CH550774A - Dotriacontapeptide calcitonin analogues - with hypocalcaemic activity - Google Patents

Abstract

Peptides of formula (I): R-CH-CO-Gly-Asn-Leu-Ser-Thr-Cys-X-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe- -Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-- NH2 (I) (where R is H or free or acylated amino; X is a valine norvaline, leucine, isoleucine, morleucine or alpha-aminobutyric acid residue) and the corresp. cpds. in which >=1 of the Asn or Gln residues are replaced by Asp and Glu respectively and/or the Asp residue is replaced by Asn. and their acid-addn. salts and Zn complexes, are new and can be prepd. by standard methods from the appropriate protected amino acids, the disulphide bridge being formed by oxidn. and the terminal COOH gp. being amidated.

Description

  

  
 



   The invention relates to a process for the production of new hypocalcemically active peptides of the formula I
EMI1.1
 X-Leu-Gly-Thr-Tyr Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-val-Gly-Ala-pro-NH2 wherein R represents hydrogen, a free or acylated amino radical and X represents the valine, norvaline, leucine, isoleucine, norleucine or α-aminobutyric acid radical and corresponding compounds in which one or more of the asparagine and glutamine radicals are replaced by the aspartic acid or . Glutamic acid residue and / or the aspartic acid residue are replaced by the aspartic residue, as well as the acid addition salts and complexes of the peptides mentioned.



   As acid addition salts, particular mention should be made of salts of therapeutically applicable acids such as hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid and sulfonic acids such as lower alkanesulfonic acids, benzene or toluenesulfonic acid.



   Acyl groups for the acylation of the Na amino group are the residues of carboxylic acids, such as aliphatic, aromatic, araliphatic, heterocyclic and heterocyclylaliphatic carboxylic acids, in particular of lower alkanoic acids such as formic acid, acetic acid, propionic acid, butyric acids, of monocyclic aromatic carboxylic acids and of substituted benzoic acids such as unsubstituted unsubstituted and aryl-substituted aryl-lower alkylcarboxylic acids such as phenylacetic acid, of 5- to 6-membered heterocyclic acids with nitrogen, sulfur and / or oxygen as heteroatoms, such as pyridinecarboxylic acids, thiophene carboxylic acids, or of heterocyclyl-lower alkanoic acids such as pyridylacetic acid, imidazole rings, in which the rings are zylacetic acid. B. halogen atoms, nitro groups, lower alkyl or lower alkoxy groups or lower carbalkoxy groups.

  Next, as acyl residues, especially acyl residues of amino acids, especially a-amino acids, such as. B. the pyroglutamyl radical, also acyl radicals derived from carbonic acid or thiocarbonic acid or their esters or amides, z. B. lower alkyloxycarbonyl groups such as ethoxycarbonyl, tert-butyloxycarbonyl, and also unsubstituted and substituted benzyloxycarbonyl, carbamoyl and thiocarbamoyl and N-substituted carbamoyl and thiocarbamoyl, e.g. B.



  N-lower alkylcarbamoyl, N-phenylcarbamoyl, N-phenylthiocarbamoyl.



   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, pyrophosphates and polyphosphates 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 carhoxymethyl 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. B. protamine or polyglutamic acid.



   The new compounds 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.



   The compounds also lower the plasma calcium level of the blood in humans with intravenous administration of 0.01 to 5 mg in 0.1 M acetate buffer of pH 4.6 and can accordingly be used in hypercalcemia.



   The process according to the invention for the preparation of the new compounds of the formula I and corresponding compounds in which one or more of the asparagine and glutamine residues are replaced by the aspartic acid or glutamic acid residue and / or the aspartic acid residue is replaced by the asparagine residue, or their acid addition salts. that the corresponding acids or their activated derivatives are condensed and the terminal carboxyl group is amidated in any chronological order with intermediate protection from the reaction to be excluded from the reaction and with the formation of the disulfide bridge from the two mercapto groups. The compounds obtained can be converted into their acid addition salts or complexes.



   In the synthesis of the end products as well as all intermediate products, 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. B. by hydrolysis, reduction, aminolysis or hydrazinolysis.



   So one uses z. B. as protecting 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 activated by the action of nucleophilic reagents, e.g. B. sulfites, thiosulfates, cf.



  English patent 1104 271, are split off) optionally substituted, such as. B. by lower alkoxy groups, especially o- or p-methoxy groups substituted benzyl, or diphenyl or triphenylmethyl groups or groups derived from carbonic acid such as, if appropriate, z. B. benzyl or benzhydryloxycarbonyl groups substituted by halogen atoms such as chlorine or bromine, nitro groups or lower alkoxy groups, e.g. B.

  Carbobenzoxy, p-bromo- or p-chlorocarbobenzoxy. p-Nitrocarbobenzoxy or p-methoxycarbobenzoxy, colored benzyloxycarbonyl groups such as p-phenylazo-benzyloxycarbonyl and p- (p 'methoxy-phenylazo) -benzyloxycarbonyl, aliphatic oxycarbonyl groups such as adamantyloxycarbonyl, cyclopentyalarbloxycarbonyl, above all cyclo-pentyalarbloxycarbonyl, trichloxy-carbonyl-butyloxycarbonyl, trichlo-carbonyl-butyloxy-carbonyl, trichloxy-carbonyl-tert-butyloxy-butyloxycarbonyl, above all such as 2-phenyl-isopropyloxycarbonyl, 2-tolyl-isopropyloxycarbonyl and, in particular, 2-p-diphenyl-isopropyloxycarbonyl (cf. Swiss Patent No. 509 266). The amino groups can also be formed by the formation of enamines. obtained by reaction of the amino group with 1,3-diketones. z. 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 z. B. by the 3,4-dimethoxybenzyl or bis (p-methoxyphenyl) methyl group.



  the hydrazide group e.g. B. by the carbobenzoxy group, the trichloroethyloxycarbonyl group, the trifluoroacetyl group, the trityl group, the tert-butyloxycarbonyl group or the 2-p-diphenyl-isopropyloxycarbonyl group. Suitable for esterification are, for. B. lower optionally substituted alkanols such as methanol, ethanol, cyanomethyl alcohol, benzoylmethyl alcohol or especially tert. Butanol, also aralkanols such as aryl lower alkanols, e.g. 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 optionally substituted by electron-withdrawing substituents such as thiophenol, thiocresol, p-nitrothiophenol, 2,4,6-trimethylbenzyl alcohol , 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 radicals in the esterification, for. B.



  Lower alkanoyl radicals such as acetyl, aroyl radicals such as benzoyl and, above all, radicals derived from carbonic 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), 3838-3849 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 alkylation are, for. B. 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. 101 (1968), 681. The imino group of histidine does not necessarily need to be protected, but it can be advantageous to protect it, e.g. B. by benzyl, trityl, carbobenzoxy, adamantyloxycarbonyl or the above-mentioned Weygand groups.



   The tert-butyl ester group is preferably used to protect the carboxyl group of the side chain, the tert-butyl ether group to protect the amino group of the side chain, and the tert-butyl ether group for the hydroxyl groups of the serine, threonine and tyrosine radicals, if they are protected at all and, if desired, to protect the imino group of the histidine, the 2,2,2-tri fluoro-1-tert.-butyloxycarbonylaminoethyl group. All of these protecting groups can, if desired, in one step by acid hydrolysis, e.g. B. by means of trifluoroacetic acid, are split off. When building up the protected dotriacontapeptide amide used as starting material 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. B. be oxidized with iodine in glacial acetic acid, with Diiodäthan 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 No. 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 decapeptide 1-10, or at the stage of the Dotriacontapeptide.



   In the preparation of the Na-acyl derivatives, the acyl group can be used as an α-amino protecting group.



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



   For the purpose of preparing Na acyl derivatives, the peptide with a free α-amino group can be N-acylated in the usual way, e.g. B. by reaction with a mixed anhydride or acid azide containing the acyl radical in question or, above all, an activated ester such as phenyl or substituted phenyl ester.



   Complexes with inorganic substances such as sparingly soluble metal, e.g. B. aluminum or zinc compounds are known in an analogous manner as for ACTH, z. B. by reaction with a soluble salt of the metal in question, e.g. B.



  Zinc chloride or zinc sulfate, and precipitation with an alkali metal phosphate and / or hydroxide. 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. It is advisable to work according to the method used for the production of long-chain peptides, taking into account the disulfide bridge. suitable linking methods, as they are known from the literature.



   The amino acid and / or peptide units are therefore linked e.g. 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 α-amino group and protected terminal carboxyl group with an amino acid or a peptide with free terminal carboxyl group and protected α-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-Trichlorophenylester, Pentachlorphenylester, N-Hydroxysuccinimidester, N-Hydroxyphthalimidester, 8-Hydroxyquinolinester, N-FIydroxypiperidinester, or by reaction by means of a carbodiimide (optionally with the addition of N-Hydroxysuccinimide) or N, N ' Carbonyldiimidazole, or isoxazolium salt, e.g. B. Woodward reagent, the amino group can be activated for example by reaction with a phosphite.

  The most common methods are the carbodiimide method, the Weygand Wünsch method (carbodiimide in the presence of N-hydroxysuccinimide), the azide method, the activated ester method and the anhydride method, as well as the Merrifield method and the N-carboxyanhydride or N method -Thiocarboxy anhydrides.



   The preparation of the starting materials, especially the peptide fragment containing the disulfide bridge and its linkage with the remaining part of the peptide, is a special subject of the invention. It is advantageous to start from a sequence which comprises the first 10 N-terminal amino acids and with this N-terminus to condense the entire remaining sequence.



   But you can also link the mentioned N-terminal sequence with the fragment up to the 28th amino acid (glycine) with a free C-terminal carboxyl group and condense the octacosapeptide with the tetrapeptide of amino acids 2W32. This condensation is carried out, for example, according to the Weygand-Wünsch method.



  If the condensation of the sequence 1-10 with the C-terminal sequence 11-32 is carried out, the carbodiimide method or the Weygand-Wünsch method is preferably used. The production of the N-terminal decapeptide (1-10) is explained in more detail below.



   It can e.g. B. from the sequences 1-4 and 5-10 or 1-5 and 910 or 16 and 7-10 or 1-7 and 8-10, as can be seen from FIGS. 1-8; however, other fragments can also be used to build the sequence 1-10. The protective group for the α-amino group on the cysteine is preferably the tert-butyloxycarbonyl group or an equivalent group which can be split off by acid hydrolysis, or if an Na-acylated Dotriacontapeptide is to be prepared, the corresponding acyl z. B. acetyl group. In addition, it is expedient to use mercapto protecting groups which can be split off selectively with respect to the N "-amino protecting group (e.g. tert-butyloxycarbonyl group) which can be split off by acid hydrolysis, e.g. the benzyl or trityl group.

  The terminal carboxyl group of the decapeptide need not necessarily be protected, e.g. B. not if condensations are carried out by the azide or anhydride method. However, this group can also be protected by esterification, as indicated above, e.g. B. by esterification with methanol or ethanol (cleavage of the ester group with dilute sodium hydroxide solution) or with benzyl alcohol or analogs (cleavage of the ester group, e.g. with sodium in liquid ammonia). The amino groups of the intermediates are protected by means of the customary protecting groups, e.g. B. carbobenzoxy, trityl, tert-butyloxycarbonyl or 2-para-diphenyl-isopropyloxycarbonyl. If necessary, the carboxyl groups of the intermediates are esterified in the usual manner. The hydroxyl groups of the serine and threonine residues can be etherified, e.g. B. 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, especially p-nitro phenyl ester (ONP) or hydroxysuccinimide ester (OSU) 4) the carbodiimide method 5) the method acc. to Weygand-Wünsch BOC tert-butyloxycarbonyl DPC p-diphenyl-isopropyloxycarbonyl Z carbobenzoxy TRI trityl Bzl benzyl OtBu tert-butyl ester OBzl benzyl ester ONB p-nitrobenzyl ester ONP p-nitrophenyl ester T-acetic acid ethyl ester tBu
The p-nitrobenzyl ester and benzyl ester groups are split off in methionine-containing sequences with sodium in liquid ammonia, otherwise by hydrogenolysis in the presence of palladium carbon, the carbobenzoxy group also by hydrogenolysis, the N-trityl group with aqueous acetic acid,

   the tert-butyloxycarbonyl group with trifluoroacetic acid, the diphenylisopropyloxycarbonyl group with aqueous acetic acid or z. B. with a mixture of glacial acetic acid, formic acid (82.8%) and water (7: 1: 2) as described in Swiss Patent No. 509 266. The p-nitrobenzyl or methyl ester can be 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, any benzyl or p-nitrobenzyl ester groups that may be present being split off at the same time. The ring closure to the disulfide takes place z.

  B. by oxidation with 1,2-diiodoethane, that of the S-trityl-protected compounds with iodine in methanol.



   The C-terminal sequence to be connected to the N-terminal sequence comprising the 11th to 32nd or 11th to 28th amino acid is composed, for example, of the sequences 11-16, 17-20, 21-28 and 29-32, as Fig. 9 shows.



   In this scheme the hydroxyl groups of the threonine residues and the tyrosine residue are protected, this is not absolutely necessary. Other partial sequences can also be combined with one another and other protective groups can be used.



   10 shows the structure of the hexapeptide (in the form of the hydrazide) of amino acids 11-16. It can be linked with the sequence 17-28 or 17-32 by the azide method.



   The sequence 17-28 can be constructed from the fragments 17-20 and 21-28 by the azide method.



   FIG. 11 shows the synthesis of the tetrapeptide hydrazide of amino acids 17-20, FIG. 12 the structure of the octapeptide 21-28. After the two sequences have been linked, the α-amino protective group is split off (the carbobenzoxy group by hydrogenolysis in the presence of palladium carbon) and the dodecapeptide with protected side chains thus obtained is condensed by the azide method with the hexapeptide hydrazide 11-16 (FIG. 10).



   The sequence 11-28 thus obtained can be linked to the tetrapeptide amide of amino acids 29-32, the preparation of which is shown in FIG. 13, e.g. B. by the Weygand-Wünsch method. The protected docosapeptide amide 11-32 is then obtained. The α-amino protective group can be split off from this (carbobenzoxy e.g. hydrogenolytically, DPC with 90% acetic acid or glacial acetic acid [82.8%] - water [7: 1: 21) and the compound thus obtained with free After removing the acetic acid, link the a-amino group with the N-terminal decapeptide (Fig. 1-8), for example by the mixed anhydride method, the activated ester (OSU) method or according to Weygand-Wünsch.

 

   But you can also link the sequence 11-28 with a free C-terminal carboxyl group after cleavage of the a-amino protective group in the manner mentioned, with the N-terminal decapeptide by the method of mixed anhydrides and the product thus obtained with the tetrapeptide amide 29-32 condense, e.g. B. after Weygand Wünsch.



   Another way to build up the C-terminal sequence 1132 is z. B. in that they are composed of the partial sequences 11-19 and 20-32, which are shown in Fig. 14 and 15, preferably by the method of mixed anhydrides or according to Weygand-Wünsch.



   The C-terminal sequence with a free carboxyl group can also be produced according to the scheme of FIG. In this case z. B. the tert-butyl ester group present at amino acid 32 is not split off and converted into the amide group, but retained up to step J. The condensation of the sequence 20-32 with a C-terminal free carboxyl group with the sequence 11-19 of FIG. B. by the mixed anhydride method, as well as the linkage of the sequence 11-32 thus obtained with the N-terminal sequence 1-10.



   The protective groups are split off from the protected dotriacontapeptide amide, for example with trifluoroacetic acid or with corzentriener hydrochloric acid.



   The dotriacontapeptide with free or trityl-protected SH groups to be used for the method according to variant 2) or 3) can be prepared in a manner analogous to the protected dotriacontapeptide described above, with the difference that the protected SH groups are retained until the end of the synthesis will.



  Only after all the other protective groups have been removed from the protected dotriacontapeptide are the SH protective groups or the trityl-protected compound oxidized directly as mentioned above.



   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.

  B. 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 , Fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, hydroxymaleic acid, dihydroxymaleic acid, benzoic acid, phenylacetic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, anthranilic acid, cinnamic acid, mandelic acid, salicylic acid, 4-amino-salicylic acid, 2oxybenzoic acid, , Methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic 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 45: sec-butanol-3% aqueous ammonia (70:30) System 52: n-butanol-glacial acetic acid-water (75: 7.5: 21) System 79: n -Butanol-pyridine-water (34:33:33) System 87: isopropanol-glacial acetic acid-water (77: 4:

  : 19)
EMI5.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> A <SEP> BOC --- OH <SEP> BOC-OH <SEP> BOC-OH <SEP> H-N2E2-Z <SEP> BOC-OH <SEP> H-ONB <SEP> BOC-OH <SEP> BOC-OH <SEP> BOC-OH <SEP> H-OBzl
 <tb> <SEP> 3) <SEP> 1) -5) <SEP> 1) -5)
 <tb> B <SEP> BOC ------ N2E2-Z <SEP> BOC --- ONB <SEP> BOC --- OBzl
 <tb> C <SEP> H ------ N2E2-Z <SEP> H-ONB <SEP> H --- OBzl
 <tb> <SEP> 1) -5) <SEP> 1) -5)
 <tb> D <SEP> BOC ----- N2E2-Z <SEP> BOC --------- OBzl
 <tb> E <SEP> BOC ----- N2H3 <SEP> H --------- OBzl
 <tb> <SEP> 1) <SEP> Bzl <SEP> 1) -5)
 <tb> F <SEP> BOC ----------------------------- ONB <SEP> BOC -------------------------- OBzl
 <tb> <SEP> Bzl
 <tb> G <SEP> H ----------------------------- ONB <SEP> H -------------------------- OBzl
 <tb>

    <SEP> Bzl <SEP> 1) -5)
 <tb> H <SEP> BOC ------------------------------------- ONB <SEP> -24 <SEP> Bzl
 <tb> I. <SEP> BOC ------------------------------------- N2H3
 <tb> <SEP> Bzl <SEP> 1) <SEP> Bzl
 <tb> J <SEP> BOC ---------------------------------------------- ------------------------- OBzl
 <tb> <SEP> SH <SEP> SH
 <tb> K <SEP> BOC ---------------------------------------------- -------------------------OH
 <tb> L <SEP> BOC ---------------------------------------------- -------------------------OH
 <tb> <SEP> 1 <SEP> 1 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> Fig.

   1
EMI6.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <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
 <tb> <SEP> 1) -5) <SEP> 1) -5)
 <tb> D <SEP> BOC -------------- OBzl <SEP> BOC -------- OBzl
 <tb> E <SEP> H -------------- OBzl <SEP> H -------- OBzl
 <tb> <SEP> 3) <SEP> Bzl <SEP> 1) -5)
 <tb> F <SEP> BOC ---------------------- OBzl <SEP> BOC ----------------- OBzl
 <tb> G <SEP> H ---------------------- OBzl <SEP> H ----------------- OBzl
 <tb> H <SEP> Z ------------------------------- OBzl
 <tb> I. <SEP>

   H ------------------------------- OH
 <tb> <SEP> Bzl <SEP> 3) <SEP> 5)
 <tb> J <SEP> BOC ------------------------------------------ OH
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> K <SEP> BOC ---------------------------------------------- ---------------------- OBzl
 <tb> <SEP> SH <SEP> SH
 <tb> L <SEP> BOC ---------------------------------------------- ----------------------OH
 <tb> M <SEP> BOC ---------------------------------------------- ----------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5FIG. 2 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb>
EMI7.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> BOC ---- OH <SEP> Z-OH <SEP> Z-ONP <SEP> H-OMe <SEP> DPC ---- OH <SEP> H --- OMe <SEP> TRI --- OH <SEP>

   BOC - OH <SEP> z - OH <SEP> H-OMe
 <tb> <SEP> 3) <SEP> tBu <SEP> tBu <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> 1) -5) <SEP> 1) -5)
 <tb> D <SEP> Z ----------------- OMe <SEP> BOC --------------- OMe
 <tb> E <SEP> H ----------------- OMe <SEP> H --------------- OMr
 <tb> <SEP> TRI <SEP> 4)
 <tb> F <SEP> TRI ------------------------- OMe
 <tb> <SEP> TRI
 <tb> G <SEP> H ------------------------- OH
 <tb> <SEP> TRI <SEP> 4) <SEP> tBu <SEP> tBu <SEP> 2) <SEP> TRI
 <tb> H <SEP> BOC -------------------------- OMe <SEP> DPC ------------------------------------------- OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> I. <SEP> BOC -------------------------- N2H3 <SEP> H ------------------------------------------- OH
 <tb> <SEP> TRI <SEP> 2) <SEP> tBu <SEP> tBu <SEP>

   TRI
 <tb> J <SEP> BOC ---------------------------------------------- -------------------------------OH
 <tb> <SEP> SH <SEP> tBu <SEP> tBu <SEP> SH
 <tb> K <SEP> BOC ---------------------------------------------- -------------------------------OH
 <tb> <SEP> tBu <SEP> tBu
 <tb> L <SEP> BOC ---------------------------------------------- -------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> Fig.

   3
EMI8.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> BOC --- OH <SEP> Z-OH <SEP> Z-ONP <SEP> H-OMe <SEP> DPC --- OH <SEP> DPC --- OH <SEP> TRI ---- OH <SEP> BOC-OH <SEP> Z-OH <SEP> H-OMe
 <tb> <SEP> 3) <SEP> 1) -5)
 <tb> B <SEP> Z --------- OMe <SEP> Z --------- OMe
 <tb> C <SEP> H --------- OMe <SEP> H --------- OMe
 <tb> <SEP> 1) -5) <SEP> 1) -5)
 <tb> D <SEP> Z ------------------ OMe <SEP> BOC --------------- OMe
 <tb> E <SEP> H ------------------ OMe <SEP> H --------------- OMe
 <tb> <SEP> TRI <SEP> 4) <SEP> TRI <SEP> 5)
 <tb> F <SEP> BOC --------------------------- OMe <SEP> TRI --------------------------- OMe
 <tb> <SEP> TRI <SEP> TRI
 <tb> G <SEP> BOC --------------------------- N2H3 <SEP>

   H --------------------------- OMe
 <tb> <SEP> tBu <SEP> TRI <SEP> 1) -5)
 <tb> H <SEP> DPC ----------------------------------- OMe
 <tb> <SEP> tBu <SEP> TRI
 <tb> I. <SEP> H ----------------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> TRI <SEP> 1) -5)
 <tb> J <SEP> DPC -------------------------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> K <SEP> H -------------------------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> L <SEP> H -------------------------------------------- OH
 <tb> <SEP> TRI <SEP> 1) <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> M <SEP> BOC ---------------------------------------------- --------------------------------OH
 <tb> <SEP> tBu <SEP> tBu
 <tb> N <SEP> BOC ---------------------------------------------- --------------------------------OH
 <tb> <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <RTI

    ID = 8.1> Fig. 4
EMI9.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> TRI <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 ---- OMR <SEP> TRI --- OH <SEP> BOC - OH <SEP> Z - H <SEP> H ---- OMe
 <tb> <SEP> 3)
 <tb> B <SEP> Z --------- OMe
 <tb> C <SEP> H --------- OMe
 <tb> D <SEP> Z ------------------ OMe
 <tb> E <SEP> H ------------------ OMe
 <tb> <SEP> TRI <SEP> 4)
 <tb> F <SEP> TRI --------------------------- OMe
 <tb> <SEP> TRI
 <tb> G <SEP> H --------------------------- OMe
 <tb> <SEP> TRI
 <tb> H <SEP> Ac --------------------------- OMe
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI <SEP> like <SEP> Fig.

   <SEP> 3I <SEP> or <SEP> 4L
 <tb> I. <SEP> Ac --------------------------- N2H3 <SEP> H ---------------------------------------------- --OH
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> J <SEP> Ac ---------------------------------------------- -------------------------------OH
 <tb> <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 <SEP> 10
 <tb> Fig.

   5
EMI10.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> TRI <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> A <SEP> BOC ---- OH <SEP> Z --- OH <SEP> Z - ONP <SEP> H --- OMeDPC --- OH <SEP> H --- OMe <SEP> TRI --- OMe <SEP> BOC - OH <SEP> BOC - OH <SEP> H --- OMe
 <tb> <SEP> tBu <SEP> 1) -5) tBu <SEP> TRI <SEP> 1) -5)
 <tb> B <SEP> DPC ---------- OMe <SEP> H --- OMe <SEP> BOC --------- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> C <SEP> DPC ---------- N2H3 <SEP> H -------- OMe
 <tb> <SEP> tButBu <SEP> 1) <SEP> TRI <SEP> BOC ------------------ OMe
 <tb> D <SEP> DPC ---------- OMe <SEP> BOC ------------------ OH
 <tb> <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> E <SEP> DPC ---------- OH <SEP> H ------------------ OH
 <tb> <SEP> TRI <SEP> like <SEP> Fig.

   <SEP> 4G <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> F <SEP> BOC ---------------------------- N2H3 <SEP> H ---------- OH
 <tb> <SEP> TRI <SEP> 2) <SEP> tBu <SEP> tBu <SEP> TRI
 <tb> G <SEP> BOC -------------------------------------------- OH
 <tb> <SEP> tBu <SEP> tBu
 <tb> H <SEP> BOC -------------------------------------------- OH
 <tb> <SEP> tBu <SEP> tBu <SEP> 3)
 <tb> I. <SEP> BOC ---------------------------------------------- ---------------------------------OH
 <tb> Fig.

   6th
EMI11.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> A <SEP> BOC --- OSU <SEP> BOC - ONF <SEP> BOC - OH <SEP> BOC - ONP <SEP> BOC --- OH <SEP> H --- OBzl <SEP> H - OBzl <SEP> BOC - N2H3 <SEP> Z -------- OEt
 <tb> <SEP> 2) <SEP> 4) <SEP> 5)
 <tb> B <SEP> BOC ---------- OBzl <SEP> H -------- OEt
 <tb> <SEP> 1)
 <tb> C <SEP> H ---------- OBzl <SEP> BOC ---------------- OEt
 <tb> <SEP> 3)
 <tb> D <SEP> BOC ------------------- OBzl <SEP> BOC ---------------- OE
 <tb> E <SEP> H ------------------- OBzl <SEP> H ---------------- OE
 <tb> <SEP> 2) <SEP> 4) <SEP> 5)
 <tb> F <SEP> BOC ------------------- OBzl
 <tb> G <SEP> H ------------------- OBzl
 <tb> <SEP> 3)
 <tb> H <SEP> BOC ------------------------------------ OBzl
 <tb> I. <SEP>

   BOC ------------------------------------ OH
 <tb> <SEP> 2) 5) <SEP> Bzl
 <tb> J <SEP> BOC ------------------------------------------- OBzl
 <tb> <SEP> Bzl
 <tb> K <SEP> E -------------------------------------------- OBzl
 <tb> <SEP> Bzl <SEP> 3) <SEP> Bzl
 <tb> L <SEP> BOC ---------------------------------------------- ------- OBzl
 <tb> K <SEP> BOC ---------------------------------------------- -------OH
 <tb> N <SEP> BOC ---------------------------------------------- -------OH
 <tb> <SEP> 2)
 <tb> O <SEP> BOC ---------------------------------------------- -----------------------------OH
 <tb> Fig.

   7th
EMI12.1

   <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
 <tb> <SEP> Cys <SEP> Gly <SEP> Asn <SEP> Leu <SEP> Ser <SEP> Thr <SEP> Cys <SEP> X <SEP> Leu <SEP> Gly
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> A <SEP> BOC ---- CSU <SEP> BOC --- OXP <SEP> BOC - OH <SEP> BOC-ONP <SEP> BOC-OH <SEP> H --- OBzl <SEP> H - N2H2Z <SEP> BOC - N2H3 <SEP> Z ---------- OEt
 <tb> <SEP> 2) 4) 5)
 <tb> B <SEP> BOC -------- OBzl <SEP> H ---------- OEt
 <tb> C <SEP> H -------- OBzl <SEP> BOC ------------------ OEt
 <tb> <SEP> 3)
 <tb> D <SEP> BOC ---------------- OBzl <SEP> H ------------------- OH
 <tb> E <SEP> H ----------------- OBzl
 <tb> <SEP> 2) 4) 5)
 <tb> F <SEP> BOC ------------------------ OBzl
 <tb> G <SEP> H ------------------------- OBzl
 <tb> <SEP> 3)
 <tb> H <SEP> BOC ---------------------------------- OBzl
 <tb> I. <SEP> BOC ---------------------------------- OH
 <tb> <SEP> 2) <SEP> 5) <SEP>

   Bzl
 <tb> J <SEP> BOC ------------------------------------------ N2H2-Z
 <tb> <SEP> Bzl
 <tb> K <SEP> H ------------------------------------------- N2H2-Z
 <tb> <SEP> Bzl <SEP> Bzl
 <tb> L <SEP> BOC ---------------------------------------------- ------- N2H2-Z
 <tb> M <SEP> BOC ---------------------------------------------- ------- N2H3
 <tb> X <SEP> BOC ---------------------------------------------- ------- N2H3
 <tb> <SEP> 1)
 <tb> O <SEP> BOC ---------------------------------------------- --------------------------------OH
 <tb> Fig.

   8th
EMI13.1

 10 <SEP> 11 <SEP> 12 <SEP> 13 <SEP> 14 <SEP> 15 <SEP> 16 <SEP> 17 <SEP> 18 <SEP> 19 <SEP> 20 <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24 <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28 <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> <SEP> Thr <SEP> Tyr <SEP> Thr <SEP> Eqs <SEP> Asp <SEP> Phe <SEP> Asn <SEP> Lys <SEP> Phe <SEP> His <SEP> Thr <SEP> Phe <SEP> Pro <SEP> Eqs <SEP> Thr <SEP> Ala <SEP> Ile <SEP> Gly <SEP> Val <SEP> Gly <SEP> Ala <SEP> Pro
 <tb> <SEP> tBu <SEP> tBu <SEP> OH
 <tb> <SEP> Z ----------- OH <SEP> H ------------------- OMe
 <tb> <SEP> tBu <SEP> tBu
 <tb> <SEP> Z ----------------------------------- OH
 <tb> <SEP> BOC <SEP> tBu <SEP> 2) <SEP> tBu
 <tb> <SEP> Z -------------- N2H3 <SEP> H ----------------------------------- OH
 <tb> <SEP> BOC <SEP> 1) <SEP> tBu <SEP> tBu
 <tb> <SEP> Z ---------------------------------------------- ---------OH
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu

    <SEP> tBu <SEP> BOC <SEP> tBu <SEP> tBu
 <tb> <SEP> Z {---------------------- N2H3 <SEP> H ---------------------------------------------- ---------OH
 <tb> <SEP> DPC
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu <SEP> tBu <SEP> 1) <SEP> BOC <SEP> tBu <SEP> tBu
 <tb> <SEP> Z {--------------------------------------------- ---------------------------------------OH <SEP> H --------- NH2
 <tb> <SEP> DPC
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu <SEP> tBu <SEP> BOC <SEP> tBu <SEP> tBu <SEP> 4) 5)
 <tb> <SEP> Z {--------------------------------------------- -------------------------------------------------- --NH2
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu <SEP> tBu <SEP> BOC <SEP> tBu <SEP> tBu
 <tb> <SEP> H ---------------------------------------------- -------------------------------------------------- --NH2
 <tb> Fig.

   9
EMI14.1

   <SEP> 11 <SEP> 12 <SEP> 13 <SEP> 14 <SEP> 15 <SEP> 16
 <tb> <SEP> Thr <SEP> Tyr <SEP> Thr <SEP> Eqs <SEP> Asp <SEP> Phe
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu <SEP> OtBu
 <tb> A <SEP> Z {---- OH <SEP> Z ---- OH <SEP> Z --- OH <SEP> Z --- ONP <SEP> Z ------ ONP <SEP> H ----- OMe
 <tb> <SEP> DPC
 <tb> <SEP> OtBu <SEP> 3)
 <tb> B <SEP> Z ---------------- OMe
 <tb> <SEP> OtBu
 <tb> C <SEP> H ---------------- OMe
 <tb> <SEP> 3) <SEP> OtBu
 <tb> D <SEP> Z ------------------------ OMe
 <tb> <SEP> OtBu
 <tb> E <SEP> H ------------------------ OMe
 <tb> <SEP> tBu <SEP> 3) <SEP> OtBu
 <tb> F <SEP> Z -------------------------------- OMe
 <tb> <SEP> tBu <SEP> OtBu
 <tb> G <SEP> H -------------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> OtBu
 <tb> H <SEP> Z ----------------------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> OtBu
 <tb> I. <SEP>

   H ----------------------------------------- OMe
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu <SEP> OtBu
 <tb> J <SEP> Z {--------------------------------------------- ----- OMe
 <tb> <SEP> DPC
 <tb> <SEP> tBu <SEP> tBu <SEP> tBu <SEP> OtBu
 <tb> K <SEP> Z {--------------------------------------------- ----- K2H3
 <tb> <SEP> DPC
 <tb> Fig. 10
EMI15.1


 <tb> <SEP> 17 <SEP> 18 <SEP> 19 <SEP> 20
 <tb> <SEP> Asn <SEP> Lys <SEP> Phe <SEP> His
 <tb> <SEP> BOO
 <tb> A <SEP> Z <SEP> --ONP <SEP> Z1 - ONP <SEP> H- <SEP> OMe <SEP> H-OMe
 <tb> <SEP> BOC
 <tb> B <SEP> 1 <SEP> 3) <SEP> OMe
 <tb> <SEP> BOC
 <tb> C <SEP> Z <SEP> NO
 <tb> <SEP> BOO
 <tb> D <SEP> e.g. <SEP> l <SEP> 1) <SEP> OMe
 <tb> <SEP> BOC
 <tb> E <SEP> H1 <SEP> - <SEP> OMe
 <tb> <SEP> BOC
 <tb> F
 <tb> <SEP> BOC
 <tb> G <SEP> Z <SEP> { <SEP> NO
 <tb> Fig.

   11
EMI16.1

   <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24 <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28
 <tb> <SEP> Thr <SEP> Phe <SEP> Pro <SEP> Eqs <SEP> Thr <SEP> Ala <SEP> The <SEP> Gly
 <tb> <SEP> tBu <SEP> tBu
 <tb> A <SEP> Z ---- OH <SEP> Z ----------- OH <SEP> Z ----- ONP <SEP> Z ------ OH <SEP> Z ------ ONP <SEP> Z --- OH <SEP> H --- OMe
 <tb> <SEP> 1) -5)
 <tb> B <SEP> H ----------- OH <SEP> Z ------------ OMe
 <tb> <SEP> tBu <SEP> 2)
 <tb> C <SEP> Z -------------------- OH <SEP> H ------------ OMe
 <tb> <SEP> 3)
 <tb> D <SEP> Z ------------------------ OMe
 <tb> E <SEP> H ------------------------ OMe
 <tb> <SEP> tBu <SEP> 2)
 <tb> F <SEP> Z --------------------------------- OMe
 <tb> <SEP> tBu
 <tb> G <SEP> H --------------------------------- OMe
 <tb> <SEP> tBu
 <tb> H <SEP> H --------------------------------- OH
 <tb> <SEP> tBu <SEP> 3)
 <tb> I. <SEP> Z -------------------------------------------- OH
 <tb> <SEP> tBu
 <tb> J

    <SEP> H -------------------------------------------- OH
 <tb> <SEP> tBu <SEP> 2) <SEP> tBu
 <tb> K <SEP> Z ---------------------------------------------- -------------------------OH
 <tb> <SEP> tBu <SEP> tBu
 <tb> L <SEP> Z ---------------------------------------------- -------------------------OH
 <tb> Fig. 12
EMI17.1


 <tb> <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> <SEP> Val <SEP> Gly <SEP> Ala <SEP> Pro
 <tb> A <SEP> ZONP <SEP> e.g. <SEP> OH <SEP> Z <SEP> ONP <SEP> H <SEP> NH2
 <tb> B <SEP> e.g. <SEP> 3) <SEP> NR2
 <tb> C <SEP> H- <SEP> iN <SEP> 2
 <tb> D <SEP> Z <SEP> 1) -5) <SEP> NH2
 <tb> 3 <SEP> g <SEP> 'H2
 <tb> F <SEP> n <SEP> 3) <SEP> NR2
 <tb> G <SEP> H --------------------------- <SEP> H <SEP> NH2
 <tb> Fig.

   13
EMI18.1

   <SEP> 11 <SEP> 12 <SEP> 13 <SEP> 14 <SEP> 15 <SEP> 16 <SEP> 17 <SEP> 18 <SEP> 19
 <tb> <SEP> Thr <SEP> Tyr <SEP> Thr <SEP> Eqs <SEP> Asp <SEP> Phe <SEP> Asn <SEP> Lys <SEP> Phe
 <tb> <SEP> OtBu <SEP> BOC
 <tb> A <SEP> Z ----- OH <SEP> BOC - OH <SEP> H --- ONB <SEP> H --- N2H20BOC <SEP> Z ---- OH <SEP> H ---- OMe <SEP> Z --- ONP <SEP> Z --- OH <SEP> H --- OBzl
 <tb> <SEP> 4) <SEP> OtBu <SEP> 4) <SEP> BOC <SEP> 4)
 <tb> B <SEP> BOC --------- ONB <SEP> Z ------------ OMe <SEP> Z ---------- OBzl
 <tb> <SEP> OtBu <SEP> BOC
 <tb> C <SEP> H ---------- ONB <SEP> Z ------------ N2H3 <SEP> H ---------- OH
 <tb> <SEP> 4) <SEP> BOC <SEP> 3)
 <tb> D <SEP> Z -------------------- ONB <SEP> Z ------------------ OH
 <tb> <SEP> BOC
 <tb> E <SEP> Z -------------------- N2H3 <SEP> H ------------------ OH
 <tb> <SEP> 1) <SEP> OtBu <SEP> 1) <SEP> BOC
 <tb> F <SEP> Z -------------------------------- N2H2-BOC <SEP>

   Z -------------------------------- OH
 <tb> <SEP> OtBu <SEP> BOC
 <tb> G <SEP> Z -------------------------------- N2H3 <SEP> H -------------------------------- OH
 <tb> <SEP> OtBu <SEP> BOC
 <tb> H <SEP> Z ---------------------------------------------- ----------------------------OH
 <tb> Fig.

   14th
EMI19.1

   <SEP> 20 <SEP> 21 <SEP> 22 <SEP> 23 <SEP> 24 <SEP> 25 <SEP> 26 <SEP> 27 <SEP> 28 <SEP> 29 <SEP> 30 <SEP> 31 <SEP> 32
 <tb> <SEP> ice cream <SEP> Thr <SEP> Phe <SEP> Pro <SEP> Eqs <SEP> Thr <SEP> Ala <SEP> Ile <SEP> Gly <SEP> Val <SEP> Gly <SEP> Ala <SEP> Pro
 <tb> A <SEP> Z - N2H3 <SEP> Z - N2H3 <SEP> Z - ONP <SEP> Z - OH <SEP> H - N2H2-BOC <SEP> BOC-N2H3 <SEP> Z-OH <SEP> Z-ONP <SEP> H - OMe <SEP> Z - ONP <SEP> Z-ONP <SEP> Z - OH <SEP> H - OtBu
 <tb> <SEP> 2) <SEP> 3) <SEP> 2)
 <tb> B <SEP> Z --------- N2H2-BOC <SEP> Z ---------- OMe <SEP> Z --------------- OtBu
 <tb> C <SEP> H --------- N2H2-BOC <SEP> H ---------- OMe <SEP> H --------------- OtBu
 <tb> <SEP> 3) <SEP> 2) <SEP> 3)
 <tb> D <SEP> Z ---------------- N2B2-BOC <SEP> Z ----------------- OMe <SEP> Z --------------------------- OtBu
 <tb> E <SEP> H ---------------- N2H2-BOC <SEP> H ----------------- OMe <SEP>

   Z --------------------------- OtBu
 <tb> <SEP> 1) <SEP> 1) <SEP> 3)
 <tb> F <SEP> Z ------------------------ N2H2-BOC <SEP> BOC ------------------------ OMe <SEP> Z ------------------------------------- OtBu
 <tb> G <SEP> H ------------------------ N2H2-BOC <SEP> BOC ------------------------ OH <SEP> Z ------------------------------------- OH
 <tb> <SEP> 1) <SEP> 2) + NH3
 <tb> H <SEP> Z -------------------------------- N2H2-BOC <SEP> Z ------------------------------------- NH2
 <tb> I. <SEP> Z -------------------------------- N2H3 <SEP> H ------------------------------------- NH2
 <tb> <SEP> 204) 5)
 <tb> J <SEP> BOC ---------------------------------------------- --------------------- NH2
 <tb> K <SEP> H ---------------------------------------------- ---------------------- NH2
 <tb> <SEP> 1)
 <tb> L <SEP>

   Z ------------------------------------------------- -------------------------------------------------- ------------ NH2
 <tb> M <SEP> H ---------------------------------------------- -------------------------------------------------- --------------- NH2
 <tb> Fig. 15 Example 1
EMI20.1
 Nle-Leu-Gly-Thr-Tyr Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr Ala-Ile-Glv-Val-Giv-Ala-Pro-NH, (Nle8-calcitonin M).
EMI20.2




     Asn-Lys (BOC) -Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro; NH, are introduced into 10 ml of ice-cold, concentrated hydrochloric acid with vigorous stirring. After 10 minutes at 0, 50 ml of water and 5 ml of glacial acetic acid are added and the solution is filtered through a column of Merck ion exchanger No. II, weakly basic, acetate form, to remove chlorine ions. It is then evaporated to dryness on a rotary evaporator with the addition of n-octanol, the residue is washed several times with petroleum ether to remove octanol, while decanting, dried, dissolved in 0.1N formic acid and cleaned on a column (3.8 cm; 120 cm) Chromatographed by Bio-Gel P6. Fractions of 10 ml each are collected.



  These are checked for purity by means of thin-layer chromatography on aluminum oxide (systems 52, 79 and 45); the uniform fractions are combined and lyophilized. In the thin-layer chromatogram on silica gel, Rf6 = 0.44; on aluminum oxide, Rfs2 = 0.55; Rf79 = 0.64; Rf45 = 0.45.



   The starting material can be prepared as follows: 1. Z-Ala-Pro-OtBu
68.85 g of Z-Ala-OH are dissolved in 550 ml of dimethylformamide and then 43.3 ml of triethylamine are added. At -15 ", 30.8 ml of ethyl chloroformate are added and the temperature is increased to -10" within 10 minutes. A solution, cooled to -10 ", of 52.65 g of H-Pro-OtBu in 105 ml of dimethylformamide is then added.

  The mixture is stirred for 30 minutes at -10 "and 2.5 hours at 20, left to stand in the refrigerator overnight and then the solvent is evaporated off in vacuo at 40". The residue is taken up in aqueous ethyl acetate, the solution is washed with 0.1N hydrochloric acid, 10% sodium bicarbonate and 5% sodium bicarbonate, dried over sodium sulphate and evaporated to dryness in vacuo. In thin layer chromatography on silica gel, Rf2 = 0.6.



  2. H-Ala-Pro-OtBu, acetate
109 g of Z-Ala-Pro-OtBu are dissolved in 649 ml of methanol and hydrogenated in the presence of 4.1 ml of glacial acetic acid and 3.1 g of palladium carbon (10%). After filtration and evaporation of the solution to dryness, the product shows Rf7 = 0.5.



  3. Z-Gly-Ala-Pro-OtBu
56.75 g of Z-Gly-OH are dissolved in 675 ml of tetrahydrofuran.



  The mixed anhydride is formed with 47.5 ml of triethylamine and 27 ml of ethyl chloroformate and this is reacted with 74 g of H-Ala-Pro-OtBu, acetate as in 1. The oil obtained is triturated with ether. The product melts at 87-89; [a] D20 = -62.9 (c = 1 in dimethylformamide).



  In the thin-layer chromatogram on silica gel, Rf2 = 0.50.



  4. H-Gly-Ala-Pro-OtBu
80 g of Z-Gly-Ala-Pro-OtBu are dissolved in 800 ml of methanol and hydrogenated as described under 2, but without glacial acetic acid. The product shows Rf5 = 0.30 in a thin-layer chromatogram on silica gel.



   5. Z-Val-Gly-Ala-Pro-OtBu
54.7 g of H-Gly-Ala-Pro-OtBu are suspended in 722 ml
Ethyl acetate, cool to -10 "and add 71.6 g of Z-Val-ONP.



  The mixture is stirred for 1 hour at -10, 1 hour at 10 and 20 hours at 20. The reaction mixture is washed with 0.1N hydrochloric acid, salt water, 20% potassium carbonate and again with salt water. The organic layer is dried and the solvent evaporated in vacuo. The residue (oil) is washed with ethyl acetate-hexane. In the thin-layer chromatogram on silica gel, Rf2 = 0.4.



  6. Z-Val-Gly-Ala-Pro-OH
102 g of Z-Val-Gly-Ala-Pro-OtBu are dissolved in 690 ml of dry ethyl acetate and hydrochloric acid gas is allowed to flow through the solution for 1.5 hours. The solvent is evaporated, the residue is redissolved in ethyl acetate, hexane is added and the mixture is stirred at 100 for 1 hour. Then it is filtered.



  The product melts at 111 "(dec.). [A] D20 = -38.3" (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf = 0.50.



  7. Z-Val-Gly-Ala-Pro-NH2
64.4 g of Z-Val-Gly-Ala-Pro-OH are dissolved in 644 ml of dry tetrahydrofuran, the mixed anhydride is formed with 18.9 ml of triethylamine and 13.5 ml of ethyl chloroformate as described under 1, then 25 ml of liquid ammonia are added and works up the reaction mixture as under 1. The product melts the 208-211 ". [A] D20 = -32.7 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf7 = 0.7.



  8. H-Val-Gly-Ala-Pro-NH2, sulfate
27.5 g of Z-Val-Gly-Ala-Pro-NH2 are suspended in 550 ml of methanol and, after addition of 3 ml of 96% sulfuric acid, hydrogenated as described under 2. The product shows Rf5 = 0.2 in a thin-layer chromatogram on silica gel.



  9. Z-Ile-Gly-OMe
84.2 g of H-Gly-OMe and 247 g of Z-Ile ONP are suspended in 2.4 liters of dimethylformamide, cooled to -10 "and 99 ml of triethylamine are added. The reaction mixture is worked up as described under 5. The product crystallizes from ethyl acetate-hexane. F. 126-128 ". [a] D20 = 270 (c = 3 in methanol). In the thin-layer chromatogram on silica gel, Rf = 0.50.



  10. H-Ile-Gly-OMe, sulfate
193 g of Z-Ile-Gly-OMe are dissolved in 4860 ml of methanol and 30 ml of 96% strength sulfuric acid. The hydrogenation is carried out as described under 2. The product shows Rf5 = 0.56 in a thin-layer chromatogram on silica gel.



  11. Z-Ala-Ile-Gly-OMe
125.3 g of Z-Ala-OH are dissolved in 1250 ml of dimethylformamide, the mixed anhydride is formed with 79.3 ml of triethylamine and 55.7 ml of ethyl chloroformate, and sulfate is added to a solution of 168 g of H-Ile-Gly-OMe 835 ml of dimethylformamide and 158.3 ml of triethylamine as described under 1 to. The dimethylformamide is removed in vacuo to 100 ml and the solution is poured into 500 ml of water. The product melts to 191.5-192.5. [a] D20 = -9.2 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rfz = 0.65.

 

  12. H-Ala-Ile-Gly-OMe, t / 2 H2SO4
100 g of Z-Ala-Ile-Gly-OMe are suspended in 824 ml of dimethylformamide and 6.66 ml of 96% sulfuric acid and hydrogenated as described under 2. The product shows Rf5 = 0.5 in a thin-layer chromatogram on silica gel.



  13. BOC-Thr-Ala-lle-Gly-OMe
57.8 g of BOC-Thr-NH-NH2 are dissolved in 340 ml of dimethylformamide and cooled to -20 ". 250 ml of 1.989N hydrochloric acid and then 33.5 ml of iso-amyl nitrite are added to the solution.



  The solution is stirred vigorously for 10 minutes. In the meantime, 105 ml of triethylamine are added to a solution of 79.1 g of H-Ala Ile-Gly-OMe in 1500 ml of dimethylformamide, cooled to -10 ", this solution is cooled to -20" and the above solution of BOC-Thr- N3 added. The mixture is left to react for 70 hours at 0, the solvent is then evaporated off in vacuo at 40, the residue is taken up in ethyl acetate, the solution is washed with 0.1N hydrochloric acid, water, 5% sodium bicarbonate and water and dried. Then the ethyl acetate is removed in vacuo and the residue is crystallized from ethyl acetate-hexane. The product melts at 187-189 ". [A] D20 = -13.20 (c = 2 in dimethylformamide).



  In the thin-layer chromatogram on silica gel, Rf = 0.68.



  14. BOC-Thr-Ala-Ile-Gly-NH-NH2
87 g of BOC tetrapeptide ester are dissolved in 870 ml of methanol, the solution is cooled to 0 and 37.6 ml of hydrazine hydrate are added. The mixture is stirred for 18 hours at room temperature and 1 hour at 0 and then filtered. The precipitate is recrystallized twice from methanol. F. 231-232.5 ".



     [a] D20 = -5.90 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf = 0.7.



  15. BOC-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2
27.1 g of BOC-Thr-Ala-Ile-Gly-NH-NH2 are suspended in 250 ml of dimethylformamide. The azide is prepared as described under 13 with 59.9 ml of 2.23N hydrochloric acid and 9.1 ml of iso-amyl nitrite. The solution is stirred for 15 minutes.



  24.2 g of H-Val-Gly-Ala-Pro-NH2, sulfate are dissolved in 120 ml of dimethylformamide, cooled to 0 and then 28 ml of triethylaline are added. After mixing with the azide solution, the pH is brought to 7. The mixture is left to react for 70 hours. The peptide is crystallized from ethyl acetate-hexane. F. 226-227.5. [a] D20 = -25.6 "(c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf, = 0.7.



  16. H-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2, trifluoroacetate
51 g of BOC-Thr-Ala-Ile-Gly-Val-Gly-Ala Pro-NH2 are dissolved in 153 ml of 10% aqueous trifluoroacetic acid, the solution is left to stand for 1 hour and then 1530 ml of dry ether are added. The precipitate is filtered off and washed 3 times with ether. F. 221-223. In the thin-layer chromatogram on silica gel, Rf = 0.5.



  17. Z-Pro-Gln-NH-NH-BOC
67.23 g of Z-Pro-OH are dissolved in 670 ml of dimethylformamide and the mixed anhydride is formed using 38.9 ml of triethylamine and 26 ml of ethyl chloroformate. 70.2 g of H-Gln-NH-NH-BOC are allowed to react with this as described under 1. The residue is crystallized from hot ethyl acetate. F. 169-171 ". [A] D20 = -41.30 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rfl = 0.45.



  18. H-Pro-Gln-NH-NH-BOC
94 g of the derivative described under 17 are dissolved in 564 ml of dimethylformamide and hydrogenated as described under 2, but without acid. The product shows Rf5 = 0.3 in the thin-layer chromatogram on silica gel.



  19. Z-Phe-Pro-Gln-NH-NH-BOC
80.4 g of Z-Phe-ONP, 68 g of H-Pro-Gln-NH-NH BOC and 9.4 ml of glacial acetic acid are dissolved in 560 ml of dimethylformamide as described under 5. The product melts at 107 ".



     [a] D20 = -35.9 "(c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf, = 0.7.



  20. H-Phe-Pro-Gln-NH-NH-BOC
103 g of Z-Phe-Pro-Gln-NH-NH-BOC are dissolved in 618 ml of dimethylformamide and hydrogenated as described under 2, but without acid. The product shows Rf6 = 0.5 in a thin-layer chromatogram on silica gel.



   21. Z-Thr-Phe-Pro-Gln-NH-NH-BOC
46.1 g of Z-Thr-NH-NH2 are suspended in 550 ml of dimethylformamide and the azide is formed as described under 13 with 177 ml of 1.958N hydrochloric acid in tetrahydrofuran and 24 ml of iso-amyl nitrite. The azide is coupled with 81.4 g of H-Phe Pro-Gln-NH-NH-BOC, dissolved in 1200 ml of dimethylformamide and 48.5 ml of triethylamine. After crystallization from ethyl acetate-hexane, the product melts at 129-134 ".



   [a] D20 = -34.8 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf7 = 0.85.



  22. H-Thr-Phe-Pro-Gln-NH-NH-BOC
23.6 g of the product described under 21 are dissolved in
188 ml of dimethylformamide and hydrogenated as described under 2, but without acid. The product shows Rf7 = 0.7 in a thin-layer chromatogram on silica gel.



  23. Z-His-Thr-Phe-Pro-Gln-NH-NH-BOC
9.68 g of Z-His-NH-NH2 are dissolved in 100 ml of dimethylformamide. The azide is formed with 36.3 ml of 2.67N hydrochloric acid in tetrahydrofuran and 4.32 ml of iso-amyl nitrite and with
19.32 g of H-Thr-Phe-Pro-Gln-NH-NH-BOC, dissolved in 300 ml of dimethylformamide and 13.8 ml of triethylamine, as described under 13, coupled. The reaction mixture is filtered, the filtrate is concentrated in vacuo to 100 ml and poured into 1100 ml of ethyl acetate. The product melts at 95-133 ".



  In the thin-layer chromatogram on silica gel, Rf6 = 0.7.



  24. Z-His-Thr-Phe-Pro-Gln-NH-NH2, HCI
114 g of Z-His-Thr-Phe-Pro-Gln-NH-NH-BOC are dissolved in 57 ml of 90% trifluoroacetic acid, the solution is left to stand for 1 hour and then 9.8 ml of 4N hydrochloric acid are added. The solution is poured into 570 ml of dry ether and the residue is dried in vacuo over potassium hydroxide. The product shows Rf5 = 0.25 in a thin-layer chromatogram on silica gel.



  25. Z-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NR2
11 g of the hydrazide described under 24 are dissolved in 47 ml of dimethylformamide and the azide is formed with 15.6 ml of 2,267N hydrochloric acid in tetrahydrofuran and 1.57 ml of amyl nitrite as described under 13. The azide is allowed to react with 6 g of the octapeptide amide described under 16, dissolved in 70 ml of dimethylformamide and 5.5 ml of triethylamine. The pH is 6.5. After 7 days, the reaction mixture is filtered, the filtrate is poured into 710 ml of ethyl acetate and the residue is stirred twice with ethyl acetate. The product melts at 186 with decomposition. In the thin-layer chromatogram on silica gel, Rf7 = 0.6.

 

  26. Z-Lys (BOC) -Phe-OBzl
261.6 g of Z-Lys (BOC) -OH, dicyclohexylammonium salt are suspended in 6.4 l of acetonitrile, 137.6 g of H-Phe-OBzl, HCl are added and the suspension is stirred for 30 minutes. After cooling to 5, 96 g of dicyclohexylcarbodiimide are added. The suspension is stirred for 20 hours at room temperature, filtered and the residue is washed with acetonitrile.



  The filtrate is evaporated in vacuo, the residue is taken up in ethyl acetate, the solution is washed with 0.1N hydrochloric acid, water, 5% sodium bicarbonate and water, dried and evaporated. The residue crystallizes from ethyl acetate-hexane. F. 101-103. [a] D20 = -12.5 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf2 = 0.55.



  27. h-Lys (BOC) -Phe-OH, HCl
105.4 g of the above dipeptide ester are dissolved in 630 ml of dimethylformamide and hydrogenated as described under 2, but no glacial acetic acid is added, instead 150 ml of 1.115 N hydrochloric acid in tetrahydrofuran is added after 21/2 hours. The hydrogenation takes 20 hours. The product shows Rf4 = 0.7 in a thin-layer chromatogram on silica gel.



  28. Z-Asn-Lys (BOC) -Phe-OH
76.2 g of Z-Asn-ONP and 73.6 g of H-Lys (BOC) Phe-OH, HCl in 850 ml of dimethylformamide and 150 ml of tetrahydrofuran are dissolved, cooled to 100 and 47.8 ml of triethylamine are added as described under 5. After 20 hours at 20, the mixture is concentrated to 320 ml and the concentrate is poured into 1435 ml of ethyl acetate and 359 ml of 0.1N hydrochloric acid. The precipitate is washed with water and ether. F. 167-168 [a] 20 D = -11.7 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf7 = 0.7.



  29. H-Asn-Lys (BOC) -Phe-OH, HCI
34 g of the above product are dissolved in 240 ml of methanol and hydrogenated as described under 27, but with the addition of 13.3 ml of 2N hydrochloric acid in tetrahydrofuran after 21/2 hours and a further 13.3 ml of 2N hydrochloric acid in tetrahydrofuran after 20 hours. The solution is processed further directly. The product shows Rf7 = 0.6 in a thin-layer chromatogram on silica gel.



  30. Z-Asp (OtBu) -Phe-OMe
100.84 g of Z-Asp (OtBu) -OH, dicyclohexylammonium salt are dissolved in 880 ml of methylene chloride, 43.16 g of H-Phe-OMe, HCl are added and the mixture is stirred for a further 10 minutes.



  The suspension is then cooled to -5, 41.2 g of dicyclohexylcarbodiimide in 120 ml of methylene chloride are added and the process is carried out as described under 26. The residue is washed with methylene chloride. The product shows Rfi = 0.9 in a thin-layer chromatogram on silica gel.



  31. Z-Asp (OtBu) -Phe-NH-NH2
79.2 g of the above dipeptide ester are dissolved in 1320 ml of methanol, the solution is cooled to 0 "and 21.1 ml of hydrazine hydrate are added as described under 14, but left to react for 3 days. The residue is washed twice with water. F. 143-145 <. [a] D20 = -22.2 "(c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf = 0.65.



  32. Z-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-OH
54.4 g of Z-Asp (OtBu) -Phe-NH-NH2 are dissolved in 325 ml of dimethylformamide and the azide is prepared as described under 13 with 111 ml of 1.98N hydrochloric acid in tetrahydrofuran and 14 ml of iso-amyl nitrite. 53.4 g of H-Asn-Lys (BOC) Phe-OH, HCl are dissolved in 280 ml of dimethylformamide and 50 ml of triethylamine, the mixture is cooled to -10 "and the azide solution is added.



  The pentapeptide derivative is precipitated by mixing the residue with ethyl acetate and the precipitate is washed with water-ethanol (15: 1). F. 189-190 <. [a] D20 = -21.8 <(c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf6 = 0.77.



     33. H-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-OH
9.6 g of the protected pentapeptide obtained under 32 are dissolved in 192 ml of dimethylformamide and hydrogenated as described under 27, but with 4.43 ml of 2.3N hydrochloric acid in tetrahydrofuran. The product shows Rf5 = 0.8 in a thin-layer chromatogram on silica gel.



  34. BOC-Tyr-Thr-ONB
69.68 g of H-Thr-ONB, HCl are suspended in 480 ml of acetonitrile, 32.84 ml of triethylamine are added, the mixture is cooled to -5 "and 67.52 g of BOC-Tyr-OH and 960 ml of cold acetonitrile are then added 49.44 g of dicyclohexylcarbodiimide were added.



  The suspension is stirred at 3 for 1 hour and worked up as described under 26. The dipeptide derivative is isolated as an oil. In the thin-layer chromatogram on silica gel, Rf1 = 0.60.



  35. H-Tyr-Thr-ONB, HCI
Dissolve 121 g of BOC-Tyr-Thr-ONB in 1537 ml of methylene chloride and 384 ml of nitromethane and proceed as described under 6. After HCl gas has been passed through for 45 minutes, the suspension is stirred for 1 hour and then filtered. The product melts at 226-227 <. In the thin-layer chromatogram on silica gel, Rf = 0.35.



  36. Z-Thr-Tyr-Thr-ONB
57.4 g of Z-Thr-NH-NH2 are suspended in 320 ml of dimethylformamide and the azide is formed with 220 ml of 1.953N hydrochloric acid in tetrahydrofuran and 29 ml of iso-amyl nitrite as described under 13. The azide solution is added to a solution of 98 g of H-Tyr-Thr-ONB, HCl in 760 ml of dimethylformamide and 91 ml of triethylamine. The tripeptide derivative crystallizes from ethyl acetate-hexane. F. 111-118 ". [A] D20 = -4.4 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf1 = 0.6.



  37. Z-Thr-Tyr-Thr-NH-NH2
114.2 g of the above tripeptide ester are dissolved in 480 ml of methanol and 48 ml of hydrazine hydrate are added as described under 14. After 4 hours, 1440 ml of ethyl acetate are added and after a further 11/2 hours the suspension is filtered. The residue is stirred with 1 liter of ethyl acetate for 20 hours. The product melts at 219-221 ". [A] D20 = -6.6 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf6 = 0.85.



  38. Z-Thr-Tyr-Thr-Gln-NH-NH-BOC
89 g of Z-Thr-Tyr-Thr-Gln-NH-NH2 are suspended in 890 ml of dimethylformamide and the azide is formed as described under 13 with 197 ml of 2.02N hydrochloric acid in tetrahydrofuran and 26.8 ml of iso-amyl nitrite. The azide solution is added to a solution, cooled to -10 ", of 39.3 g of H-Gln-NH-NH-BOC and 56.2 ml of triethylamine in 750 ml of dimethylformamide, and the suspension is stirred for 2 hours. The crystals are then filtered off and Stir it with 400 ml of 0.05N hydrochloric acid (2x). The product melts at 216-218. [a] D20 = -8.3 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rfjo = 0.8.

 

  39. Z-Thr-Tyr-Thr-Gln-NH-NH2
53.5 g of the derivative described under 38 are dissolved in 110 ml of 90% strength trifluoroacetic acid, stirred for 11/2 hours and then 880 ml of ether are added as described under 16. The precipitate is stirred (2x) with 200 ml of methanol-ether (1: 4). The product melts at 200-203. [a] D20 = -4.6 (c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rfs = 0.75.



  40. Z-Thr-Tyr-Thr-Gln-Asp (OtBu) -Phe-Asn-Lys (BOC)
Phe-OH
5 g of Z-Thr-Tyr-Thr-Gln-NH-NH2 are suspended in 95 ml of dimethylformamide and the azide is formed as described under 13 with 11.94 ml of 2.122N hydrochloric acid in tetrahydrofuran and 2.28 ml of iso-amyl nitrite. 8.6 g of H-Asp (OtBu) Phe-Asn-Lys (BOC) -Phe-OH are dissolved in 210 ml of dimethylformamide and 7.5 ml of triethylamine. This solution is cooled to -10 "and added to the azide solution. It is left to react at 0" for 3 days. It is then filtered, the filtrate is evaporated and the residue is stirred with 300 ml of 0.1N hydrochloric acid. The product crystallizes from dimethylformamide 0.1N hydrochloric acid (1: 3.5). It melts at 206 ". [A] D20 = -19.3 (c = 1 in dimethylformamide).

  In the thin-layer chromatogram on silica gel, Rf5 = 0.87.



  41. H-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly
Ala-Pro-NH2
32 g of the BOC tridecapeptide amide described under 25 are dissolved in 1200 ml of dimethylformamide and 12 ml of 4N hydrochloric acid and hydrogenated as described under 2 for 20 hours. The filtrate is concentrated to 250 ml and stirred with 1500 ml of dry ether. The crude product is purified by countercurrent distribution according to Craig in the n-butanol-glacial acetic acid-water system (4: 1: 5). K = 0.11. The product has [a] D20 = -39.4 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf6 = 0.2.



  42. Z-Thr-'Fyr-Thr-Glu-Asp (OtBu) -Phe-Asn-Lys (BOC) -
Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly
Ala-Pro-NH2
7.3 g of the protected nonapeptide described under 40 are dissolved in 75 ml of dimethylformamide, 1.33 g of N-hydroxysuccinimide are added and the solution is cooled to -22 ". A likewise cooled solution of 1.195 g of dicyclohexylcarbodiimide in 15 ml of dimethylformamide is added (solution A). After stirring for one hour at -22 "and 5 hours at +4", the suspension is cooled back down to -22 ". A cooled solution of 6.9 g of the tridecapeptide amide described under 41 in 80 ml of dimethylformamide is then added to solution A, brought to pH 6.7 to 7 with triethylamine.

  After a reaction time of 14 days, the suspension is added to a new solution A, prepared from 0.73 g of the nonapeptide described under 40.



  The mixture is left to react for a further 7 days at 0 ", then the suspension is filtered and the filtrate is evaporated to dryness in vacuo. The residue is purified by column chromatography on silica gel in the system n-butanol-glacial acetic acid-water (4: 1: 5) The product shows Rf5 = 0.32 in the thin-layer chromatogram on silica gel.



  43. H-Thr-Tyr-Thr-Gln-Asp (OtBu) -Phe-Asn-Lys- (BOC)
Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly
Ala-Pro-NH2
2 g of the product described under 42 are dissolved in 150 ml of dimethylformamide and hydrogenated. After 21/2 hours, 0.3 ml of 4.844N hydrochloric acid are added. After 21 hours, it is filtered and the filtrate is evaporated. The residue is dissolved in 25 ml of dimethylformamide, 0.73 ml of 0.1N hydrochloric acid is added, mixed with 40 ml of ethyl acetate and cooled to 0 ". After the precipitate has been filtered off, it is taken up in tert-butanol-water and lyophilized. The product shows [a] D20 = -25.2 <(c = 2 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf6 = 0.32.



  44. BOC-Ser-Thr-OBzl
47.0 g of H-Thr-OBzl, HCI in 300 ml of methylene chloride are added to a solution of 73.8 g of BOC-Ser-OH, dicyclohexylammonium salt in 500 ml of methylene chloride, the mixture is stirred for 10 minutes at room temperature and then cooled to -5 ".



  At this temperature, a solution of 40.1 g of dicyclohexylcarbodiimide in 90 ml of methylene chloride is added dropwise. The mixture is stirred for 3 hours at -5 "and all night at room temperature. After the dicyclohexylurea and dicyclohexylamine hydrochloride have been filtered off, 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 dried twice with 20% sodium chloride solution and over sodium sulfate. The solution is concentrated to about 600 ml, cooled to 5 "and further dicyclohexylurea is filtered off. After evaporation to dryness, the residue is crystallized from ethyl acetate-hexane for purification.

  F. 110111 <; [aj = -8.5 "(c = 2 in dimethylformamide); Rf2 = 0.33 (on silica gel).



  45. H-Ser-Thr-OBzl, TFA
65.7 g of BOC-Ser-Thr-OBzl are dissolved in 100 ml of 90% trifluoroacetic acid and the solution is left at 20 "for one hour. It is then dripped into 1000 ml of dry ether while stirring, stirred for one hour and kept overnight at - 10 "left. The resulting precipitate is filtered off and washed three times with dry ether and dried in vacuo over caustic soda; F. 128-129; Rf, = 0.65; Rf4 = 0.50 (on silica gel).



  46. BOC-Leu-Ser-Thr-OBzl
52.5 g of H-Ser-Thr-OBzl, TFA are dissolved in 145 ml of dimethylformamide and a solution of 48.0 g of BOC-Leu-ONP is added at 0 ". 21 ml of triethylamine and 0.75 ml of glacial acetic acid are then added The reaction mixture is stirred for 3 hours at 0 "and overnight at room temperature. After adding 2.1 liters of ethyl acetate, the solution is shaken out, twice with water, twice with 0.1N hydrochloric acid, twice with 10% sodium chloride solution, eight times with 20% potassium carbonate solution, twice with 10% and once with 30% iger saline solution.

  After drying over sodium sulfate, the solution is concentrated to about 1.4 liters. The protected tripeptide of F. 114-116 "; [a] D = -14" (c = 2 in dimethylformamide) crystallizes overnight in the refrigerator; Rf2 = 0.25 (on silica gel).



  47. H-Leu-Ser-Thr-OBzl, TFA
46.1 g of BOC-Leu-Ser-Thr-OBzl are dissolved in 92.5 ml of 90% trifluoroacetic acid and left at 20 "for 1 hour. Dry ether (925 ml) is then added while stirring, and the mixture is stirred at 0" for one hour and left to stand overnight at -10 ". The resulting precipitate is filtered off, washed three times with dry ether and dried in vacuo over caustic soda. F. 168-171; Rf, = 0.80 (on silica gel).



  48. BOC-Asn-Leu-Ser-Thr-OBzl
20.8 g of BOC-Asn-OH are slurried in 208 ml of acetonitrile, 22.7 g of Woodward's reagent K are added and the mixture is stirred for 30 minutes at 20 ". Then 12.6 ml of triethylamine are added dropwise with stirring so that the internal temperature + 32 <does not exceed, cools to 20 "and stirs at this temperature for another 50 minutes. The almost clear solution is cooled to 0" and with a solution of 39.1 g of H-Leu-Ser-Thr-OBzl, which is also cooled to 0 " , TFA and 10.5 ml of triethylamine in 257 ml of dimethylformamide were added.

  The reaction mixture, which soon solidifies, is stirred overnight at room temperature, cooled to -10 "and stirred for a further 2 hours at -10. The crystalline precipitate is then suction filtered and the tetrapeptide derivative is washed once with cold acetonitrile, once with ethyl acetate and three times with water, until it is chloride-free. The product obtained is crystallized from 370 ml of dimethylformamide, 3.7 ml of glacial acetic acid and 370 ml of acetonitrile. F. 225-226; [a] D = -36 "(c = 2 in dimethylformamide); Rf6 = 0.85 (on silica gel).



  49. H-Asn-Leu-Ser-Thr-OBzl, TFA
32 g of BOC-Asn-Leu-Ser-Thr-OBzl are dissolved in 192 ml of 90% trifluoroacetic acid and left at 200 for 45 minutes. The solution is then concentrated to about 40 ml, 400 ml of ether are added while stirring and the mixture is stirred under reflux for 20 minutes at 35. The crystal suspension is then cooled to -10 "and left to stand at -10" overnight.



  The precipitate is filtered off, washed three times with ether and dried in vacuo over caustic soda. M.p. 125-127 "; Rf4 = 0.33 (on silica gel).



   50. B OC-Gly-Asn-Leu-Ser-Thr-OBzl
26.3 g of H-Asn-Leu-Ser-Thr-OBzl, TFA are in 100 ml
Dimethylformamide dissolved, cooled to 00, one after the other with
7.5 ml of triethylamine, 0.54 ml of glacial acetic acid and a solution of
14.4 g of BOC-Gly-ONP in 100 ml of dimethylformamide are added, the mixture is stirred at room temperature until the mixture solidifies and left to stand for two days. 300 ml of ethyl acetate are then added with stirring and the mixture is left to stand overnight at -10 ". The precipitate is filtered off, washed twice with ethyl acetate and twice with ether, stirred with 200 ml of water for half an hour, filtered off, washed with water and in vacuo dried. m.p. 227-228 "; [a] D = -23 "(c = 2 in dimethylformamide); Rf9 = 0.50 (on silica gel).



  51. BOC-Gly-Asn-Leu-Ser-Thr-OH
17.0 g of BOC-Gly-Asn-Leu-Ser-Thr-OBzl are dissolved in 340 ml of dimethylformamide with heating. After cooling to room temperature, 3.4 g of 10% palladium carbon are added and the mixture is hydrogenated. The reduction is complete after 4 hours. After filtering off the catalyst, the solution is concentrated in a high vacuum. Trituration with ether three times gives a pentapeptide which is uniform according to thin-layer chromatogram. F. 181-183 "; [aln = -16.5" (c = 2 in dimethylformamide); Rf, = 0.65 (on silica gel).



   52. B OC-Gly-Asn-Leu-Ser-Thr-Cys (Bzl) -N2H2-Z
9.2 g of H-Cys (Bzl) -N2H2-Z, HCl are dissolved in 300 ml of freshly distilled dimethylformamide, with 12.2 g of BOC
Gly-Asn-Leu-Ser-Thr-OH is added and the mixture is stirred at room temperature until it is dissolved. The solution is then cooled to 0, 3.28 ml of triethylamine and 4.88 g of N-hydroxysuccinimide in 100 ml of dimethylformamide are added, the mixture is cooled further to -22 "and 4.36 g of dicyclohexylcarbodiimide in 30 ml of dimethylformamide are added stirs for one hour at -22 ", then allows the internal temperature to rise gradually, and stirs for a further 3 days at room temperature, filtered off the precipitated dicyclohexylurea and evaporated to dryness in a high vacuum.

  The residue is stirred with a mixture of ethyl acetate and 5% citric acid solution, the precipitate is filtered off, washed with water, dried in vacuo, stirred with dry ether, filtered and dried in a high vacuum. F. 173-178 [ajn = -25.5 "(c = 2 in dimethylformamide); Rft = 0.21 (on silica gel).



  53. H-Gly-Asn-Leu-Ser-Thr-Cys (Bzl) -N2H2-Z, TFA
13 g of Boc-Gly-Asn-Leu-ser-Thr-cys (Bzl) -N2H2-z are dissolved in 130 ml of 90% strength trifluoroacetic acid and the solution is left to stand at 22 for 2 hours. The solution is then concentrated and the residue is stirred three times with ether and dried over caustic soda in vacuo.



  M.p. 159-161 ". Rf6 = 0.63 (on silica gel).



  54.BOC-Cys (Bzl) -Gly-Asn-Leu-Ser-Thr-Cys (Bzl)
N2H2-Z
6.69 g of BOC-Cys (Bzl) -OSU and 12.2 g of H-Gly-Asn-Leu Ser-Thr-Cys (Bzl) -N2H2-Z, 1.22 TFA are dissolved in 100 ml of dimethylformamide. From a solution of 150 mmol of triethylamine in 100 ml of dimethylformamide, enough triethylamine is added dropwise with stirring until the reaction mixture shows a pH value of 6.4 on moist 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 300 ml of ethyl acetate, then three times with a mixture of 150 ml of ethyl acetate and 30 ml of 5% citric acid solution, filtered off, dried in vacuo and crystallized from dimethylformamide-ethyl acetate.



  F. 191-193 "; [a] D = -31.5" (c = 2 dimethylformamide); Rf8 = 0.35 (on silica gel).



  55. BOC-Cys-Gly-Asn-Leu-Ser-Thr-Cys-N2H3
6 g of B OC-Cys (Bzl) -Gly-Asn-Leu-Ser-Thr-Cys (Bzl) -N2H2-Z are dissolved in 700 ml of dry liquid ammonia at 40 ". While stirring, 973 mg of sodium are obtained at the boiling point of the ammonia added in such a way that the color of the reaction mixture becomes only light blue.



  The reduction is complete after 25 minutes. The mixture is stirred for a further 10 minutes while maintaining the blue color, then 2.4 ml of glacial acetic acid are added and the mixture is evaporated to dryness in a high vacuum (about 1 mm). The residue is stirred with 12 ml of water, 3.2 ml of glacial acetic acid and 20 ml of ethyl acetate for one hour at 0, the precipitate is filtered off, washed twice with 10 ml of 1% acetic acid solution and once with 10 ml of ethyl acetate and dried in a high vacuum.



     Rf5 = 0.65 (on silica gel).



  56.BOC
EMI24.1
 N2H3
1.0 g of BOC-Cys-Gly-Asn-Leu-Ser-Thr-Cys-N2H3 is dissolved in 100 ml of dimethylformamide and 500 ml of water which is 1.23 mEq. Contains hydrogen chloride. The pH of the solution is adjusted to 6.8 with 3.7 ml of a 0.43N potassium hydroxide solution. While maintaining this pH value, 247 ml of a 0.01M potassium ferricyanide solution and 4.9 ml of a 0.43N potassium hydroxyl solution are simultaneously added dropwise over the course of 90 minutes while stirring. The mixture is stirred for a further 90 minutes at room temperature, then the pH of the solution is brought to 4.0 with 0.7 ml of glacial acetic acid. The solution is then stirred with 50 ml of Dowex-2-X8, acetate form, then with 13 ml of Dowex-SOW-X8, H + form, filtered and evaporated to dryness. The residue is dissolved in 50% t-butanol, lyophilized and dried in a high vacuum. The product contains approximately 15% potassium acetate.

  It is uniform in thin-layer chromatograms on silica gel; Rf5 = 0.63; Rf6 = 0.70; Rf7 = 0.75.



  57. H-Leu-Gly-OEt, HC1
14 g of Z-Leu-Gly-OEt [shown according to J.R.



  Vaughan & R.L. Osato, J. Am. Chem. Soc. 73, 5553 (1951)] in 150 ml of absolute ethanol, mixed with 11.5 ml of a 6.9N hydrogen chloride solution in ethanol and hydrogenated in the presence of 2.8 g of palladium-carbon (10% Pd). After 1 hour, the catalyst is filtered off with suction and evaporated in vacuo at a bath temperature. The residue is an oil and is processed further directly. Rf1 = 0.50 (on silica gel).



  58. BOC-Nle-Leu-Gly-OEt
The mixed anhydride is prepared from 4.63 g of BOC-Nle-OH, 2.82 ml of triethylamine and 1.9 ml of ethyl chloroformate in 50 ml of tetrahydrofuran as described under 1 and it is coupled with 5.05 g of H-Leu-Gly- OEt, HCl in 50 ml tetrahydrofuran. The residue is crystallized from ethyl acetate petroleum ether (40:60). F. 126-127. [a] D70 = -28.3 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf7 = 0.7.



  59. BOC-Nle-Leu-Gly-OH
3.25 g of the ester described under 55 are dissolved in 100 ml of methanol and 21 ml of 0.53N sodium hydroxide are added.



     After stirring for 1 1/2 hours, the solution is acidified and evaporated in vacuo. The residue is dissolved in water and extracted with ethyl acetate. The organic layer is dried and evaporated. The product melts at 71-72. [a] D70 = -28.4 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf7 = 0.75.



  60. H-Nle-Leu-Gly-OH
2.42 g of BOC-Nle-Leu-Gly-OH are dissolved in 24 ml of 90% strength trifluoroacetic acid as described under 16. After an hour the solution is evaporated. The product melts at 94-96. In the thin-layer chromatogram on silica gel, Rf = 0.56.



  61. BOC
EMI25.1
   NIe-Gly-OH
1 g of the hydrazide described under 56 is dissolved in 35 ml of dimethylformamide and the azide is formed as described under 13 with 0.85 ml of 3.64N hydrochloric acid in tetrahydrofuran and 0.155 ml of iso-amyl nitrite. The azide solution is added to a solution of 0.955 g of H-Nle-Leu-Gly-OH and 0.324 g of triethylamine in 25 ml of dimethylformamide. The reaction mixture is evaporated and stirred twice with ethyl acetate-citric acid solution. The product shows Rfs = 0.71 in a thin-layer chromatogram on silica gel.



  62.BOC
EMI25.2
   NIe-Leu-Gly-Thr-
Tyr-Thr-Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe
His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Al Pro-NH2
440 mg of the BOC decapeptide described under 61 and 109 mg of N-hydroxysuccinimide are dissolved in 35 ml of dimethylformamide, the solution is cooled to -22, 98 mg of dicyclohexylcarbodiimide are added and the mixture is stirred for a further 1 1/2 hours at -22 and 3 1/2 hours at 0. Then it is cooled again to -22 and 850 mg of the docosapeptide amide described under 43 are added and the pH is adjusted to 6.6 with triethylamine.

  After 7 days at 0 and 5 days at 22, the solution is evaporated to dryness in vacuo and the residue is chromatographed over silica gel in the n-butanol-glacial acetic acid-water system (4: 1: 5). The product shows in the thin-layer chromatogram on silica gel Rfs = 0.29-
EMI25.3
 Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe Ala-Ile-Glv-Val-Glv-Ala-Pro-NH, (Val8-calcitonin M).
EMI25.4




  Gly-Thr-Tyr-Thr-Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) Phe-His-Thr-Phe-Pro-Gln-thr-Ala-Ile-Gly-Val-Gly-Al Pro- NH2 are dissolved in 3 ml of ice-cold, 95% trifluoroacetic acid and the solution is warmed to 25. After 1 hour at 25, 70 ml of ice-cold ether is poured and the resulting precipitate is suction filtered. It is washed several times with ether, dried and then dissolved in 5% acetic acid.



  Then, to remove trifluoroacetate ions, it is filtered through a column of Merck ion exchanger No. II (weakly basic, acetate form). The eluate is lyophilized. The dot triacontapeptide obtained in this way is not entirely uniform according to thin-layer chromatography (aluminum oxide plates, systems 52 and 45). It can be cleaned by countercurrent distribution in the n-butanol-acetic acid-water (4: 1: 5) system. In the thin-layer chromatogram on silica gel, Rf6 = 0.45; on aluminum oxide, Rf52 = 0.53; Rf45 = 0.42.



   The starting material can be prepared as follows: 1. BOC-Val-Leu-Gly-OEt
4.35 g of BOC-Val-OH are dissolved in 50 ml of tetrahydrofuran and the mixed anhydride is formed as described in Example 1 under 58 and coupled with 5.06 g of Hleu-Gly-OEt (Example 1, 57). The product melts at 112-113. [a] D20 = -27.3 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf = 0.7.



  2. BOC-Val-Leu-Gly-OH
BOC-Val-Leu-Gly-OEt is saponified in the same way as described in Example 1 under 59. The product melts at 78. [a] D20 = ¯24.7 (c = 1 in dimethylformamide). In the thin-layer chromatogram on silica gel, Rf7 = 0.71.



  3. H-Val-Leu-Gly-OH
2.9 g of BOC-Val-Leu-Gly-OH are dissolved in 24 ml of 90% trifluoroacetic acid as described in Example 1 under 60.



  The product melts at 75. In the thin-layer chromatogram on silica gel, Rf = 0.5.
EMI25.5




   1 g of the hydrazide described in Example 1 under 56 is converted into the azide as described in Example 1 under 61 and this is condensed with 0.933 g of H-Val-Leu-Gly-OH. The product shows Rfs = 0.71 in a thin-layer chromatogram on silica gel.
EMI25.6




   Tyr-Thr-Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe
His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala
Pro-NH2
440 mg of the Docosa nentidamids described in Example 1 under 43 are described in the same way as in Example 1 under 62 with
EMI25.7
 Leu-Gly-OH condensed. The product shows Rfs = 0.29 in a thin-layer chromatogram on silica gel.



   Example 3
EMI25.8
   Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-
Ala-Ile-Glv-Val-Gly-Ala-Pro-NH2 (Leu8-calcitonin M).
EMI25.9




  Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-His Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-Val-Gly Ala -Pro-NH2 with 3.5 ml of ice-cold, 95% trifluoroacetic acid, heats the mixture to 30 after the peptide derivative has completely dissolved and lets the solution stand for 1 hour. Then it is poured into 200 ml of ice-cold ether. The fine precipitate is sucked off. after drying in vacuo over sodium hydroxide, dissolved in dilute acetic acid and filtered to remove trifluoroacetate ions over a column of Merck ion exchanger No. II. weakly basic. The eluate is lyophilized. The acetic acid salt of Leu8-calcitonin M is obtained.

 

   The starting material can be made as follows:
EMI25.10
 Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Cys (BOC) Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-V Pro-NII2 100 mg N- Hydroxysuccinimide, 120 mg of dicyclohexylcarbodiimide and 6 ml of dimethylformamide are stirred for 3 hours at 45 ". The mixture is then poured into 300 ml of ether and the precipitate is filtered off. The crude product is purified by countercurrent distribution in the methanol-buffer-chloroform-carbon tetrachloride 11 system: 3: 6: 7; (buffer as in example 1); K = 0.7.



   The decapeptide used as starting material is prepared as described in British Patent No. 1,287,601 for the corresponding Met8 decapeptide. One starts with H-Leu-Gly-OMe, reacts with Z-leucine-N-hydroxysuccinimide ester, hydrogenates the Z-Leu-Leu-Gly-OMe obtained, and condenses the H-Leu-Leu-Gly-OMe, HCI with it TRI-Cys (TRI) -OH as described under 9) in Example 1 of the above-mentioned British patent and proceeds with the tetrapeptide derivative obtained analogously to that described in the above-mentioned patent under 10 to 18.



   Example 4
EMI26.1
   Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-
Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 (Nva8-Calcitonin M).
EMI26.2




  Nva-Leu-Gly-Thr (tBu) -Tyr (tBu) -Thr (tBu) - (iln -Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro Gln-Thr (tBu) -Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 are mixed with 0.2 ml of 95% trifluoroacetic acid and left to stand for 1 hour at 30. 5 ml of ether are then added, the Precipitation is washed off with a little ether and dried. The dot-triacontapeptide amide obtained as a trifluoroacetic acid salt can be converted into the acetate by ion exchangers.



   The starting material can be prepared as follows: 1. Z-Nva-Leu-Gly-OMe:
4.3 g of ZL-Nva-2,4,5-trichlorophenyl ester (J. chem. Soc. (C) 1968, 715) and 2.2 g of H-Leu-Gly-OMe are mixed with 15 ml of dimethylformamide and the mixture is stirred Reaction mixture for 48 hours at 35 "Then it is diluted with a lot of chloroform, cooled in ice and shaken three times with 5% ice-cold potassium carbonate solution and three times with ice-water.



  The chloroform solution is dried with sodium sulfate and then evaporated to dryness, finally at 0.1 mm Hg to remove the remaining dimethylformamide. The residue is then dissolved in chloroform and chromatographed on a column of silica gel prepared in chloroform, using chloroform + methanol (1: 1) (1 liter each) for the elution of a linear, graded chloroform. 100 ml fractions are collected, evaporated to dryness and the purity of the residue is checked by thin-layer chromatography on silica gel plates and the pure fractions are combined. The product has Rf = 0.4 in toluene-acetone (1: 1).



  2. H-Nva-Leu-Gly-OMe
1.7 g of the above tripeptide are dissolved in methanol and hydrogenated with vigorous stirring in the presence of 400 mg of palladium carbon (10% Pd). After the uptake of hydrogen has ended, the catalyst is filtered off and the eluate is evaporated to dryness. A colorless resin is obtained which is used directly for further reaction.



  3. TRI-Cys (TRI) -Nva-Leu-Gly-OMe
40 ml of acetonitrile are added to 2.5 g of TRI-Cys (TRI) -OH and 1.1 g of the above H-Nva-Leu-Gly-OMe, and 1.3 g of dicyclohexylcarbodiimide are then added with stirring. After stirring for 20 hours at 27, the precipitated product is filtered off with suction and extracted with 1 liter of chloroform. The eluate is then evaporated to dryness and the residue is triturated with petroleum ether. After the petroleum ether solution has been poured off, the insoluble product is dried and 3 ml of petroleum ether is reprecipitated for purification. The tetrapeptide derivative is obtained as an amorphous, solid powder. It shows Rf = 0.62 in chloroform-methanol (98: 2) by thin layer chromatography on silica gel plates.



  4. H-Cys (TRI) -Nva-Leu-Gly-OMe
980 mg TRI-Cys (TRI) -Nva-Leu-Gly-OMe are dissolved at 30 in 25 ml of 75% acetic acid and left to stand for 1 hour. It is then evaporated to dryness and the residue is triturated several times with ether. The acetate of H-Cys (TRI) -Nva-Leu-Gly-OMe, which is insoluble in ether, is dried in vacuo at 35 and further processed directly.



  5. DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) -Nva-Leu-Gly-OMe
750 mg of DPC-Ser (tBu) -Thr (tBu) -NHNH2 are dissolved in 6 ml of dimethylformamide, cooled to 200 and 3.5 ml of 1N hydrogen chloride in ethyl acetate are added. 0.16 ml of t-butyl nitrite are then added and the reaction mixture is left to stand at 100 for 15 minutes. The solution, cooled to 100, of 500 mg of H-Cys (TRI) -Nva-Leu-Gly-OMe in 4 ml of dimethylformamide and 0.7 ml of triethylamine is then added and the mixture is left to stand for 30 minutes at 100 and 18 hours at 0.



  Then it is evaporated in a high vacuum at a bath temperature to a volume of about 5 ml and the product is precipitated by adding ice water. After thorough trituration, it is filtered off with suction, washed with ice water and the crude hexapeptide derivative is dried over calcium chloride in a desiccator. For purification, it is reprecipitated three times from ethyl acetate cyclohexane. The product shows Rf = 0.47 in toluene-acetone (7: 3) by thin layer chromatography on silica gel plates.



  6. H-S er (tBu) -Thr (tBu) -Cys (TRI) -Nva-Leu-Gly-OH
430 mg of DPC-Ser (tBu) -Thr (tBu) -Cys (TRI) Nva-Leu-Gly-OMe are dissolved in 10 ml of 80% acetic acid, left to stand for 7 hours at 28 ", evaporated on a rotary evaporator at 35" bath temperature to dryness, dry for 3 hours at 35 and 0.01 torr, then dissolve in 30 ml of methanol are 3 ml
Add 1.0N sodium hydroxide solution, stir for 1 hour at 28, then add 2 ml of glacial acetic acid and evaporate to dryness on a rotary evaporator at a bath temperature of 35. The residue is triturated with ice water, the pH being adjusted to 6-7, suction filtered and washed with ice water and dried in a desiccator over calcium chloride.



   7. BOC-Cys (TRI) -Gly-Asn-Leu-S er (tBu) -Thr (tBu) -Cys (TRI) -Nva-Leu-Gly-OH
270 mg of BOC-Cys (TRI) -Gly-Asn-Leu-
NH-NH2 with 4 ml of dimethylformamide, cools to -20 "and adds 1.2 ml of 1N hydrogen chloride in ethyl acetate. Then 0.044 ml of t-butyl nitrite is added and the mixture is left to stand at -10" for 15 minutes. A solution of 230 mg H-Ser (tBu) -Thr (tBu) -Cys (TRI) -Nva-Leu, cooled to -10 ", is then added
Gly-OH in 4 ml of dimethylformamide, 1 ml of water and
0.25 ml of triethylamine. It is left at 0 for 18 hours, then evaporated at 0.01 torr. and 35 bath temperature to a small volume and the decapeptide precipitates by adding water. The precipitate is crushed, suction filtered and dried over calcium chloride in a desiccator.

  The powder is suspended in 7 ml of methanol at 0, ground thoroughly and then filtered off with suction.
EMI27.1




   Leu-Gly-OH
105 mg of the decapeptide obtained under 7 are dissolved in 20 ml of dimethylformamide and this solution is added dropwise over the course of 1 hour to the vigorously stirred solution of 150 mg of iodine in 100 ml of methanol. The mixture is stirred for a further 1 hour, then decolorized at 0 by adding 1N sodium thiosulphate solution, then evaporated to a volume of about 20 ml on a rotary evaporator and precipitated by adding 300 ml of ether.



  It is suction filtered, dried in vacuo at 30 and then the powder is triturated with water. It is filtered off with suction and dried over calcium chloride in a desiccator.



  9. Z-Asp (OtBu) -Phe-OCH3
30.3 g of Z-Asp (OtBu) -ONP and 18.3 g of H-Phe-OCH3, HCl are dissolved together in 150 ml of dimethylformamide, and 11.8 ml of triethylamine are added dropwise to the clear solution.



  The resulting suspension is stirred for 20 hours at room temperature, whereupon it turns deep yellow. It is then concentrated in vacuo to about 100 ml and extracted three times in 1 liter of ethyl acetate / chloroform (4: 1) with 5% citric acid, 19 ml with about 2N sodium carbonate and with saturated saline solution until a neutral reaction is obtained. The crude product, a yellow oil, is treated with activated charcoal in ether and, after inoculation, crystallized from 650 ml of ether / hexane (1: 1) in the refrigerator. Colorless needles with a diameter of 74.5-76.5 are formed. In the thin-layer chromatogram on silica gel, the Rf value in the chloroform-methanol system (95: 5) = 0.74 in chloroform-acetone (75:27) = 0.65.



  10. H-Asp (tBu) -Phe-OCH3
48.6 g of Z-Asp (OtBu) -Phe-OCH3 are decarbobenzoxylated in 700 ml of methanol after the addition of 33.5 ml of 3N hydrogen chloride in dioxane and 5 g of 10% palladium catalyst on charcoal in a shaking duck at room temperature.



  After the uptake of hydrogen has ended, it is filtered and evaporated. 38.7 g of a white foam are obtained. In the thin-layer chromatogram on silica gel in chloroform methanol (9: 1), Rf = 0.60; in chloroform-acetone (1: 1), Rf = 0.58; Rfl02E = 0.42. The product is used for the subsequent condensation without further purification.



  11. Z-Gln-Asp (OtBu) -Phe-OCH3
38.6 g of the H-Asp (OtBu) -Phe-OCH3, HCI obtained are dissolved with 42.0 g of Z-Gln-ONP in 170 ml of dimethylformamide to give a clear, slightly yellow solution and, with stirring, with 13.9 ml of triethylamine slowly moved. An orange suspension is formed which is stirred for 24 hours at a bath temperature of 30-35. During this time, a further 40 ml of dimethylformamide and also 1.39 ml of triethylamine are added.



   For work-up, the batch is dissolved in 4 liters of chloroform and in a 20-stage countercurrent distribution apparatus (phase volume: lower phase 400 ml, upper phase 200 rr.l per vessel) with 1 liter of 5% citric acid solution, 400 ml of saturated saline solution, 6 liters of about 2n Soda and 2.8 liters of saturated saline solution. After drying and evaporation, the tripeptide derivative slowly crystallizes from 1.8 l of ethanol in the refrigerator. Z-Gln-Asp (OtBu) -Phe-OCH3 from F. 1861880 is obtained.



   The following Rf values are obtained in the thin-layer chromatogram on silica gel: in chloroform-methanol (9: 1): Rf = 0.39; in chloroform-acetone (1: 1): Rf = 0.24; Rf102E = 0.69, Rf89 = 0.46, Rf43A = 0.65 [a] D20 = -28 # 1 (c = 1.3% in dimethylformamide).



   12. H-Gln-Asp (OtBu) -Phe-OCH3
7.55 g of Z-Gln-Asp (OtBu) -Phe-OCH3 are dissolved in 400 ml of methanol, 4.1 ml of 3N hydrogen chloride in dioxane are added and the mixture is hydrogenated in the presence of 2 g of palladium carbon (10% Pd). After filtering off the catalyst with suction and evaporation, H-Gln-Asp (OtBu) -Phe-OCH3, HCI is obtained in the form of a colorless foam. The following Rf values are obtained in the thin-layer chromatogram on silica gel: in chloroform-methanol (9: 1); Rf = 0.13; in chloroform-acetone (25:75): Rf = 0.14; Rf102E = 0.22.



   13. Z-Thr (tBu) -Gln-Asp (OtBu) -Phe-OCH3
The entire amount of the hydrochloride of 12 is dissolved together with 7.4 g of Z-Thr (tBu) -OSU in 14 ml of dimethylformamide at room temperature, and 1.72 ml of triethylamine are added dropwise to this solution while cooling in an ice bath. The brownish suspension is then stirred at room temperature for 20 hours. After the usual work-up in a lot of ethyl acetate (washing three times each with 5% citric acid and about 2N sodium carbonate, washing neutral with saturated sodium chloride solution, drying over sodium sulfate and evaporation in vacuo at 30-40), the crude product is treated in ethanol with activated charcoal and made from 90 ml Ethanol crystallizes in the refrigerator. F. 155-161 ".



   The following Rf values are found in the thin-layer chromatogram on silica gel: in chloroform-methanol (9: 1): Rf = 0.52; in cyclohexane-acetone (3: 7): Rf = 0.48; Rf89 = 0.48, Rf12IA = 0.76; [aj¯20 = -4 +0.5 (c = 2.3% in dimethyllormamide).



   14. H-Thr (tBu) -Gln-Asp (OtBu) -Phe-OCH3
478 mg of Z-Thr (tBu) -Gln-Asp (OtBu) -Phe-OCH3 are neutralized in 150 ml of methanol with 100 mg of palladium-carbon (10%) hydrogenated at room temperature. 395 mg of a colorless foam of H-Thr (tBu) -Gln-Asp (OtBu) -Phe OCH3, which is used for the subsequent condensation without further purification, are obtained.



   The following Rf values are obtained in the thin-layer chromatogram on silica gel: in chloroform-methanol (1: 1): Rf = 0.75; in chloroform-methanol (9: 1): Rf = 0.17; in acetone Rf = 0.18; Rf102E = 0.23; Rf89 = 0.12.



   15. Z-Tyr (tBu) -Gln-Asp (OtBu) -Phe-OCH3
687 mg of Z-Tyr (tBu) -OH-dicyclohexylammonium salt are extracted by shaking with aqueous citric acid in chloroform, and 0.139 ml of N-methylmorpholine is added to the free acid obtained, a clear oil, in 6.5 ml of tetrahydrofuran. At -22, 0.170 ml of isobutyl chloroformate is added and the mixture is stirred for half an hour at -22 to -10 ". The above-described H-Thr (tBu) Gln-Asp (OtBu) -Phe-OCH3 is then dissolved in 15 ml of tetrahydrofuran and Pre-cooled dropwise and rinse with 5 ml of the same solvent.After 1/2 hour at 100, the mixture is stirred for 15 hours at room temperature.Then it is concentrated in vacuo and worked up in ethyl acetate as usual (cf.

 

  13). The crude product is dissolved in 15 ml of ethyl acetate, precipitated with 40 ml of ether and then crystallized from methanol in a refrigerator. Short, thick needles that weather when dried in a high vacuum at 50. F. 169-173.



   The following Rf values are obtained in the thin-layer chromatogram on silica gel: in chloroform-methanol (9: 1) Rf = 0.46; in chloroform-methanol (1: 1) Rf = 0.95; in chloroform-acetone (1: 1) Rf = 0.44: Rf89 = 0.61; RfAcetOn = 0.68, Rf102E = 0.73: [a] D21 = -54 # 0.5 (c = 2.0% in dimethylformamide).



  16. H-Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe-OCH3
2.36 g of Z-Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe OCH3 are hydrogenated in 450 ml of methanol with 500 mg of 10% strength palladium carbon as usual at room temperature. A colorless foam is obtained which is uniform according to thin layer chromatography and which is used as such.



  In the thin-layer chromatogram on silica gel, the following
Rf values obtained: in chloroform-methanol (95: 5)
Rf = 0.22; Rf89 = 0.42.



   17. Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) - Phe-OCH3
The product of 16 is dissolved together with 1.48 g of Z-Thr (tBu) -OSU in 3 ml of dimethylformamide and stirred for 21 hours at room temperature. After diluting the
Reaction solution with a lot of ethyl acetate is worked up as usual (cf. 13). The crude product is dissolved in 30 ml of ethyl acetate
Methanol (9: 1) dissolved warm and after cooling in an ice bath with
80 ml of ether like. The product is obtained as a colorless, amorphous powder with a melting point of 146-148 ". The following Rf values are obtained in a thin-layer chromatogram on silica gel: in chloroform-methanol (9: 1): Rf = 0.55; in chloroform, acetone (1 : 1): Rf = 0.60; Rf89 = 0.43; [a] D21 = + 6 + 0.5 (c = 2.0 in dimethylformamide).



   18.Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -
Phe-NH-NH2 1.91 g of Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe-OCH3 are dissolved in 80 ml of methanol and mixed with
8 ml of hydrazine hydrate mixed. After standing at room temperature for 22 hours, the precipitated product is isolated and dried at 60 in a high vacuum. The microcrystalline hydrazide from F. 226-229 "is obtained (decomposition).



   The following Rf values are found in the thin-layer chromatogram on silica gel: In chloroform-methanol (9: 1)
Rf = 0.32; in cyclohexane-acetone (3: 7) Rf = 0.23; Rf89 =
0.34. [a] D20 = +4 + 1 (c = 1.0 in dimethylformamide).



   19. Z-Lys (BOC) -Phe-OMe
25.0 g of Z-Lys (BOC) -ONP and 10.7 g of H-Phe-OMe, HCI in 70 ml of dimethylformamide are mixed with 6.9 ml of triethylamine while stirring at room temperature
Stirred for 18 hours. After dilution with ethyl acetate, it is washed free of nitrophenol with potassium carbonate solution, with
0.1m citric acid and water extracted, dried over sodium sulfate and evaporated to dryness in vacuo. The protected dipeptide of F. 78800 crystallizes from ethyl acetate-hexane. Rf = 0.45 in thin-layer chromatography on silica gel in the system chloroform-acetone (8: 2).



   20. Z-Lys (BOC) -Phe-NH-NH2
27 g of the above dipeptide methyl ester are dissolved in 135 ml of warm methanol, 25 ml of hydrazine hydrate are added at room temperature and the mixture is left to stand for 16 hours.



   The crystals are mixed with 135 ml of water, suction filtered and washed well with water. F. 173-174 "after recrystallization from methanol-water. Rf = 0.3 in the thin-layer chromatogram on silica gel in the chloroform-methanol system 5).



  21. Z-Lys (BOC) -Phe-His-OMe
5.4 g of Z-Lys (BOC) -Phe-NH-NH2 in 40 ml of dimethylformamide are mixed with 6.8 ml of 3.66N HCl in dioxane and then with 1.5 ml of t-butyl nitrite at -16 " Minutes at -10 "to -15" 3.5 ml of triethylamine are added.



  3.64 g of H-His-OMe, 2 HCl are added in solid form, then 4.2 ml of triethylamine are added dropwise. The mixture is allowed to warm to 0 within 1 hour, a pH of about 7 being set by adding a total of 0.8 ml of triethylamine.



  After stirring overnight at 0, it is poured into 250 ml of water and the greasy product is obtained in powder form by trituration with water. In the thin-layer chromatogram on silica gel in chloroform-methanol (9: 1), Rf = 0.4.



   22. H-Lys (BOC) -Phe-His-OMe
6.8 g of Z-Lys (BOC) -Phe-His-OMe in 140 ml of methanol are hydrogenated in the presence of 1 g of 10% Pd-carbon.



  After the hydrogenation has ended, the catalyst is filtered off, the filtrate is evaporated to dryness and the residue is immediately processed further.



   23. Z-Asn-Lys (BOC) -Phe-His-OMe
5.4 g H-Lys (BOC) -Phe-His-OMe and 4.5 g Z-Asn
ONP are stirred in 20 ml of dimethylformamide for 20 hours at room temperature. The peptide derivative is precipitated by adding ethyl acetate, filtered off and washed with ether. After recrystallization from methanol it melts
Product at 182-183. In the thin layer chromatogram is
Rf100 = 0.57 (on silica gel). [α] 20 D = -28 (c = 1 in dimethylformamide).



   24. Z-Asn-Lys (BOC) -Phe-His-NH-NH2
3.97 g of Z-Asn-Lys (BOC) -Phe-His-OMe are dissolved in 8 ml of warm dimethylformamide and 12 ml of methanol. 2.5 ml of hydrazine hydrate are added to the solution, which is still warm, and it is left to stand for 20 hours at room temperature. The peptide hydrazide is precipitated by adding water, filtered off and washed with water
Washed hydrazine-free. The product is recrystallized from ethanol, F. = 200-201 ". In thin-layer chromatography on silica gel, Rf43C = 0.5.



   25. H-Phe-Pro-OH
Z-Phe-Pro-OH is converted into the free dipeptide by catalytic reduction (Pd-carbon) in methanol-water (4: 1). After concentrating the hydrogenation solution freed from the catalyst to a small volume by adding acetone, this is obtained in crystallized form, namely as a dipeptide
Monohydrate from F. 125-128.



   26. Z-Thr (tBu) -Phe-Pro-OH
20.2 g of Z-Thr (tBu) -OSU, 13.3 g of H-Phe-Pro-OH (monohydrate) and 6.54 ml of triethylamine are dissolved in 80 ml of dimethyl formamide, left to stand overnight at about 20 and then concentrated in a high vacuum until a sticky mass was formed. This is dissolved in 500 ml of ethyl acetate and washed five times with 100 ml of 5% tartaric acid solution each time and then with water until neutral. The organic
Phase is concentrated to dryness and the remaining solid foam is pulverized and dried in a high vacuum at 40 ge.

  Twice reprecipitation from ethyl acetate-petroleum ether gives 13.3 g of amorphous, chromatographically pure
Tripeptide derivative with an unsharp melting range at about 75-85 ". In the thin-layer chromatogram on silica gel, Rf115 = 0.68; Rfl2lA = 0.57.

 

   27. Z-Ile-Gly-OMe
2.23 g of Z-Ile-OH-dicyclohexylammonium salt are in
Suspended ethyl acetate and acidified with 0.2m citric acid. The ethyl acetate solution obtained is washed neutral, dried and evaporated to dryness. The residue is dissolved in 15 ml of acetonitrile to become the solution
750 mg of H-Gly-OMc, HCl were added and 0.84 ml of triethylamine were added at 0 while stirring. After 10 minutes, 1.24 g are added
Dicyclohexylcarbodiimide and stirred overnight at 0 "
Precipitation is filtered off and the filtrate is evaporated to dryness, the residue is taken up in 30 ml of ethyl acetate and filtered. The ethyl acetate solution is washed with 0.2m citric acid and saturated sodium bicarbonate solution, dried and concentrated in vacuo to about 10 ml.

  After adding 25 ml of hexane, the protected dipeptide crystallizes out, mp 120-122 ". Rf = 0.53 in the chloroform-methanol system (95: 5) in the thin-layer chromatogram on Slicagel.



  28. H-Ile-Gly-OMe
3.36 g of Z-Ile-Gly-OMe are dissolved in 100 ml of methanol and 10 ml of 1N hydrochloric acid and hydrogenated in the presence of 0.5 g of Pd-carbon (10%). After filtering off the catalyst, the solvent is completely evaporated. The foam obtained is uniform in the thin-layer chromatogram on silica gel; Rf = 0.26 in chloroform-methanol (95: 5).



  29. Z-Ala-Ile-Gly-OMe
2.39 g of the above dipeptide ester hydrochloride and 3.78 g of Z-Ala-ONP in 40 ml of dimethylformamide are mixed with 1.4 ml of triethylamine while stirring, and the resulting suspension is stirred at room temperature overnight.



  After dilution with ethyl acetate, it is washed free of nitrophenol with dilute potassium carbonate solution, then washed with 0.1M citric acid and water. A part of the tripeptide derivative remains undissolved during the shaking out; it is filtered off. The ethyl acetate solution is completely evaporated after drying. The residue also consists of pure product. F. 1901910, Rf = 0.5 in the thin-layer chromatogram on silica gel in the chloroform-methanol system (95: 5).



  30. H-Ala-Ile-Gly-OMe
2.0 g of Z-Ala-Ile-Gly-OMe are dissolved in 40 ml of methanol with gentle heating, then hydrogenated in the presence of 300 mg of Pd-charcoal (10%). When the hydrogenation has ended, the catalyst is filtered off and the filtrate is evaporated completely to dryness. The residue, which is uniform in the thin layer chromatogram, is processed further immediately.



  31. Z-Thr (tBu) -Ala-Ile-Gly-OMe
1.36 g of H-Ala-Ile-Gly-OMe and 2.5 g of Z-Thr (tBu) -OSU are stirred in 3 ml of dimethylformamide for 20 hours at room temperature. The tetrapeptide derivative is precipitated by ether, filtered off and washed with ether. After recrystallization from ethanol, F. = 229-2300. [a] D20 = -43 "(c = 1 in methanol). Rf = 0.55 in the system chloroform methanol (95: 5) on silica gel.



  32. H-Thr (tBu) -Ala-Ile-Gly-OMe
5.66 g of the above carbobenzoxy compound are dissolved in 400 ml of warm methanol and hydrogenated in the presence of 1 g of Pd-charcoal (10%). After filtering off the catalyst, the methanol is evaporated at 40 in vacuo. The solid residue is processed further immediately. Rf = 0.2 in the chloroform-methanol (95: 5) system on silica gel.



  33. Z-Gln-Thr (tBu) -Ala-Ile-Gly-OMe
4.6 g of the H-Thr (tBu) -Ala Ile-Gly-OMe described under 32 in 30 ml of dimethylformamide are mixed with 3.5 g of Z-Gln-ONP and stirred at room temperature until the mixture solidifies. After standing overnight, it is diluted with ether, the precipitate is filtered off and washed with ether to remove nitrophenol. The protected pentapeptide shows in the thin-layer chromatogram on silica gel Rf = 0.14 in the chloroform-methanol system (95: 5). Q .:> 250.



  34. H-Gln-Thr (tBu) -Ala-Ile-Gly-OMe, HCI
14.4 g of the pentapeptide derivative described under 33 are suspended in 800 ml of 80% methanol and heated to 50 for some time. The suspension is cooled to room temperature, 20.8 ml of hydrochloric acid and 3 g of Pd-charcoal (10% mg) are added and the mixture is hydrogenated until the uptake of hydrogen has ended and the starting material has dissolved.



  After filtering off the catalyst, the filtrate is evaporated in vacuo at 40 and the residue is dehydrated by evaporation twice with dimethylformamide in a high vacuum. The residue is used without further purification. Rfloo = 0.33 in the thin-layer chromatogram on silica gel.



  35. Z-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-OMe
12.0 g of the pentapeptide methyl ester hydrochloride described under 34 are dissolved in 80 ml of dimethylformamide. 13.3 g of Z-Thr (tBu) -Phe-Pro-OH and 5.75 g of N-hydroxysuccinimide are added one after the other with stirring at room temperature, then 2.76 ml of triethylamine and 6.2 g of dicyclohexylcarbodiimide are added at 0. Stir at 00 until the mixture becomes thick, then leave at 0 overnight. After concentration in a high vacuum to about 50 ml, the product is precipitated with 300 ml of ether. The isolated material is washed with 0.05 M citric acid and water and dried at 40 in a high vacuum. It is purified by recrystallization from about 11 isopropanol.

  18.2 g of the protected octapeptide derivative are obtained. Rfôg = 0.27 in the thin layer chromatogram on silica gel.



  36. Z-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-OH
10.9 g of the octapeptide methyl ester described under 35 are dissolved in 190 ml of 90% methanol with heating to 70 ". After cooling to room temperature, 30 ml of 1N sodium hydroxide solution and 10 minutes later 160 ml of water are added in small portions. Then it is filtered clear The product is precipitated from the filtrate by pouring it into 600 ml of 0.05N ice-cold hydrochloric acid. The precipitate is filtered off and washed neutral with water. The product, which is uniform in the thin-layer chromatogram on silica gel (Rfloo = 0.45) can be recrystallized from methanol-water.



  37. H-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-OH
3.6 g of the Z-Thr (tBu) -Phe-Pro Gln-Thr (tBu) -Ala-Ile-Gly-OH obtained under 36 are dissolved in 100 ml of 80% acetic acid and the solution is in the presence of 0, 5 g palladium carbon (10% Pd) hydrogenated. When the uptake of hydrogen has ended, the catalyst is suction filtered and the solution is evaporated to dryness. The acetic acid salt of the octapeptide derivative obtained as a colorless firn is dried in a high vacuum. In the thin-layer chromatogram on silica gel, Rfloo = 0.21.



  38. Z-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro-Gln-
Thr (tBu) -Ala-Ile-Gly-OH
11.25 g of Z-Asn-Lys (BOC) -Phe-His-hydrazide, dissolved in 65 ml of dimethylformamide, are mixed with 8.4 ml of 4.2N hydrogen chloride in dioxane at -20 to 250 in 2 minutes. Subsequently, at -15 to -20 ", 2.1 ml of tert. Butyl nitrite are added and left to stand for 15 minutes at -15". After cooling to -20 ", 4.8 ml of triethylamine are added, then a solution of 9.0 g of the product described under 37 in 210 ml of 90% strength dimethylformamide. The internal temperature is kept at 150 by vigorous cooling.

 

  In the course of an hour, the mixture is warmed to 0, the pH being kept at 7-8 by the occasional addition of triethylamine. A total of 3.5 ml of triethylamine are added. After stirring overnight at 0, ether is poured into 3 liters, the flaky precipitate is filtered off and washed twice with ether and once with water. The crude product is dissolved in 500 ml of warm methanol and reprecipitated by pouring it into 1.5 1 of 1% strength acetic acid. The product which is filtered off and washed twice with water is reprecipitated again in the same way. Rftoo = 0.33 (on silica gel plates).



  39. H-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro-Gln
Thr (tBu) -Ala-Ile-Gly-OH
1.7 g of the above protected dodecapeptide are dissolved in 100 ml of 80% acetic acid and hydrogenated as usual in the presence of 0.5 g of palladium carbon (10% Pd). After the catalyst has been filtered off, it is concentrated strongly at 30 in a high vacuum and the residue is lyophilized with tert-butanol. The product, which is obtained in quantitative yield and is uniform in the thin layer chromatogram (Rfloo = 0.1 on silica gel), is processed further immediately.



  40. Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -
Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro-Gln
Thr (tBu) -Ala-Ile-Gly-OH
1.73 g of the peptide derivative described under 18 are dissolved in 10 ml of dimethylformamide at 50. After cooling to -20 ", 0.9 ml of 4.2N hydrogen chloride in dioxane are added dropwise. 0.22 ml of tert-butyl nitrite is then added at 150 and allowed to react for 15 minutes at -15". It is then cooled again to -20 "and allowed to add dropwise 0.53 ml of triethylamine and then a solution of 1.6 g of the peptide derivative described under 39 in 40 ml of 90% strength dimethylformamide.



  The temperature is increased to 0 within 1 hour, the pH being kept at 7-8 by adding triethylamine in portions. A total of 0.25 ml of triethylamine are added. After a further 15 hours of stirring at 0, the product is precipitated by pouring it into ether, the precipitate is filtered off and washed with ether and water. For cleaning, it is reprecipitated once from dimethylformamide / ethyl acetate and once from dimethylformamide 0.02N hydrochloric acid. The pure material shows in the thin layer chromatogram on silica gel Rfloo = 0.40.



  41. Z-Ala-Pro-NH2
2.28 g of H-Pro-NH2 and 7.57 g of Z-Ala-ONP are dissolved in 20 ml of dimethylformamide and the yellow solution becomes
Left to stand at room temperature for 18 hours. It is then evaporated to dryness in a high vacuum, ether is added to the residue and the resulting powder is thoroughly triturated. After suction filtration and drying, 5.5 g of Z-Ala Pro-NH2 of F. 167.5-168.5 "are obtained. [A] D20 = 740 (c = 1 in methanol).



  42. Z-Gly-Ala-Pro-NH2
27.1 g of the above dipeptide derivative are dissolved in 425 ml of ethanol and, after addition of 85 ml of 1.0N aqueous hydrochloric acid, hydrogenated in the presence of 4.25 g of palladium-carbon (10% Pd). After the hydrogenation has ended, the solution is evaporated at 40-50 "bath temperature in vacuo.



  The residue is dissolved in 40 ml of dimethylformamide at 40 "and the solution is cooled to 20. Then 29.1 g of Z-Gly-ONP are added and, after dissolving, 11.2 ml of absolute triethylamine are added dropwise with stirring over the course of 45 minutes The suspension is evaporated at 40 and 0.01 torr and the residue is distributed between 600 ml of water and 300 ml of ether. The aqueous layer is mixed twice with 300 ml of ether each time and the ether layer is twice with 300 ml of water each time The aqueous fractions are evaporated together in vacuo at 40-50 and freed from water several times by adding chloroform and evaporation.

  The well-dried residue is taken up in 500 ml of ethyl acetate at 40-50 ", insoluble triethylamine hydrochloride is filtered off and 10 ml of ether are added to the filtrate at 30-40", whereupon crystallization occurs.



  After standing for about 20 hours at + 5 to + 10, the crystals are isolated, washed and dried. F .: 105-106.



  The product contains 3% triethylamine hydrochloride. It can be implemented further in this form. For purification, 5.0 g of the above crude crystals are dissolved in 10 ml of water and, after the addition of 20 ml of methylene chloride, 15 ml of saturated potassium carbonate solution are added. The organic layer is separated, extracted with 10 ml of saturated potassium carbonate solution, the aqueous layer is extracted with 15 ml of methylene chloride. The combined methylene chloride solutions are dried using anhydrous sodium sulfate and evaporated. The residue is crystallized from 50 ml of ethyl acetate.

  4.4 g of tripeptide derivative, mp 109-112 "are obtained. Crystallization from acetone-methanol ether (10: 4: 6) gives crystals of mp 144.5-145.5" (crystal polymorphism). [a] D20 = -93 "(c = 1.0 in methanol). On thin layer chromatography on silica gel in chloroform + methanol (8: 2), Rf = 0.38.



  43. H-Gly-Ala-Pro-NH2
18.8 g of the crude tripeptide amide derivative obtained under 42 are dissolved in 400 ml of dimethylformamide and hydrogenated in the presence of 1.0 g of palladium-carbon (10So Pd).



  After the hydrogenation has ended, it is filtered and, after brief degassing, the solution is used in the next stage.



  44. Z-Val-Gly-Ala-Pro-NH2
20.1 g of Z-Val-p-nitrophenyl ester are added to the tripeptide amide solution obtained under 43 (550 ml) and the mixture is left to stand at room temperature for 18 hours. It is then evaporated to dryness at 50-60 "and 0.01 Torr, the residue is triturated with 300 ml of ether and filtered. The filter residue is dried and stirred in 210 ml of absolute ethanol for 15 minutes at 70-80", cooled to 0 and filtered. The residue is crystallized from a mixture of 170 ml of absolute tetrahydrofuran, 25 ml of water and 110 ml of ether. F. = 209-211 ".



  Rf on silica gel plates = 0.42 in chloroform-methanol (8: 2).



  45. H-Val-Gly-Ala-Pro-NH2
1.1 g of Z-Val-Gly-Ala-Pro-NH2 are dissolved in 50 ml of 80% methanol and the solution is hydrogenated in the presence of 0.3 g of palladium-carbon (10So Pd). After the uptake of hydrogen has ended, the catalyst is filtered off with suction, the solution is evaporated and the residue is dried in a high vacuum at a bath temperature. The tetrapeptide H-Val-Gly-Ala-Pro-NH2 is obtained as a colorless substance, Rf value = 0.20 (silica gel plates, chloroform / methanol system = 1: 1).

 

  46. Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) - Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro-Gln-
Thr (tBu) -Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2
800 mg of the peptide derivative described under 40, 500 mg of H-Val-Gly-Ala-Pro-NH2 and 170 mg of N-hydroxysuccinimide are dissolved in 10 ml of dimethylformamide, concentrated to about half in a high vacuum and mixed with 250 mg of dicyclohexylcarbodiimide. After stirring overnight at room temperature, it is precipitated with ether and the material is isolated. Purification is carried out by Craig partitioning in a mixture of methanol-buffer (as in Example 1) -chloroform-carbon tetrachloride (10: 3: 5: 6), K = 0.29. The pure product isolated from the distribution shows in the thin-layer chromatogram Rf1o7 = 0.62 (on silica gel plates).



  47. H-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -
Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro-Gln
Thr (tBu) -Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2
300 mg of the above peptide derivative are dissolved in 30 ml of 80% acetic acid and hydrogenated in the presence of 100 mg of palladium-carbon (10% Pd). After the decarbobenzoxylation is complete, the catalyst is filtered off, the filtrate is strongly concentrated in a high vacuum at 30 and the residue is lyophilized from tert-butanol. The residue is dissolved in 10 ml of methanol, made weakly alkaline by adding sodium bicarbonate and precipitated by dropping into 0.1N sodium carbonate. The isolated product is immediately reprecipitated again.

  In the thin-layer chromatogram on silica gel, Rfloo = 0.34.
EMI31.1
 Leu-Gly-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe Pro-Gln-Thr (tBu) -Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2
EMI31.2
   Nva-Leu-Gly-OH, 35 mg H-Thr (tBu) -Tyr (tBu) -Thr (tBu)
Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) -His-Phe-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-Val-Gly-Ala Pro-NH2 , 7 mg of N-hydroxysuccinimide, 8 mg of dicyclohexylcarbodiimide and 0.5 ml of dimethylformamide are stirred for 4 hours at 45 ". Then 15 ml of ether are added and the fine precipitate is filtered off with suction.

  For purification, the crude product is multiply distributed over 35 stages in the methanol-buffer-chloroform-carbon tetrachloride solvent system (11: 3: 6: 7; buffer as in Example 1.



      Reieniel S
EMI31.3
   Thr-Gln-Asp-Phe-Asn -Lys-Phe-His-Thr -Phe-Pro-Gln-Thr-
EMI31.4
   Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly Val-Gly-Ala-Pro-NH2 in 50 ml ice-cold, concentrated hydrochloric acid, stir for 10 minutes at 0, then add 300 ml ice-cold, 10% acetic acid and filter through a column of Merck ion exchanger No. II, acetate form. The solution is then evaporated on a rotary evaporator at a bath temperature of 40 with the addition of n-octanol, the evaporation residue is washed with petroleum ether to remove n-octanol, dried at 40, dissolved in water and lyophilized.

  The dotriacontapeptide amide is obtained as an almost colorless, light powder.



   The AusPansmateriai represents one w1e follows from 20 BC
EMI31.5
   Gly-OH, 3.2 g H-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp- (OtBu) -Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro Gln-Thr (tBu) -Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 and 500 mg of N-hydroxysuccinimide are mixed with 35 ml of dimethylformamide. A solution of 600 mg of dicyclohexylcarbodiimide in 5 ml of dimethylformamide is then added with stirring and the mixture is stirred for a further 4 hours at 45 ". The mixture is then cooled to 0 and the reaction mass is introduced into 600 ml of ether.

  It is left to stand at 0 for 3 hours, then suction filtered and the crude product is dried in vacuo at 35 ". For purification, it is treated with methanol, filtered off in a little dicyclohexylurea and chromatographed on a column of Sephadex LH-20 prepared in methanol (4, 8 cm; 120 cm).



  Fractions of 20 ml are collected, evaporated to dryness and the purity of the fractions is evaluated by thin-layer chromatography on silica gel plates in systems 52A and 100. The pure fractions are combined.



   The decapeptide derivative used as starting material is z. B. shown as follows:
Z-Abu-OH is condensed using the mixed anhydride method (mixed anhydride with pivaloyl chloride) with H-Leu-Gly-OMe, and the tripeptide derivative Z-Abu-Leu-Gly-OMe, which has been purified after chromatography on silica gel, is hydrogenated to H-Abu-Leu -Gly-OMe and then proceeds analogously as described for H-Met-Leu-GIy-OMe in British Patent No. 1,287,601 in Example 1 under 9 to 18.



   Example 6
EMI31.6

Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr
Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 (Val8-Calcitonin M).



   Dissolve 12 mg H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Val Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr Phe-Pro- Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 in 10 ml of water, adjust the resulting solution to pH 6.5 by adding about 2N aqueous ammonia and passing air through the solution until no more free SH groups can be detected with sodium nitroprusside reagent. It is then acidified to pH = 4 with dilute acetic acid and the solution is lyophilized. The residue is freed from ammonium acetate by drying at 40 and 0.01 mm Hg and thus almost pure Val8-calcitonin M.



   In the thin-layer chromatogram on silica gel, Rf6 = 0.45; on aluminum oxide, Ruf52 = 0.53; Rf45 = 0.42.



   The starting material can be prepared as follows: 1. BOC-Cys (TRI) -Gly-Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-Gly-Thr (tBU) -Tyr (tBu) -Thr (tBu)
Gln-Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Ile-Gly-Val-Gly
Ala-Pro-NH2
30 mg BOC-Cys (TRI) -Gly-Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-Gly-OH, 10 mg N-hydroxysucciniimide, 50 mg H-Thr ( tBu) -Tyr (tBu) -Thr (tBu) -Gln Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe Pro-Gln-Thr (tBu) -Ala-Ile -Gly-Val-Gly-Ala-Pro-NH2 and 1 ml of dimethylformamide are stirred at 50 for 1 hour.



  A solution of 15 mg of dicyclohexylcarbodiimide in 0.2 ml of dimethylformamide is then added and the mixture is stirred at 4045 for 6 hours. Then one enters into 15 ml of ether, left to stand for 5 hours at 0 and filtered off the precipitate. For purification, the dried, finely pulverized crude product is suspended in 3 ml of ice-cold methanol, stirred for 2 hours and the sparingly soluble, protected dotriacontapeptide amide is centrifuged off. The process is repeated and the purified Dotriacontapeptide is dried in a vacuum desiccator over calcium chloride.



   In the thin-layer chromatogram on silica gel, Rfzoo = 0.54; Rf52A = 0.61.

 

  2. H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Thr-Tyr
Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln
Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2
19 mg BOC-Cys (TRI) -Gly-Asn-Leu-Ser (tBu) -Thr (tBu) -Cys (TRI) -Val-Leu-Gly-ThrtBu) -Tyr (tBu) -Thr (tBu) -Gln -Asp (OtBu) -Phe-Asn-Lys (BOC) -Phe-His-Thr (tBu) -Phe-Prn-Gln-Thr (tBu) -Ala-fle-Gly-Val-Gly-Ala-Pro-NH2 are dissolved in 0.3 ml of 95% trifluoroacetic acid and left to stand at 25 for 60 minutes. It is then cooled to 0 and poured into an ice-cold mixture of 10 ml of water and 3 ml of pyridine. It is filtered under nitrogen, the aqueous phase is washed with 10 ml of ethyl acetate, then treated with 0.5 ml of acetic acid and filtered under nitrogen through a column of Merck ion exchanger No. II (weakly basic, acetate form).

  The eluate is evaporated to dryness on a rotary evaporator at a bath temperature of 40 and the residue is freed from pyridine acetate by drying at 40 and 0.01 mm Hg.



   PATENT CLAIM 1
Process for the production of new peptides of the formula I.
EMI32.1
 Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 wherein R is hydrogen, a free or acylated amino radical and X stands for the valine, norvaline, leucine, isoleucine, norleucine or α-aminobutyric acid residue, and corresponding compounds in which one or more of the asparagine and glutamine residues are replaced by aspartic acid or. Glutamic acid residue and / or the aspartic acid residue are replaced by the asparagine residue, and their acid addition salts, characterized in that the corresponding acids or acids are added in any chronological order.



  their activated derivatives condensed with intermediate protection from reactive functional groups to be excluded from the reaction and with formation of the disulfide bridge from the two mercapto groups by oxidation and the terminal carboxyl group amidated.



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



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



   4. The method according to claim I or dependent claims 1, 2 or 3, characterized in that a compound of the formula I is prepared in which R is hydrogen and X has the meaning given.



   5. The method according to claim I or subclaims 1, 2 or 3, characterized in that the Dotriacontapeptidamid of the formula
EMI32.2
   Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile, Gly-Val-Gly-Ala-Pro-NH2 where X for the valine, norvaline, leucine -, isoleucine, norleucine or α-aminobutyric acid residue, or its acid addition salts.



   6. The method according to claim I or dependent claims 1, 2 or 3, characterized in that the Dotriacontanentidamid the format
EMI32.3
   Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro -NH2 or its acid addition salts.



   7. The method according to claim I or dependent claims 1. 2 or thereby # these
EMI32.4
   Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH, where X is the valine , Norvaline, leucine, isoleucine, norleucine or α-aminobutyric acid residue, or its acid addition salts.

 

   8. The method according to claim I, characterized in that obtained compounds of the formula I in which R represents a free amino group and X has the meaning given, N -acylated.



   PATENT CLAIM II
Use of peptides obtained 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, polyphosphate and / or hydroxide.

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



   

 

Claims (1)

** WARNING ** Beginning of CLMS field could overlap end of DESC **. Acetate form). The eluate is evaporated to dryness on a rotary evaporator at a bath temperature of 40 and the residue is freed from pyridine acetate by drying at 40 and 0.01 mm Hg. PATENT CLAIM 1 Process for the production of new peptides of the formula I. EMI32.1 Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 wherein R is hydrogen, a free or acylated amino radical and X stands for the valine, norvaline, leucine, isoleucine, norleucine or α-aminobutyric acid residue, and corresponding compounds in which one or more of the asparagine and glutamine residues are replaced by aspartic acid or. Glutamic acid residue and / or the aspartic acid residue are replaced by the asparagine residue, and their acid addition salts, characterized in that the corresponding acids or acids are added in any chronological order. their activated derivatives condensed with intermediate protection from reactive functional groups to be excluded from the reaction and with formation of the disulfide bridge from the two mercapto groups by oxidation and the terminal carboxyl group amidated. SUBCLAIMS 1. The method according to claim I, characterized in that 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 amino groups to be excluded from the reaction are protected by the tert-butyloxycarbonyl group. 3. The method according to claim I and dependent claim 1, characterized in that hydroxyl groups in the side chains to be excluded from the reaction are protected by the tert-butyl group. 4. The method according to claim I or dependent claims 1, 2 or 3, characterized in that a compound of the formula I is prepared in which R is hydrogen and X has the meaning given. 5. The method according to claim I or subclaims 1, 2 or 3, characterized in that the Dotriacontapeptidamid of the formula EMI32.2 Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile, Gly-Val-Gly-Ala-Pro-NH2 where X for the valine, norvaline, leucine -, isoleucine, norleucine or α-aminobutyric acid residue, or its acid addition salts. 6. The method according to claim I or dependent claims 1, 2 or 3, characterized in that the Dotriacontanentidamid the format EMI32.3 Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro -NH2 or its acid addition salts. 7. The method according to claim I or dependent claims 1. 2 or thereby # these EMI32.4 Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH, where X is the valine , Norvaline, leucine, isoleucine, norleucine or α-aminobutyric acid residue, or its acid addition salts. 8. The method according to claim I, characterized in that obtained compounds of the formula I in which R represents a free amino group and X has the meaning given, N -acylated. PATENT CLAIM II Use of peptides obtained 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, polyphosphate and / or hydroxide.