chlorohydrate Rf = 0.45 in chloroform-acetone (1: 1) are obtained.



  21. Z-Ala-Tyr (tBu) -Try-OMe
20.8 g of H-Tyr (tBu) -Try-OMe, HCl are dissolved in 40 ml of dimethylformamide, 16.7 g of Z-Ala-ONP and 6.1 ml of triethylamine are added and the mixture is stirred until the mixture crystallizes. After standing overnight, 300 ml of ethyl acetate are added, the insoluble light material is filtered off and the filtrate is washed free of nitrophenol at 0 with dilute potassium carbonate solution, then extracted with 0.1 M citric acid and water. After the solution has been dried and the ethyl acetate has evaporated, the residue is dissolved in 25 ml of chloroform, drawn up on a column of 200 g of silica gel and eluted with chloroform. The product obtained is recrystallized from ethyl acetate-hexane. The Z-Ala-Tyr (tBu) -Try-OMe melts from 1080 with slow decomposition.

  Rf = 0.5 in chloroform-acetone (1: 1).



  22. H-Ala-Tyr (tBu) -Try-OMe
10.3 g of the carbobenzoxy compound of 21 are hydrogenated in 200 ml of methanol in the presence of 0.5 g of 10% palladium-carbon as previously shown. 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 of DPC-Ser (tBu) -OH (released from the cyclohexylammonium salt with citric acid) in 25 ml of ethyl acetate are mixed with 2.7 ml of triethylamine and 2.7 ml of isobutyl chloroformate at -100.



  After 5 minutes at -100, the solution of 8.1 g of H-Ala-Tyr (tBu) -Try-OMe in 20 ml of dimethylformamide is added to the mixed anhydride and for 10 minutes at -100, 2 hours at 0 and 12 hours at room temperature touched. The mixture is diluted with ethyl acetate, extracted at 0 with 0.1 M citric acid and saturated potassium carbonate solution, then washed with water at room temperature, dried and evaporated to dryness in vacuo. The residue is recrystallized from ethyl acetate-hexane. 10.5 g of DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-OMe with a melting point of 148-149 (decomposition) are obtained. Rf = 0.7 in the chloroform-methanol (9: 1) system.



  24. DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-NH-NH,
1.34 g of the tetrapeptide derivative of 23 are dissolved in 7.5 ml of methanol, treated with 0.75 ml of hydrazine hydrate and left to stand for 24 hours at room temperature. By adding 7.5 ml of methanol and 15 ml of water, the DPC-Ser (tBu) -Ala Tyr (tBu) -Try-NH-NH2 crystallizes out. The product is filtered off with suction, washed well with 50% methanol and water and dried over concentrated sulfuric acid in a high vacuum. The product melts from 1360 with slow decomposition. Rf = 0.35 in the chloroform-methanol (9: 1) system.

 

  25. Z-Asn-Phe-NHNH-BOC
1.4 g of H-Phe-NHNH-BOC, 1.94 g of Z-Asn-ONP and 3 ml of dimethylformamide are stirred at room temperature until the mixture solidifies. After standing overnight, it is triturated with ether, the dipeptide derivative is filtered off and washed free of nitrophenol with ether.



  The product begins to melt with decomposition at 2110. 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 who, dissolved in 1 liter of methanol, hydrogenated in the presence of 2.9 g of 10% palladium-carbon at room temperature. After the decarbobenzoxylation is complete, the catalyst is filtered off and the filtrate is evaporated to dryness. The product melts at 160-1610.



  Rf = 0.15 in chloroform-methanol (8: 2).



  27. Z-Asn-Asn-Phe-NH-SH-BOC
20.93 g of H-Asn-Phe-NH-NH-BOC are dissolved in 45 ml of dimethylformamide with heating, 22.7 g of Z-Asn-ONP are added at room temperature and the mixture is stirred until the mixture solidifies. After standing overnight, the solid mass is dissolved in 160 ml of dimethylformamide with warming, precipitated by dropping into 1 liter of ether, the product is filtered off and washed free of nitrophenol 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 the decarbobenzoxylation is complete, the catalyst is filtered off, the dimethylformamide solution is concentrated to about 100 ml and the H-Asn-Asn-Phe NHNH-BOC is precipitated by dropping it into 1 liter of ether. Rfloo = 0.35 on silica gel.



  29. Z-Leu-Asn-Asn-Phe-NHNH-BOC
26.5 g of H-Asn-Asn-Phe-NHNH-BOC are dissolved in 58 ml of warm dimethylformamide, a solution of 24.2 g of Z-Leu-ONP in 16 ml of dimethylformamide is added at room temperature and the mixture is stirred until the mixture solidifies . After leaving to stand overnight, it is triturated with ether, filtered off, dissolved in 190 ml of dimethylphenamide and the product is reprecipitated by dropping it into 1.5 liters of ether. It is filtered off and washed free of nitrophenol with ether. Rf = 0.45 in the chloroform-methanol (7: 3) system on silica gel.



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



  31. Z-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC
27.9 g of H-Leu-Asn-Asn-Phe-NH-NH-B OC are dissolved in 145 ml of dimethylformamide, a solution of 21.6 g of Z-Asn-ONP in 20 ml of dimethylformamide is added at room temperature and the mixture is stirred until the mixture solidifies. After standing overnight, it is worked up as described for 29. 34.2 g = 88% of theory are obtained. Th. Of the pentapeptide derivative.



     Rfroo = 0.5.



  32. H-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC
34.2 g of the pentapeptide derivative described under 31 are hydrogenated and worked up as described under 30. The H-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC shows Rfioo = 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, while cooling with cold water, 21.2 ml of 3.6N hydrochloric acid in dioxane are added, and solution occurs. 28.2 g of the 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, HC1, then 17.4 g of dicyclohexylcarbodiimide. The mixture is stirred for 24 hours, the dicyclohexylurea which has crystallized out is filtered off and the filtrate is concentrated to about 250 ml. The crude product is precipitated by dripping this solution into 2 liters of ether. This is dissolved in 280 ml of hot dimethylformamide by adding it with stirring.



  After cooling to room temperature, the product is precipitated with 870 ml of saturated sodium chloride solution and 1740 ml of water. The precipitated product is filtered off with suction and washed well with semi-saturated sodium chloride solution. The moist suction filter is dissolved in 1 liter of warm dimethylformamide and azeotropically dehydrated by concentrating the solution twice. The precipitated common salt is filtered off, the filtrate is added dropwise to 2.4 liters of acetonitrile, the resulting precipitate is filtered off after a few hours, washed with acetonitrile and ether and dried in vacuo at 400. The Z-Arg-Asn-Leu-Asn-Asn-Phe-NH-NH-BOC hydrochloride has Rfloo = 0.25.



  34. ZArg-Asn-Leu-Asn-Asn-Phe-NHNH2-
Hydrochloride
10.6 g of the hexapeptide derivative described under 33 are dissolved in 50 ml of 90% strength trifluoroacetic acid with stirring and ice cooling, warmed to room temperature and left at this temperature for 20 minutes. The solution is then added dropwise to 500 ml of ether while stirring and cooling with ice, the precipitate is filtered off and washed acid-free with ether. The product is introduced into 90 ml of warm dimethylformamide, stirred for some time and then precipitated in powder form with 1 liter of ether. This crude product is stirred with 170 ml of isopropanol for 1 hour at room temperature, filtered off, washed with isopropanol and ether and dried in vacuo at 400. The Z-Arg-Asn-Leu-Asn-Asn-Phe-NHN112 hydrochloride is obtained, which has Rfs6 = 0.38 on silica gel.



  35. Z-Phe-Ser (tBu) -OCH3
27.0 g (90 mmol) of Z-Phe-OH are dissolved in 250 ml of absolute tetrahydrofuran together with 12.5 ml (90 mmol) of triethylamine and cooled. At -180, 14.7 ml (112 mmol) of isobutyl chlorocarbonate are added dropwise and for half an hour <-100 stirred. 18.1 g of H-Ser (tBu) -OCH3 hydrochloride (85.5 mmol) in finely powdered form are then added to the white suspension (triethylamine hydrochloride), diluted with 350 ml of tetrahydrofuran and then 15.0 ml (108 mmol) Add triethylamine in 100 ml of tetrahydrofuran dropwise. The mixture is stirred for a further 3 hours at about -100 and then overnight at room temperature.

  The suspension is then concentrated in vacuo to about 250 ml, taken up in a lot of ethyl acetate and extracted with dilute citric acid solution (3 x 200 ml), dilute sodium carbonate solution (3 x 200 ml) and saturated sodium chloride solution (5 x 200 ml), dried over sodium sulfate and evaporated. The crude product is dissolved in 200 ml of ethyl acetate-hexane (1: 1). When the solution cools, white drusen crystallize out, which are isolated and dried. The product melts at 92.5-94.



       [α] D24 = + 3 0.5 (c = 2.2% in methanol).



  When using thin layer chromatography on silica gel plates
On thin-layer chromatography on silica gel plates, the product had the following Rf advertising: 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 of Z-Phe-Ser (tBu) -OCH3 (55.7 mmol) are dissolved in 250 ml of methanol, and 28 ml (575 mmol) of hydrazine hydrate are added while cooling with ice. The solution is left in the refrigerator for 67 hours, a thick crystal cake forming. The product is isolated and dried. It melts at 158.5-159.5.



     [] 2r) = + 160 # 0.50 (c = 2.3% in glacial acetic acid).



   In the thin-layer chromatogram on silica gel layers, Rf = 0.27 in chloroform-methanol (95: 5); Rfl3.s = 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 41.6 ml of hydrogen chloride in ether (2.43 n., 101 mmol) are added at -200 . 7.15 ml of t-butyl nitrite (59 mmol) in 40 ml of dimethylformamide are added to the clear solution and the mixture is rinsed with 20 ml of dimethylformamide.



  After 10 minutes at temperatures below -9, 14.0 ml (101 mmol) of triethylamine in 60 ml of dimethylformamide are added and 8.85 g of H-Gly-OCH3-hydrochloride (70 mmol) are added. Three portions of 5.6 ml each of triethylamine are added at intervals of 10 minutes (121 mmol in total). The pH is then about 8.5. The cooling mixture is then removed and stirred for three hours at 00, then overnight at room temperature. After concentrating the reaction mixture in vacuo to a thick paste, this is taken up in a large amount of ethyl acetate and the solution is washed successively with 3 portions of dilute citric acid solution, 3 portions of dilute sodium carbonate solution and 5 portions of saturated saline solution.

  The crude product obtained after drying and evaporation of the solution is dissolved in ethyl acetate, filtered through a glass frit and treated with about 800 ml of ether. The protected tripeptide ester crystallizes out very slowly, mp 140-1410.



     [Ia] 20 = + 130 + 0.50 (c = 2.3 in glacial acetic acid).



   In the thin-layer chromatogram on silica gel layers in chloroform-methanol (95: 5) Rf = 0.61; in chloroform-acetone (9: 1) Rf = 0.28; Rflo2E = 0.82; Rfisc = (), 72.



  38. H-Phe-Ser (tBu) -Gly-OCHo
7.4 g of Z-Phe-Ser (tBu) -Gly-OCH3 are decarbobenzoxylated in methanol with hydrogen in the presence of 1.0 g of palladium-carbon (10% Pd). When the uptake of hydrogen has ended, it is filtered and evaporated, and the resulting crude product is processed further directly. The product has the following Rf values in a thin-layer chromatogram on silica gel layers: in chloroform-methanol (95: 5): Rf = 0.26; Rfu = 0.60; Rfse = 0.51.



  39. Z-Arg-Phe-Ser (tBu) -Gly-OCH3 hydrobromide
5.3 g of Z-Arg-OH (17.3 mmoles) are suspended in 15 ml of dimethylformamide and, after cooling with an ice-sodium chloride mixture, 5.9 ml of hydrobromic acid in methanol (3.06%, 18 mmoles) are added. A clear, colorless solution results after brief stirring. To this end, a solution of 5.6 g of H-Phe Ser (tBu) -Gly-OHC3 (14.4 mmol) in 15 ml of dimethylformamide is added dropwise at -180 and a further 7 ml of dimethylformamide are added. A solution of 3.85 g of dicyclohexylcarbodiimide in 5 ml of methylene chloride is then added, after 1 hour the cooling mixture is replaced by ice water and, after a further three hours, the mixture is stirred overnight at room temperature.

  The precipitated dicyclohexylurea precipitate is then filtered off, the filtrate concentrated to half in vacuo, cooled to 0 and stirred into 600 ml of ether. The resulting precipitation is decanted off, the precipitate is washed with ether, dissolved in about 50 ml of methanol and reprecipitated with 600 ml of ether. After redissolving in methanol, it is evaporated and dried in a high vacuum. The product shows in a thin chromatogram on silica gel layers:
Rf52 = 0.48; Rfloo = 0.54; on Cellulose Selekta
Rf25 = 0.81 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 and treated with 2.9 ml of hydrogen chloride in dioxane (3.09-n., 8 , 9 mmol) added. It is hydrogenated in the presence of 1.2 g of 10% palladium-carbon at room temperature and atmospheric pressure. When the uptake of hydrogen has ended, it is filtered and evaporated, the residue obtained is dissolved in 30 ml of methanol and the solution is stirred into 700 ml of ether. This creates a yellowish paste that is dissolved and precipitated in the same way. Finally the product is redissolved in methanol, filtered and evaporated. The product shows the following values in a thin-layer chromatogram on silica gel layers:
Rf59 = 0.12; Rf96 = 0.38; Rfloo = 0.13.



  41. Z-His-Arg-Phe-Ser (tBu) -Gly-OCH3
7.6 g of Z-His-NH-NH2 (25 mmol) are slurried in 60 ml of dimethylformamide, 31 ml of hydrogen chloride in ether (2.43-n., 75.3 mmol) are added at - 160 units and the resulting mixture is mixed Clear, yellowish solution obtained at - -18 with 3.2 ml of t-butyl nitrite (95%, 26.5 mmol) in 10 ml of dimethylformamide. After 10 minutes, 10.4 ml of triethylamine (75 mmol) in 25 ml of pre-cooled dimethylformamide and 8.5 g of H-Arg-Phe Ser (tBu) -Gly-OCH8, 2HCl (14 mmol) in 50 ml of pre-cooled dimethylformamide are added. It is diluted with 40 ml of dimethylformamide and 4 portions of 1.0 ml each of triethylamine (total 28.7 mmol) are added at intervals of 5 minutes.

  The mixture is left to stand for a further three hours at 0 and 20 hours at room temperature, the precipitated salts are then filtered off and the reaction solution is concentrated in vacuo to about 3/4 of its volume. By repeatedly stirring the product into a large amount of ethyl acetate, a crude product is obtained which is distributed in the system in countercurrent
Methanol buffer-chloroform
Carbon tetrachloride 9: 3.5: 8: 2) is purified over 130 steps (buffer as under 18); rmax = 54; K = 0.7). The pure fractions are combined, evaporated, freed from ammonium acetate at 400 in a high vacuum and lyophilized after dissolving in water.

 

   In the thin-layer chromatogram on silica gel layers, Rf52A = 0.34; Rflola = 0.71; Rfioi = 0.76; RfltlA = 0.40.



  42. H-His-Arg-Phe-Ser (tBu) -OCH3,2 CH8-COOH
2.9 g of 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 uptake of hydrogen has ceased, it is filtered and evaporated to dryness in vacuo, 50 ml of toluene each time evaporated over the residue. The product, a white powder, shows the following Rf values in thin-layer chromatograms on silica gel plates: RflolB = 0.51; Rf96 = 0.10; RflIIA = 0.28; on cellulose Selekta Rf45 = 0.55; Rf52s = 0.44.



  43. H-His-Arg-Phe-Ser (tBu) -Gly-OH, CH3-COOH
2.84 g of H - His - Arg - Phe - Ser (tBu) - Gly - OCH 3, 2 CH 3 - COOH are dissolved in 40 ml of water and stirred with 15 ml of 2-n-piperidine solution in water for 14 minutes at room temperature. The clear, slightly yellow solution is then neutralized with 15 ml of 2N aqueous acetic acid, filtered and lyophilized. 600 piperidine acetate is stripped off from the lyophilizate, a slightly yellow deliquescent mass, in a high vacuum in a water bath. It is now dissolved in 10 ml of methanol and stirred into 400 ml of ethyl acetate. The white, flaky precipitate is filtered, 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 layers, Rf101B = 0.45; Rf121 = 0.47; on cellulose Selekta: Rf45 = 0.48; Rf55 = 0.38; Rf90 = 0.36.



   In paper electrophoresis (paper S + S 2043 6; 5 hours; 2000 volts; pH 6.3) the substance migrates 6.5 cm to the cathode. In the electrophoresis on Selekta cellulose plates (pH 6.3; 3 hours; 1000 volts) the substance migrates 8.5 cm to the cathode.



  44. Z-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-
Ser (tBu) -Gly-OH, CH3-COOH
3.77 g of Z-Arg-Asn-Leu-Asn-Asn-Phe-NH-NHe are suspended 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) added. Part of the suspension goes into solution. Now 0.5 ml of t-butyl nitrite (95%, 4.14 mmol) is added at - 100. After 5 minutes at -10 to -120, another 0.08 ml of t-butyl nitrite is added. After a total of 11 minutes of reaction time, 1.29 ml of triethylamine (9.26 mmol) are added and immediately thereafter the precooled solution of 2.40 g of 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.

  In four portions, 1.08 ml of triethylamine (7.8 mmol) are then added over a period of 2.5 hours, the pH is between 7 and 8. The batch is left at 0 overnight and the solution is equalized in the morning Kind of azide prepared as described above from 1.81 g of Z-Arg-Asn-Leu-Asn Asn-Phe-NH-NHs, then another 0.62 ml (4.47 mmol) of triethylamine. Again, the batch is left overnight at 00, and in the morning it is concentrated in vacuo at a bath temperature of 450 to about
15 ml volume and stir the batch in 800 ml of ethyl acetate. The filtered crude product is washed with ethyl acetate and dried. It becomes one
Countercurrent distribution in the n-butanol-glacial acetic acid system
Water (4: 1: 5) subjected to over 300 stages; rrna = 94; K = 0.46.

  The pure fractions determined by thin layer chromatography are combined, evaporated, dissolved in water, filtered and lyophilized.



   In a thin-layer chromatogram on silica gel layers, the product shows: RflolB = 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% strength Palladium-carbon hydrogenated at room temperature and atmospheric pressure. After 15 hours of shaking under a hydrogen atmosphere, the mixture is filtered, evaporated in vacuo, dissolved in water and lyophilized. The pure product shows the following Rf values in a thin layer chromatogram on silica gel plates:
Rf101B = 0.42; Rf101A = 0.34; on cellulose selecta: Rf45 = 0.27; on Alox-Camag: Rf53A = 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 of DPC-Ser (tBu) -Ala-Tyr (tBu) -Try-NHNH2 in 15 ml of degassed dimethylformamide are mixed with 0.6 ml of 3.6N hydrogen chloride in dioxane and then with 0.124 ml of t-butyl nitrite at - 300. After 15 minutes at -150, 1.7 ml of 2N sodium carbonate solution are added dropwise (pH 7-8) and the azide is precipitated with ice-cold water. The precipitate is filtered off with suction and washed with ice-cold water. 272 mg of H-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe are dissolved
Ser (tBu) -Gly-OH, acetate by gentle heating in 5.5 ml of dimethylformamide, gives 0.51 ml of l-m at room temperature. Triethylamine in dimethylformamide is added and the solution is treated at 0 with the solid azide prepared above.

  The suction filter is washed out with 4 ml of cold dimethylformamide and 2 ml of cold ethyl acetate. The solution is then stirred at 0, after 20 minutes 0.5 ml of water and after 2 hours 0.05 ml of 1-m. Triethylamine in dimethylformamide are added. After 4 hours, the reaction mixture is precipitated with a lot of ether. The product, which initially precipitated out as an oily product, was obtained as a flaky product after repeated trituration with ether and reprecipitation from methanol-ether. The crude product obtained is purified by Craig distribution over 150 stages in a mixture of methanol-buffer-chloroform-carbon tetrachloride (10: 3 7: 4) (buffer as under 18). The pure substance is in the distribution elements 75-112.

  These are combined, the organic solvents are removed in vacuo and the aqueous solution is lyophilized. The protected pentadecapeptide is obtained which, on chromatography on silica gel in the ethyl acetate-pyridine-water system (49: 24: 27) shows Rf = 0.4.

 

  47. Z-Gly-Phe-Gly-Pro-OtBu
15.5 g of Z-Gly-Phe-Gly-OH [represented by J. R. Vaughan & J. A. Eichler, J. Am. Chem.



  Soc. 75, 5556 (1953)] in 100 ml of absolute tetrahydrofuran and 5.3 ml of absolute triethylamine and 4.64 ml of isobutyl chlorocarbonate is added dropwise to the solution, which has been cooled to - 200, with stirring so that the internal temperature does not exceed - 150. Then will
Allowed to react for 10 minutes at -100, then cooled again to -200 and a solution of 7.6 g of H-Pro OtBu in 30 ml of dimethylformamide was added dropwise with stirring in such a way that the internal temperature does not exceed 0.



  The mixture is left to stand at 0 for 18 hours, the triethylamine hydrochloride is then filtered off with suction and the filtrate is evaporated at a bath temperature of 400. The residue is dissolved in 200 ml of ethyl acetate and the solution is washed with sodium bicarbonate solution, water, 10% strength tartaric acid solution and water, dried with sodium sulfate and evaporated. The residue is crystallized from ethyl acetate petroleum ether. F. 66-710; [a] D = -450 (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 the solution is left to stand at 220 for 20 minutes. It is then concentrated to an oily consistency on a rotary evaporator at a bath temperature of 250, then 250 ml of water are added and the viscous mass is kneaded at 0. The aqueous phase is decanted off and the residue is washed twice with 100 ml of water each time in the same way. It is then dried in a high vacuum at a bath temperature of 300. The residue obtained is mixed with 600 ml of ether-petroleum ether (1: 1), triturated, suction filtered and dried. The product is processed further directly.



  49. Z-Thr (tBu) -Pro-NH2
5.27 g of ZThr (tBu) -OH and 1.56 g of H-Pro-NH2 are dissolved in 60 ml of acetonitrile, the solution is cooled to 0 and 3.11 g of dicyclohexylcarbodiimide are added.



  After 1 hour at 0 and 20 hours at room temperature, the dicyclohexylurea is suction filtered, the filtrate is evaporated and the residue is taken up in ethyl acetate. The ethyl acetate solution is washed with dilute citric acid solution, soda solution and water, dried with sodium sulfate and evaporated. In thin-layer chromatography on silica gel plates, the colorless residue shows Ruf45 = 0.76; Rf43C = 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 1N HCl and hydrogenated in the presence of 660 mg of palladium-carbon (10% Pd). After the uptake of hydrogen has ended, the mixture is evaporated to dryness at a bath temperature of 350 and dried. The residue is crystallized from ethanol-ether; F. 170-1730.



  51. Z-Glu (OtBu) -Thr (tBu) -Pro-NHz
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 300, then diluted with 150 ml of ethyl acetate and the ethyl acetate solution is washed at 0 four times with 50 ml of saturated potassium carbonate solution, 50 ml of water, 2 x 50 ml of oily citric acid solution and 2 x 50 ml of water, dried with sodium sulfate and evaporated. The evaporation residue is crystallized from ethyl acetate-hexane, mp 159-1610.



  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 hydrogenated in the presence of 1.2 g of palladium-carbon (10% Pd). When the hydrogenation is complete, the catalyst is suction filtered 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 chloroform-methanol (9: 1) system.



  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 1.76 g are added to the solution Dicyclohexylcarbodiimide. After 12 hours at 250 °, the precipitated dicyclohexylurea is suction filtered and the filtrate is evaporated to dryness. The residue is taken up in ethyl acetate and the solution is washed with tartaric acid solution, water, sodium bicarbonate solution and water, dried with sodium sulfate and evaporated. For purification, the residue is reprecipitated twice from ethyl acetate solution by adding petroleum ether. In thin layer chromatography on silica gel plates, Rf43c = 0.52; Rfs2 = 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 the solution is hydrogenated in the presence of 600 mg of palladium-carbon (10% Pd). When the hydrogenation is complete, the catalyst is suction filtered, the filtrate is evaporated to a volume of 10 ml, 20 ml of benzene are added, the mixture is concentrated again to 10 ml and the product is precipitated with 70 ml of petroleum ether. The greasy precipitate is ground to a powder, suction filtered and dried; Yield 3.3g. In thin layer chromatography on silica gel plates, Rfac = 0.27; Rf5 = 0.24; Rf in chloroform
Methanol (8: 2) = 0.10.



  55. DPC-Met-Gly-Phe-Gly-Pro-Glu (OtBu) Thr (tBu) -Pro-NH2
3.72 g of DPC methionine (G. number 17108/67, Case 6106 / 1-3) and 5.21 g of H-Gly-Phe Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro- are suspended NH2 in 30 ml of acetonitrile and then 1.98 g of dicyclohexylcarbodiimide are added.



  After the air in the reaction vessel has been displaced by nitrogen, the mixture is stirred at 300 for 20 hours.



  The mixture is then diluted with 45 ml of methanol, warmed slightly and then the still undissolved dicyclohexylurea is suctioned off and the filtrate is concentrated in vacuo. The residue is dissolved in ethyl acetate and washed neutral at 0 as described under 52.



  The ethyl acetate solution leaves 9.12 g of colorless product on drying and evaporation, which crystallizes from ethyl acetate-hexane; F. 1820. By thin layer chromatography on silica gel plates, Rf4e = 0.64, Rf in chloroform-methanol (9: 1) = 0.30.

 

  56. H-Met-Gly-Phe-Gly-Pro-Glu (OtBu) Thr (tBu) -Pro-NH,
3.0 g of the octapeptide derivative obtained under 55 are dissolved in 35 ml of methylene chloride and 28 ml of a mixture of monochloroacetic acid and water (3: 1) are added at room temperature. After 10 minutes at room temperature, it is poured into 170 ml of ice-cold 2N soda solution. The octapeptide derivative which separates out is extracted with 200 ml of n-butanol / ethyl acetate (1: 1) and the organic phase is washed neutral with water. It is then evaporated to dryness, the octapeptide derivative having a free amino group being obtained as a colorless residue which, on thin-layer chromatography on silica gel plates, shows Rftoo = 0.23 and Rf43C = 0.51.



  57.Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-
Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH, ditosylate
258 mg of the pentadecapeptide diacetate described under 46 are dissolved in 4 ml of absolute pyridine at 400 and then 1.025 ml of a 4% solution of toluenesulfonic acid monohydrate (2 equivalents) are added at 0. The solution is evaporated to dryness in a high vacuum at a bath temperature of 350 and the residue is triturated with ether. This is followed by suction filtration and drying (at 300), the ditosylate being obtained as a solid powder.



   RflOlA = 0.58; Rfno = 0.09; Ruf57 = 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-hydroxysuccinimide are dissolved in 1 ml of dimethylformamide with heating to 600 and 0.3 ml of a dimethylformamide solution containing 30 mg of dicyclohexylcarbodiimide is added to the solution, which has been cooled to 250. The reaction mixture is left under nitrogen for 4 hours at 250 and 12 hours at 450, the deposited dicyclohexylurea is filtered off and the filtrate is precipitated with a mixture of 15 ml of benzene and 40 ml of petroleum ether. The resulting precipitate is centrifuged off, dried and, after dissolving in 2.5 ml of dimethylformamide, precipitated again by adding 30 ml of benzene petroleum ether (1: 2).



  After the solution has been decanted off, the precipitate is dried. Yield 390 mg of crude product. For purification, it is multiplicatively distributed over 200 levels in the methanol-buffer-chloroform-carbon tetrachloride system (10: 3: 6: 5; buffer as under 18). The pure product is obtained from elements 103-132, the maximum is in element 122 (K = 1.5).



  The yield is 213 mg; Roller blind = 0.36; Rf52A = 0.25; Rf96 = 0.42.



  59. H-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-Met-Gly
Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2-tritosylate
210 mg of the protected tricosapeptide amide obtained under 58 are dissolved in 10 ml of glacial acetic-formic acid (83 S) -water (7: 1: 2) and the solution is left to stand at 300 for 1 hour. The product with a free α-amino group is then precipitated by adding 100 ml of ether and centrifuged off. The supernatant is mixed with 25 ml of petroleum ether, concentrated to a volume of 30 ml, and the resulting precipitate is centrifuged off. Both precipitates are combined and dissolved in 5 ml of methanol-0.1-m. Acetic acid (2: 1) dissolved. The solution is passed through a column of Merck 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 450 to constant weight and the residue is dissolved in 10 ml of 90% pyridine. Then 27 mg of toluenesulfonic acid monohydrate are added and the solution is evaporated to dryness in a high vacuum. The residue is dissolved in 2 ml of water, the solution is lyophilized and then dried in a high vacuum at 400.



  17 mg of tritosylate of the protected tricosapeptide amide with a free α-amino group are obtained.



     Rf9s = 0.27; RflolA = 0.58; Rf87 = 0.15.
EMI33.1


 <tb>



  60. <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu)
 <tb> <SEP> Cys-Val-Leu-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn- <SEP>
 <tb> Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-NH2
EMI33.2


 <tb> <SEP> 675 <SEP> mg <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr- <SEP>
 <tb> (tBu) -Cys-Val-Leu-OH, <SEP> 1.30 <SEP> g <SEP> des <SEP> under <SEP> 59 <SEP> received
 <tb> Tritosylate of the sequence 10-32, 38 mg of N-methylmorpholine and 86 mg of N-hydroxysuccinimide are dissolved in 7 ml of freshly distilled dimethylformamide with heating to 700. The solution is then cooled to 250 and 115 mg of dicyclohexylcarbodiimide are added. The reaction mixture is left to stand at 250 for 9 hours and at 450 for 8 hours under nitrogen.

  The dicyclohexylurea is then filtered off and 80 ml of benzene and 400 ml of petroleum ether are added to the filtrate. The crude protected dotriacontapeptide amide which precipitates out is dissolved in 25 ml of methanol and ether precipitated by adding 100 ml of benzene and 250 ml of petroleum. The precipitate is then filtered off with suction 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).



   With thin layer chromatography on silica gel, Rf52 = 0.20; Rf> G = 0.44; Rftoo = 0.23; Rf87 = 0.69; on aluminum oxide, Rf45 = 0.67.
EMI33.3


 <tb>



  61. <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu)
 <tb> <SEP> Cys-Val-Leu-Ser (tBu) -Ala-Tyr (tBu) <SEP> -Try-Arg-Asn Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) -Gly-OH
EMI33.4


 <tb> <SEP> 145 <SEP> mg <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr
 <tb> (tBu) -Cys-Val-Leu-Oh <SEP> in <SEP> 0.7 <SEP> ml <SEP> degassed <SEP> dimethyl formamide and 0.116 ml of a 1-m. Solution of N-methylmorpholine in dimethylformamide are added at - 150 with 0.116 ml of a l-m. Solution of pivaloyl chloride in dimethylformamide was added. After 3 minutes at -10 to -150, a solution of 172 mg of H-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg-Asn-Leu-Asn-Asn
Phe-His-Arg-Phe-Ser (tBu) -Gly-OH, diacetate in 0.4 ml of dimethylformamide and 0.04 ml of a 1-m.



  Solution of N-methylmorpholine in dimethylformamide was added dropwise. After 30 minutes at -5 to -100, it is precipitated with ether and the solid product is isolated. This crude product is purified by Craig partition over 530 stages; System: methanol buffer-chloroform-carbon tetrachloride (10: 3: 7: 4) (buffer as under 18).

 

  The pure fractions (partition coefficient: 0.9 to 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.



     Rfg6 = 0.4; Rf43G = 0.18.
EMI33.5


 <tb>



  62. <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys Val-Leu-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
EMI34.1


 <tb> <SEP> 99 <SEP> mg <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) - <SEP>
 <tb> Thr (tBu) -Cys-Val-Leu-Ser (tBu) -Ala-Tyr (tBu) -Try Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) - Gly-OH, 2 HCl and 85 mg of the peptide of the sequence 25-32 obtained under 56 are mixed with 10 mg of N-hydroxysuccinimide, dissolved in 0.4 ml of dimethylformamide, while passing nitrogen over it, and the mixture is stirred at 400 until everything is dissolved is. After adding 12.5 mg of dicyclohexylcarbodiimide, dissolved in 0.1 ml of dimethylformamide, the mixture is stirred at 400 for a further 12 hours.

  The crude product is precipitated with 8 ml of peroxide-free ether and centrifuged off. For cleaning purposes, distribute as described under 60. The product obtained is completely identical to that described under 60.



   Example 2
EMI34.2


 <tb> H-Cys-S <SEP> er-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala- <SEP>
 <tb> Tyr-Tru-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-NH2 (thyrocalcitonin)
EMI34.3


 <tb> 300 <SEP> mg <SEP> BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val Leu-Ser (tBu) -Ala-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 as described in Example 1 with trifluoroacetic acid to split off the protective groups hydrolyzed, converted into the acetate and purified by countercurrent distribution. According to thin-layer chromatography and electrophoresis on cellulose acetate film, the product proves to be identical to the dotriacontapeptide amide obtained in Example 1. The starting material can be prepared 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 while warming, after cooling to 0 25.9 ml of absolute triethylamine are added, and the solution, cooled to - 10, with 8.64 ml of chlorocarbonic acid ethyl ester added. After 10 minutes at -100, a solution of 23.2 g (90 mmol) of H-Leu-OBzl. HC1 in 90 ml of dimethylformamide was added. The mixture is left at -10 for 30 minutes, at 0 for 3 hours and at 20 for 16 hours. The undissolved material is then filtered off and the filtrate is concentrated to dryness in a high vacuum. The residue is dissolved in ethyl acetate and the solution is washed with dilute citric acid solution, water, sodium bicarbonate solution and water, dried with sodium sulfate and evaporated.

  After the addition of petroleum ether, the product crystallizes, dried over vacuum over caustic soda.



   F.128-129.5; Rf2 = 0.65 Rf4 (c = 2 in dimethyl formamide); 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. Gaseous hydrogen chloride is passed through this solution for 1 1/2 hours. After evaporation to dryness, the residue is triturated three times with ether.



   F. 192-1950. Rf2 = 0727.



  3. BOC-Cys (Bzl) -Val-Leu-OBzl
14.2 g (32.8 mmol) BOC-Cys (Bzl) -ONP and 11.7 g (32.8 mmol) 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 0. The yellow suspension is stirred for 3 hours at 0 and overnight at room temperature.



  The precipitated triethylamine hydrochloride is then filtered off and the filtrate is evaporated to dryness in a high vacuum. The residue is dissolved in ethyl acetate and extracted by shaking, namely twice with water, twice with 0.1N hydrochloric acid, twice with 10% sodium chloride solution, six times with 10% sodium carbonate solution and twice with saturated sodium chloride solution. After drying over sodium sulfate, it is evaporated to dryness. The protected tripeptide of F. 113-115 crystallizes from ethyl acetate / petroleum ether; [α] D20 -33 (c = 1 in dimethylformamide); Rf3 = 0.30.



  4. H-Cys (Bzl) -Val-Leu-OBzl, TFA
10 g (16.3 mmol) of BOC-Cys (Bzl) -Val-Leu-OBzl are dissolved in 20 ml of 90% strength trifluoroacetic acid and left at room temperature for 1 hour. 200 ml of peroxide-free ether are then added and the mixture is stirred for 2 hours while cooling with ice / common salt. The precipitate is wiped off and washed twice with peroxide-free ether and dried in vacuo over caustic soda.



   F. 168-1730; Rf4 = 0.77.



  5. BOC-Ser-Thr-OBzl
9.4 g (38.2 mmol) of H-Thr-OBzl, HCl and 60 ml of methylene chloride are added to a solution of 14.7 g (38.2 mmol) of BOC-Ser-OH, dicyclohexylammonium salt in 100 ml of methylene chloride 10 minutes at room temperature and then cools to -5. At this temperature, a solution of 8.0 g (38.6 mmol) of dicyclohexylcarbodiimide in 18 ml of methylene chloride is added dropwise. The mixture is stirred for 3 hours at -5 and all night at room temperature. After filtering off the dicyclohexylurea and dicyclohexylamine hydrochloride, the solution is shaken out, three times with 0.1N hydrochloric acid, twice with 20% sodium chloride solution, once with 10% sodium bicarbonate solution and twice with 20% sodium chloride solution and dried over sodium sulfate.

  The solution is concentrated to about 100 ml, cooled to 5 and further dicyclohexyl nucleus is filtered off. Evaporation to dryness gives 15.8 g of ester. For purification, the product is crystallized from ethyl acetate-hexane.



   M.p. 110-111 [α] 20-8.5 (c = 2 in dimethyl formamide); @; [α] = 33 6. H-Ser-Thr-OBzl, TFA
9.3 g (23.5 mmol) of BOC-Ser-Thr-OBzl are dissolved in 13.8 ml of 90% strength trifluoroacetic acid and the solution is left at 22 for 20 minutes. It is then added dropwise to 138 ml of dry ether with stirring, stirred for one hour and left to stand at -100 for 2 days. The resulting precipitate is filtered off and washed three times with dry ether and dried over caustic soda in a vacuum.

 

   F. 12S1290; Rf * = 0.65; Rf4 = 0.50.



  7. BOC-Asn-Leu-NgHg-Z 34.8 g (150 mmol) of BOC-Asn-OH are slurried in 300 ml of acetonitrile and 38 g (150 mmol) of Woodwards reagent K are added. Then, while stirring and cooling, 21 ml (150 mmol) of triethylamine are added dropwise so that the internal temperature does not exceed +300. After the end of the introduction, stirring is continued at 250 for 45 minutes. 52.2 g (165 mmol) of H-Leu-N2H2-Z, HCl and 23 ml (165 mmol) of triethylamine in 185 ml of acetonitrile are added to the almost clear solution, the reaction mixture, which soon solidifies, is stirred overnight at room temperature, then cools down - 100 and stir for 2 hours at -10. The crystalline precipitate is then suction filtered and the dipeptide derivative is washed once with cold acetonitrile, once with ethyl acetate and eight times with water until it is free of chloride.

  The product obtained is dried at 400 in a vacuum.



   F. 194-196; [α] 30-39 (c = 2 in dimethylform amide); Rf1 = 0.45.



  8. H-Asn-Leu-NH2-Z, TFA
46.3 g (94 mmol) of BOC-Asn-Leu-N2H2-Z are dissolved in 92.6 ml of 90% trifluoroacetic acid and left at 200 for 1 hour. The solution is then added dropwise to 975 ml of ether with stirring and stirred for 15 minutes. The ether is decanted from the precipitation, another 210 ml of ether are added and the mixture is stirred under reflux at 350 for 1 hour, cooled to -100, and the product is filtered off after a few hours. It is dried over caustic soda in a vacuum.



   F. 175-1790; Rf4 = 0.46.



  9. BOC-Ser-Asn-Leu-N2H2-Z
16 g (78 mmol) of BOC-Ser-OH are dissolved in 200 ml of acetonitrile, the solution is cooled to 0 and 20 g (78 mmol) of Woodwards reagent K and 11 ml of triethylamine are added. After stirring for 11/2 hours at 0, 39.6 g (78 mmol) of H Asn-Leu-N2H2-Z, TFA and 11 ml of triethylamine in 200 ml of dimethylformamide are added to the clear solution. After stirring overnight at room temperature, the solution is evaporated in a high vacuum at 500, the oily residue is taken up in 500 ml of ethyl acetate, twice with 0.2N hydrochloric acid, twice with 10% sodium chloride solution, twice with 10% sodium bicarbonate solution and twice with 10 Siger Shaken out sodium chloride solution, dried over sodium sulfate and concentrated to 100 ml.

  After adding 20 ml of hexane, the tripeptide derivative crystallizes out. Crystallization twice from ethyl acetate 10% water gives uniform BOC Ser-Asn-Leu-N2Hs-Z.



   F. 149-151; [α] 20-18.5 (c = 2 in dimethyl formamide); Rf1 = 0.33.



  10. BOC-Ser-Asn-Leu-N2H3
15.3 g (26.7 mmol) of BOC-Ser-Leu-N2H2-Z are hydrogenated in 456 ml of dimethylformamide in the presence of 2.4 g of Pd-charcoal (10%) at room temperature.



  The reduction is complete after 5 hours. The solution is filtered off from the catalyst and concentrated in a high vacuum. Trituration with ether three times produces a white powder.



   F. 179-1810; [a] 2D0 140 (c = 2 in dimethyl formamide); Rf6 = 0.73.



  11. BOC-Ser-Asn-Leu-Ser-Thr-OBzl
8.4 g (18.7 mmol) of BOC-Ser-Asn-Leu-N2H3 in 49 ml of dimethylformamide are mixed with 19.1 ml of hydrogen chloride in tetrahydrofuran (1.97-n; 37.6 mmol) and 2, 53 ml of iso-amyl nitrite are added. After 8 minutes at -20, 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 left at 0 for 3 days.



  The precipitated triethylamine hydrochloride is then filtered off, the filtrate is concentrated to 40 ml in a high vacuum at 400, 400 ml of ethyl acetate are added while stirring, and the mixture is left at -100 overnight.



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



   F. 207-2080; [a] 20-19.50 (c = 2 in dimethylform amide); Rf6 = 0.88.



  12. BOC-Ser-Asn-Leu-Ser-Thr-OH
10 g (14.1 mmol) of BOC-Ser-Asn-Leu-Ser-Thr-OBzl are hydrogenated in 300 ml of dimethylformamide with the addition of 1.25 g of Pd-charcoal (10%) at room temperature and atmospheric pressure. The reduction is complete after 2.5 hours. The solution is filtered off from the catalyst over asbestos and concentrated in a high vacuum. Trituration of the residue three times with ether gives a pentapeptide which is uniform according to thin-layer chromatogram.



   F. 141-1440; [a] 2r) -130 (c = 2 in dimethyl formamide); Rfs = 0.34.



  13. H-Ser-Asn-Leu-Ser-Thr-OH, TFA
8.5 g (13.7 mmol) of 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 about 25 ml, mixed with 200 ml of ether while stirring and left to stand at -100 overnight. The resulting precipitate is filtered off and stirred again with 100 ml of ether for 5 hours, filtered off, washed twice with ether and dried in vacuo over caustic soda.



   F. 161-1680; [a] D 110 (c = 2 in dimethyl formamide), Rf: 0.09.



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

 

   F. 184-185; [a] 2D0 130 (c = 2 in dimethyl formamide); Rfo = 0.45.



  15. BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-Thr
Cys (Bzl) -Val-Leu-OH
3.90 g (6 mmoles) of H-Cys (Bzl) -Val-Leu-OH. 1.15TFA are dissolved in 50 ml of freshly distilled dimethylformamide, 4.48 g (5.5 mmol) of BOC-Cys (Bzl) -Ser-Asn Leu-Ser-Thr-OH are added and the mixture is stirred at room temperature until it is dissolved. The solution is then cooled to 0, 1.26 g (11 mmol) of N-hydroxysuccinimide and 0.98 ml of triethylamine in 45 ml of dimethylformamide are added, the mixture is further cooled to -220 and 1.13 g (5.5 mmol ) Dicyclohexylcarbodiimide in 10 ml of dimethylformamide. The mixture is stirred for 1 hour at -220 and then for 3 days at room temperature, precipitated dicyclohexylurea is filtered off and evaporated to dryness in a high vacuum.

  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 under high vacuum at 400.



   F. 218-2210; [a] D20 28.50 (c = 2 in dimethyl formamide); Rfg = 0.43.



  16. BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH
2.5 g (1.9 mmol) of BOC-Cys (Bzl) -Ser-Asn-Leu-Ser Thr-Cys (Bzl) -Val-Leu-OH are suspended in 250 ml of dry liquid ammonia at -400. While stirring, sodium is added at the boiling point of the ammonia in such a way that the color of the reaction mixture is only light blue. After 50 minutes the nonapeptide derivative has completely dissolved. The mixture is stirred for a further 10 minutes while maintaining the blue color, then 1 g of ammonium chloride is added and the mixture is evaporated to dryness in a high vacuum (about 1 mm). The residue is stirred with 30 ml of 0.1N hydrochloric acid, the precipitate is 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 10% strength and once with saturated sodium chloride solution, dried over sodium sulfate and evaporated to dryness.



  The product is uniform in the thin-layer chromatogram.



     Rf = 0.86; Rfs; = 0.76; Rf7 = 0.81.
EMI36.1


 <tb>



  17th <SEP> B <SEP> 0 <SEP> C-Cys-Ser-Asn-Leu-Ser-Thr-Cvs-Val-Leu-OH <SEP>
 <tb>
234 mg (0.225 mmol) of BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Vai-Leu-OH are dissolved in 100 ml of degassed dimethylformamide and treated with 1 ml of a triethylamine solution in dimethylformamide (0.95 ml of triethylamine in 10 ml of dimethylformamide, i.e. 0.675 mmol). This solution is simultaneously added dropwise with a solution of 69.8 mg (0.248 mmol) of 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 with stirring. 1 ml of cyclohexane is added and the mixture is evaporated to dryness in a high vacuum. The residue is stirred twice with a mixture of 10 ml of 2S 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.



     Rfa = 0.82; Rfs = 0.75; Rf7 = 0.78.
EMI36.2


 <tb>



  18th <SEP> BOC-Cvs-Ser-Asn-Leu-Ser-Thr-Cvs-Val-Leu- <SEP>
 <tb> 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
EMI36.3


 <tb> 412 <SEP> mg <SEP> BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val- <SEP>
 <tb> eu-OH 360 mg of the tritosylate obtained under 59 with the sequence 10-32, 15 mg of N-methylmorpholine, 50 mg of N-hydroxysuccinimide and 15 ml of dimethylformamide are stirred at 450 for 2 hours under nitrogen.



  Then cool to 0, add 8 mg of dicyclohexylcarbodiimide, stir for 2 hours at 0 under nitrogen, then add another 50 mg of dicyclohexylcarbodiimide and stir under nitrogen for 8 hours at 450. Then it is poured into 100 ml of peroxide-free ether, the precipitate is sucked off off, washes it with ether and dries it in vacuo at 350. The crude product is purified by countercurrent distribution as described under 60.



   Example 3
EMI36.4


 <tb> H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-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 (Thyrocalcitoninsulfoxyd)
25 mg of thyrocalcitonin are dissolved in 5 ml of 0.05N acetic acid. 0.5 ml of an oily aqueous hydrogen peroxide solution is added and the mixture is left to stand for 90 minutes at room temperature. Then add a trace of platinum black and stir until the development of oxygen ceases. The platinum is centrifuged off and the supernatant solution is lyophilized. The thyrocalcitonin sulfoxide obtained in this way shows Rf5 = 0.51 and Rfrol = 0.48 in thin-layer chromatography on aluminum oxide.

  In 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 4
EMI36.5


 <tb> H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg, -Phe- Ser -Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH
EMI36.6


 <tb> <SEP> 25 <SEP> mg <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu)
 <tb> Thr (tBu) -Cys-Val-Leu-Ser (tBu) -Ala-Tyr (tBu) -Try
 <tb> Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) Gly-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) Pro-OtBu become leave 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 Amberlite 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 15 hours 280 V) migration distance against the cathode 6.3 cm.



   On Cellogel (pH = 1.9, 1.5 hours, 280 V) migration distance towards 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 and treated 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 reacted in 100 ml of tetrahydrofuran. After leaving to stand at room temperature overnight, it is filtered and the filtrate in ethyl acetate, which has evaporated to dryness in a vacuum, is extracted several times with dilute aqueous citric acid solution, dilute aqueous sodium carbonate solution and saturated saline solution.

  The oily compound obtained gives in a thin-layer chromatogram on silica gel: Rf43c = 0.89; Rf45 = 0.75, Rf52 = 0.64; [α] 20 D = -44 10 (c = 1.0 in ethanol).



  2. H-Thr (tBu) -Pro-OtBu, HCl
2.7 g of Z-Thr (tBu) -Pro-OtBu are dissolved in 200 ml of methanol with the addition of 2.9 ml of aqueous 2N hydrochloric acid and 500 mg of palladium-on-carbon catalyst (10%) with hydrogen in the shaking duck at atmospheric pressure and room temperature decarbobenzoxylated. After the uptake of hydrogen, filtration and evaporation to dryness has ended, the product is crystallized from hexane. F. 142-145 (decomposition).



   Thin layer chromatogram on silica gel: Rf43o = 0.45; Rf45 = 0.70; Rf52 = 0.55; [a] =
660 + 0.50 [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 dissolved together in 15 ml of dimethylformamide, mixed with 3.2 ml of triethylamine and overnight at 27-30 touched. The solution is evaporated to dryness in vacuo, the residue is taken up in ethyl acetate and chloroform and extracted with citric acid, soda solution and sodium chloride solution as in 1 and the crude product obtained is chromatographed on silica gel. The substance can be eluted with hexane / ether 1: 1 and ether and then crystallizes from sither / hexane. F. 135-1370.



   Thin-layer chromatogram on silica gel: Rfcuci3-u0on (95: 5) = 0.53, RfCHCl3 acetone 1: 1 =
0.72, Rf45 = 0.82; [a] 2.0 = -44 + 0.50 (c = 2 in ethanol).



  4. H-Glu (OtBu) -Thr (tBu) -Pro-OtBu
777 mg of the compound described under 3 are hydrogenated in 250 ml of methanol with 300 mg of 10% palladium catalyst on carbon. A thick oil is obtained which is used for the next stage without further purification.



   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 of Z-Gly-Phe-Gly-Pro-OH are dissolved with the 608 mg of H-Glu (OtBu) -Thr (tBu) Pro (OtBu) obtained under 4 in 20 ml of acetonitrile and with the addition of 222 mg of N-hydroxysuccinimide stirred with 269 mg of dicyclohexylcarbodiimide for 16 hours at room temperature. The precipitated dicyclohexylurea is filtered off, evaporated in vacuo, the residue is dissolved in a large amount of ethyl acetate and the solution is extracted as described under 1. After drying and evaporation in vacuo, the crude product is purified by column chromatography on silica gel. It can be eluted with ethyl acetate and ethyl acetate / ethanol (4: 1). Subsequently, the ether falls over. F. 96 to 990.



   Thin-layer chromatogram on silica gel: RfclicI3-MeO (9: 1) = 0.40; RfI02A = 5.75; Rf52 =
0.80.



  6. H-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu
858 mg of Z-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu are hydrogenated in 150 ml of methanol in the presence of 200 mg of palladium catalyst (10% Pd on carbon). The product, which is obtained as an amorphous powder after trituration with ether-hexane (1: 1), shows the following values in a thin-layer chromatogram on silica gel:
RfCHCl3-MeOH (9: 1) = 0.16; Rfl02E = 0.17; Rf52 = 0.41.



  7. DPC-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu)
Pro-OtBu
410 mg DPC-Met-OH are mixed with 750 mg H-Gly Phe-Gly-Pro-Glu (OtBu) -Thr (tBu) -Pro-OtBu in 15 ml acetonitrile under nitrogen and with the addition of 152 mg N-hydroxysuccinimide with 271 mg of dicyclohexylcarbodiimide for 19 hours at room temperature. After the dicyclohexylurea has been filtered off, the filtrate is evaporated in vacuo, the residue is taken up in a large amount of ethyl acetate and extracted as in 1 and evaporated. The crude product is purified by column chromatography on silica gel.



  It can be eluted with ethyl acetate / ethanol (4 l).



   Thin-layer chromatogram on silica gel: RfHcI3-MeOH (9: 1) = 0.42; Rfx02E = 0.39; Rf53 = 0.29.



  8.H-Met-Gly-Phe-Gly-Pro-Glu (OtBu) -Thr (tBu)
Pro-OtBu
190 mg of the DPC compound obtained under 7 are 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 minutes of reaction at room temperature, the batch is poured into 20 ml of ice-cold 2N soda solution in water and extracted with three portions to 30 ml of 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.
EMI37.1


 <tb>



  9. <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) -Thr (tBu) -Cys Val-Leu-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
EMI37.2


 <tb> <SEP> 55 <SEP> mg <SEP> BOC-Cys-Ser (tBu) -Asn-Leu-Ser (tBu) - <SEP>
 <tb> Thr (tBu) -Cys-Val-Leu-Ser (tBu) -Ala-Tyr (tBu) -Try-Arg Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser (tBu) - Gly OH-ditosylate is added with 81 mg of H-Met-Gly-Phe-Gly-Pro-Glu (OtBu) Thr (tBu) -Pro-OtBu in 0.2 ml of dimethylformamide while introducing nitrogen after adding 9 mg of N-hydroxysuccinimide 400 stirred. After everything has dissolved, 7.3 mg of dicyclohexylcarbodiimide in 1 ml of dimethylformamide are added and the mixture is stirred at 400 for 22 hours. After cooling, the product is precipitated with 8 ml of peroxide-free ether and centrifuged.

  In the methanol / buffer / chloroform / carbon tetrachloride system (10: 3: 6: 5) the crude product is multiplied over 100 levels (K = 0.4). (Buffer as in Example 1, under 18). The tubes containing the product are poured together, evaporated and the ammonium acetate stripped off in a high vacuum at 400.



     Rf0-. = 0.24; Rfioo = 0.25; Rfllo = 0.65 on silica gel plates.



   Example 5 a) 1.3 g of BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-Ser (tBu) Thr (tBu) -Cys (TRI) -VaI-Leu-OH (cf. Example 1, under
17), 2.2 g of the tritosylate of the sequence 10-32 described in Example 1 under 59, 0.1 ml of N-methylmorpholine and 200 mg of N-hydroxysuccinimide are stirred in 20 ml of freshly distilled dimethylformamide at 450 for 1 hour. Then 250 mg of dicyclohexylcarbodiimide are added. The mixture is stirred under nitrogen for 3 hours at 450, then a further 100 mg of dicyclohexylcarbodiimide are added and the mixture is again stirred for 5 hours at 450. The mixture is then cooled to 0 and the reaction mixture is added to 1 liter of peroxide-free ether. It is left at 00 for 5 hours, the fine precipitate is filtered off with suction, washed with ether, dried and triturated with 100 ml of water. You suction off and dry in the desiccator.

  The resulting crude product (about 3.5 g) is subjected to a countercurrent distribution over 750 stages in the methanol-buffer-chloroform-carbon tetrachloride system (10: 3: 3: 7; K-0.5) for purification. The uniformity of the fractions obtained is checked by means of thin layer chromatography on silica gel plates (systems 108 and 53) and the uniform fractions are combined. It is dried for 18 hours in a high vacuum at 400 to drive off the ammonium acetate. Yield: 1.1 g.



   b) 610 mg of the protected dotriacontapeptide amide obtained under a) are dissolved in 15 ml of ice-cold 95% strength trifluoroacetic acid, the solution is allowed to warm to 250 under nitrogen and left to stand for 2 hours. Then (to trap the triphenylcarbonium ions) the intensely yellow solution is poured into a mixture of 300 g of ice and 300 ml of 5% aqueous pyridine solution, the precipitated triphenylcarbinol is extracted with peroxide-free ether, 50 ml of glacial acetic acid are added to the aqueous solution and the mixture is filtered to remove trifluoroacetate -Ions through a column of Merck ion exchanger No. II, weakly basic, acetate form. In doing so, nitrogen is passed over the surface of the solutions; all solvents used have previously been degassed with nitrogen.

  The eluate, which shows a strong violet color with sodium prusside solution, is lyophilized and the residue is dried in a high vacuum at 400 to remove pyridine acetate. The raw product is processed further directly.



   c) Dissolve 250 mg of H-Cys-Ser-Asn-Leu-Ser-Thr Cys-Val-Leu-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn Asn-Phe-His-Arg-Phe- Ser-Gly-Met-Gly-Phe-Gly Pro-Glu-Thr-Pro-NHW, in 100 ml of water, adjusts the resulting solution to pH 6.5 by adding 2N aqueous ammonia and passing air through until no more free SH groups can be detected with sodium nitroprusside reagent. Then it is evaporated to dryness on a rotary evaporator with the addition of n-octanol in vacuo at 400, the residue is dissolved in 0.1N aqueous formic acid and chromatographed on a column (2.485 cm) Bio-Gel P6. The elution is followed by means of a UV spectrophotometer. The fractions obtained are lyophilized and their purity is checked by means of thin-layer chromatography on aluminum oxide plates (systems 52 and 104).

  The fractions containing thyrocalcitonin (mixed with sulfoxide) are pooled and lyophilized. Yield: 210 mg.



  The product (thyrocalcitonin) shows the following Rf values: Rf45 = 0.33; Rf52 = 0.59; Calls04 = 0.56.



   The sulfoxide of I has the following Rf values: Rf45 = 0.30; Rfs2 = 0.54; Ruf104 = 0.50.



   PATENT CLAIM 1
Process for the production of new peptides of the formula
EMI38.1


 <tb> H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala Tyr-Try-Arg-A sn-LewAsn-Asn-Phe-His-Arg-Phe-Ser-Gly -Met [or Met (O)] - Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH or corresponding compounds in which one or more of the asparagine residues through the aspartic acid residue and / or the glutamic acid residue through the glutamine residue and / or the methionine residue is replaced by the valine, norvaline, leucine, isoleucine or norleucine residue, or their C-terminal amides or acid addition salts of these compounds, characterized in that the corresponding amino acids, where the proline can also be present as an amide or

   their activated derivatives with intermediate protection from the reaction to be excluded reactive functional groups and with the formation of the disulfide bridge between Cysl and Cys7 by oxidation, condensed.



   SUBCLAIMS
1. The method according to claim I, characterized in that the carboxyl groups to be excluded from the reaction are protected by the tert-butyl group.



   2. The method according to claim I and dependent claim 1, characterized in that hydroxyl groups to be excluded from the reaction are protected by the tert-butyl group.



   3. The method according to claim I, characterized in that the amino group on the cysteine radical is protected by the tert-butyloxycarbonyl group.



   4. The method according to claim I, characterized in that the C-terminal amide of the compound of formula I is prepared.



   5. The method according to claim I, characterized in that from
EMI38.2


 <tb> H-Cys-Ser-Asn-Leu-S <SEP> er-Thr-Cys-Val-Leu-Ser-Ala- <SEP>
 <tb> Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-ProNlI2, wherein the α-amino group by the tert-butyloxycarbonyl group, the r-carboxyl group by the tert. The butyl group and, if appropriate, the hydroxyl groups of the side chains are protected by tert-butyl groups, the protecting groups being split off by acid hydrolysis.

 

     
6. The method according to claim 1 or one of the dependent claims 1-5, characterized in that the product obtained with the Met2 radical is converted into the corresponding sulfoxide.



   PATENT CLAIM II
Use of peptides obtained by the process according to claim I for the production of

** WARNING ** End of DESC field could overlap beginning of CLMS **.



   

 

Claims (1)

** WARNING ** Beginning of CLMS field could overlap end of DESC **. the ammonium acetate stripped off in a high vacuum at 400. Rf0-. = 0.24; Rfioo = 0.25; Rfllo = 0.65 on silica gel plates. Example 5 a) 1.3 g of BOC-Cys (TRI) -Ser (tBu) -Asn-Leu-Ser (tBu) Thr (tBu) -Cys (TRI) -VaI-Leu-OH (cf. Example 1, under 17), 2.2 g of the tritosylate of the sequence 10-32 described in Example 1 under 59, 0.1 ml of N-methylmorpholine and 200 mg of N-hydroxysuccinimide are stirred in 20 ml of freshly distilled dimethylformamide at 450 for 1 hour. Then 250 mg of dicyclohexylcarbodiimide are added. The mixture is stirred under nitrogen for 3 hours at 450, then a further 100 mg of dicyclohexylcarbodiimide are added and the mixture is again stirred for 5 hours at 450. The mixture is then cooled to 0 and the reaction mixture is added to 1 liter of peroxide-free ether. It is left at 00 for 5 hours, the fine precipitate is filtered off with suction, washed with ether, dried and triturated with 100 ml of water. You suction off and dry in the desiccator. The resulting crude product (about 3.5 g) is subjected to a countercurrent distribution over 750 stages in the methanol-buffer-chloroform-carbon tetrachloride system (10: 3: 3: 7; K-0.5) for purification. The uniformity of the fractions obtained is checked by means of thin layer chromatography on silica gel plates (systems 108 and 53) and the uniform fractions are combined. It is dried for 18 hours in a high vacuum at 400 to drive off the ammonium acetate. Yield: 1.1 g. b) 610 mg of the protected dotriacontapeptide amide obtained under a) are dissolved in 15 ml of ice-cold 95% strength trifluoroacetic acid, the solution is allowed to warm to 250 under nitrogen and left to stand for 2 hours. Then (to trap the triphenylcarbonium ions) the intensely yellow solution is poured into a mixture of 300 g of ice and 300 ml of 5% aqueous pyridine solution, the precipitated triphenylcarbinol is extracted with peroxide-free ether, 50 ml of glacial acetic acid are added to the aqueous solution and the mixture is filtered to remove trifluoroacetate -Ions through a column of Merck ion exchanger No. II, weakly basic, acetate form. In doing so, nitrogen is passed over the surface of the solutions; all solvents used have previously been degassed with nitrogen. The eluate, which shows a strong violet color with sodium prusside solution, is lyophilized and the residue is dried in a high vacuum at 400 to remove pyridine acetate. The raw product is processed further directly. c) Dissolve 250 mg of H-Cys-Ser-Asn-Leu-Ser-Thr Cys-Val-Leu-Ser-Ala-Tyr-Trp-Arg-Asn-Leu-Asn Asn-Phe-His-Arg-Phe- Ser-Gly-Met-Gly-Phe-Gly Pro-Glu-Thr-Pro-NHW, in 100 ml of water, adjusts the resulting solution to pH 6.5 by adding 2N aqueous ammonia and passing air through until no more free SH groups can be detected with sodium nitroprusside reagent. Then it is evaporated to dryness on a rotary evaporator with the addition of n-octanol in vacuo at 400, the residue is dissolved in 0.1N aqueous formic acid and chromatographed on a column (2.485 cm) Bio-Gel P6. The elution is followed by means of a UV spectrophotometer. The fractions obtained are lyophilized and their purity is checked by means of thin-layer chromatography on aluminum oxide plates (systems 52 and 104). The fractions containing thyrocalcitonin (mixed with sulfoxide) are pooled and lyophilized. Yield: 210 mg. The product (thyrocalcitonin) shows the following Rf values: Rf45 = 0.33; Rf52 = 0.59; Calls04 = 0.56. The sulfoxide of I has the following Rf values: Rf45 = 0.30; Rfs2 = 0.54; Ruf104 = 0.50. PATENT CLAIM 1 Process for the production of new peptides of the formula EMI38.1 <tb> H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Ser-Ala Tyr-Try-Arg-A sn-LewAsn-Asn-Phe-His-Arg-Phe-Ser-Gly -Met [or Met (O)] - Gly-Phe-Gly-Pro-Glu-Thr-Pro-OH or corresponding compounds in which one or more of the asparagine residues through the aspartic acid residue and / or the glutamic acid residue through the glutamine residue and / or the methionine residue is replaced by the valine, norvaline, leucine, isoleucine or norleucine residue, or their C-terminal amides or acid addition salts of these compounds, characterized in that the corresponding amino acids, where the proline can also be present as an amide or their activated derivatives with intermediate protection from the reaction to be excluded reactive functional groups and with the formation of the disulfide bridge between Cysl and Cys7 by oxidation, condensed. SUBCLAIMS 1. The method according to claim I, characterized in that the carboxyl groups to be excluded from the reaction are protected by the tert-butyl group. 2. The method according to claim I and dependent claim 1, characterized in that hydroxyl groups to be excluded from the reaction are protected by the tert-butyl group. 3. The method according to claim I, characterized in that the amino group on the cysteine radical is protected by the tert-butyloxycarbonyl group. 4. The method according to claim I, characterized in that the C-terminal amide of the compound of formula I is prepared. 5. The method according to claim I, characterized in that from EMI38.2 <tb> H-Cys-Ser-Asn-Leu-S <SEP> er-Thr-Cys-Val-Leu-Ser-Ala- <SEP> <tb> Tyr-Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-Ser-Gly-Met-Gly-Phe-Gly-Pro-Glu-Thr-ProNlI2, wherein the α-amino group by the tert-butyloxycarbonyl group, the r-carboxyl group by the tert. The butyl group and, if appropriate, the hydroxyl groups of the side chains are protected by tert-butyl groups, the protecting groups being split off by acid hydrolysis. 6. The method according to claim 1 or one of the dependent claims 1-5, characterized in that the product obtained with the Met2 radical is converted into the corresponding sulfoxide. PATENT CLAIM II Use of peptides obtained by the process according to claim I for the production of sparingly soluble zinc complexes of these peptides, characterized in that the peptides obtained are reacted in aqueous solution with a water-soluble zinc salt and an alkali metal phosphate, pyrophosphate and / or hydroxide.