CH582653A5 - Hypocalcaemic dotriaconta peptide amides - prepd by removing protecting groups from protected peptide amides, using acid hydrolysis - Google Patents

Abstract

Dotriaconta peptide amides of general formula (I): 1 2 3 4 5 6 7 8 9 10 11 12 H-Cys-Cly-Asn-Leu-Ser-Thr-Cys-X-Leu-Gly-Thr-Tyr-13 14 15 16 17 18 19 20 21 22 23 24 Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-25 26 27 28 29 30 31 32 Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2, where X = L-methionine, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine, or L-alpha-aminobutyric acid, 26 is replaced by L-asparagine and/or 27 by L-threonine, one or more amino acids in posn. 11, 12, 16, 19, 22 and 24 are opt. substd. by other amino acids, e.g. 11 by L-lysine, 12.16 and 19 by L-leucine, 22 by L-tyrosine and 24 by L-arginine, including derivatives, acid-addition salts and complexes of these cpds are prepd. e.g. by removing protecting gps. such as OtBu, BOC or tBU from cpds. (I) where min one amino- or carbonyl gp. is protected with one of these gps. using acid hydrolysis (I) have hypocalcaemic activity and lower the plasma calcium- and phosphate level in mammals. They may be used in hypocalcaemia, osteoporosis and osteitis.

Description

  

  
 



   The invention relates to a process for the production of new hypocalcemically active peptides of the formula I
EMI1.1
 where R is hydrogen or a free or acylated amino group and X is L-methionine, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine or Ln-amino butyric acid radical and in which L-alanine26 is replaced by L-asparagine and / or L-isoleuctine27 by L-threonine and in which one or more of the amino acids in positions 11, 12, 16, 19, 22 and 24 are optionally replaced by another amino acid is, namely L-threonine11 against L-lysine, L-tyrosine12 against L-leucine, L-phenylalanint6 against L-leucine, L-phenylalanint9 against L-leucine,

   L-phenylalanine22 against L-tyrosine and L-glutamine24 against L-arginine, their acid addition salts and complexes.



   Acyl groups for the acylation of the N1 -amino group are the radicals of carboxylic acids such as aliphatic, aromatic, araliphatic, heterocyclic and heterocyclylaliphatic carboxylic acids, in particular of lower mono- or divalent alkanoic or alkenoic acids such as formic acid, acetic acid, propionic acid, butyric acid, acrylic acid, succinic acid alicyclic carboxylic acids such as cycloalkylcarboxylic acids, of mono- or dihydric monocyclic aromatic carboxylic acids such as unsubstituted and substituted benzoic acid or phthalic acid, of unsubstituted and aryl-substituted aryl-lower alkyl or alkenyl-carboxylic acids such as phenylacetic acid, of unsubstituted or substituted mono- or dihydric 5- to dihydric heterocyclic acids with nitrogen, sulfur and / or oxygen as heteroatoms, such as pyridinecarboxylic acids,

   Thiophenecarboxylic acids, or of Heterocyclylniederalkansäuren such as pyridyl acetic acid, imidazoly acetic acid, wherein the substituents of the rings z. B. halogen atoms, nitro groups, lower alkyl or lower alkoxy groups or lower carbalkoxy groups. Acyl residues of amino acids, especially of a-amino acids, such as e.g. Glycyl, L-leucyl, L-pyroglutamyl, and acyl radicals derived from carbonic acid or thiocarbonic acid or their esters or amides, e.g. 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-phenyl-thiocarbamoyl.



   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.



   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 e.g. inorganic compounds derived from metals such as calcium, magnesium, aluminum, cobalt and, in particular, zinc, especially sparingly soluble salts such as phosphates, pyrophosphates and polyphosphates and hydroxides of these metals, furthermore alkali metal polyphosphates such as e.g. Calgon N, Calgon 322, Calgon 188 or Polyron B 12. Organic substances which cause the effect to be prolonged are, for example, non-antigenic gelatin, e.g.

  Polyoxygelatine, polyvinylpyrrolidone and carboxymethylcellulose, also sulfonic acid or phosphoric acid esters of alginic acid, dextran, polyphenols and polyalcohols, especially polyphoretin phosphate and phytic acid, as well as polymers and copolymers of basic or especially acidic amino acids, e.g. Protamine or polyglutamic acid.



   The new compounds have a hypocalcemic effect. They lower the plasma calcium and phosphate content of the blood of mammals, as by experiments
Rats was detected. They can be used for hypercalcemia, osteoporosis or osteitis deformens. The excretion of electrolytes in the urine is the same as with human calcitonin, in contrast to salmon calcitonin, which has strong renal effects.



   The process according to the invention for the preparation of the new peptide amides is characterized in that the corresponding amino acids, where proline32 is optionally present as an amino acid amide and cysteine1 optionally as a deamino acid or N-acylated amino acid, or their derivatives with an activated terminal carboxy group or



  activated a-amino group, with intermediate protection of the functional groups to be excluded from the reaction, with the formation of the peptide bonds correspondingly condensed with one another, the two mercapto groups in the first part of the sequence being combined by oxidation to form the disulfide bridge.



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



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



   So one uses e.g. as amino protecting groups, e.g. aryl-lower alkyl group substituted by halogen, nitro, lower alkyl or lower alkoxy such as diphenylmethyl or triphenylmethyl groups, e.g. Benzhydryl, trityl, di-paramethoxybenzhydryl, or acyl groups such as formyl, trifluoroacetyl, phthaloyl, p-toluenesulfonyl, benzylsulfonyl, benzenesulfenyl, o-nitrophenylsulfenyl, or, above all, groups derived from carbonic acid or thiocarbonic acid, nitro groups, such as, if appropriate, in aromatic groups derived from carbonic acid or thiocarbonic acid , Lower alkyl or lower alkoxy or lower carbalkoxy groups substituted carbobenzoxy groups, e.g.



  Carbobenzoxy, p-bromo- or p-chlorocarbobenzoxy, p-nitrocarbobenzoxy, p-methoxycarbobenzoxy, colored benzyloxycarbonyl groups such as p-phenylazo-benzyloxycarbonyl and p (p'-methoxyphenylazo) -benzyloxycarbonyl, tolyloxycarbonyl, 2-tolyloxycarbonyl, 2-tolyloxycarbonyl isopropyl isopropyl isopropyl carbonyl especially 2- (para-biphenylyl) -2-propyloxycarbonyl, also aliphatic oxycarbonyl groups such as

 

  Allyloxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl, 2,2,2 trichloroethyloxycarbonyl, 2-iodoethoxycarbonyl and primarily tert-butyloxycarbonyl, further z. Carbamoyl, thiocarbamoyl, N-phenylcarbamoyl and -thiocarbamoyl.



   The amino groups can also be obtained by the formation of enamines by reaction of the amino group with 1,3 diketones, e.g. B. benzoylacetone, acetylacetone or dimedone are protected.



   Carboxyl groups are protected, for example, by amide or hydrazide formation or by esterification. The amide and hydrazide groups can optionally be substituted, the amide group e.g. by the 3,4-dimethoxybenzyl or bis (p-methoxyphenyl) methyl group, the hydrazide group z. B.



  by the carbobenzoxy group, the trichloroethyloxycarbonyl group, the trifluoroacetyl group, the trityl group, the tert. Butyloxycarbonyl group or the 2- (p-biphenyl) isopropyloxycarbonyl group. Suitable for esterification are, for example, lower optionally substituted alkanols such as methanol, ethanol, cyanomethyl alcohol, 2,2,2-trichloroethanol, 2-iodoethanol, benzoylmethyl alcohol or, in particular, tert-butanol, and 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
Phenol, thiophenol, thiocresol, p-nitrothiophenol, 2,4,5 - and
2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4 dinitrophenol, p-cyanophenol, or p-methanesulfonylphenol, further e.g. B. N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxypiperidine, 8-hydroxyquinoline.



   The hydroxy groups of the serine, threonine and tyrosine residues can, for. 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 suitable. To
Groups suitable for etherification are e.g. B. benzyl, tetrahydro pyranyl 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 1-benzyloxycarbonylaminoethyl groups (Weygand). The
However, 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, further z. B. an acylamidomethyl radical. see. Tetrahedron Letters No. 26 (1968) p. 3057 or DOS 2 060 969. Acylamidomethyl is e.g.

  B. a group of the formula -CH2-NH-CO-R, where CO-R is the acyl radical of a carboxylic acid, such as an aliphatic, aromatic, araliphatic or heterocyclic carboxylic acid or a carbonic acid derivative, such as a carbonic acid ester or carbamic acid radical. Primarily R is an optionally substituted lower alkyl radical, e.g. B. methyl, ethyl, propyl, isopropyl, n-butyl or tert-butyl radical, which z. B. chlorine, trifluoromethyl or the nitro group may contain.

  Furthermore, R is, for example, an optionally substituted cycloalkyl radical with 3-8, preferably
5-6, ring atoms such as the cyclopentyl or cyclohexyl radical or an optionally substituted aromatic or araliphatic radical, in which the aromatic ring is preferably the benzene ring, especially optionally substituted phenyl or benzyl, e.g. Phenyl or benzyl which is unsubstituted or substituted in the phenyl radical by lower alkyl, lower alkoxy, halogen or nitro, or an optionally substituted, preferably monocyclic, heterocyclyl radical, e.g. B. thienyl or furyl radical. In particular, acylamidomethyl stands for acetylamidomethyl. 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 Weygand groups mentioned above.



   These protective groups can be split off in a known manner. So you can the carbobenzoxy group by hydrogenolysis, the N-trityl group with mineral acids such as hydrohalic acids, z. B. hydrogen fluoride or preferably hydrogen chloride or an organic acid such as formic acid, acetic acid, chloroacetic acid or trifluoroacetic acid in aqueous or absolute trifluoroethanol as a solvent or with aqueous acetic acid, the tert-butyloxycarbonyl group with trifluoroacetic acid or hydrochloric acid, the 2- (p-biphenyloxmityl) isylopropylox aqueous acetic acid or e.g. B. with a mixture of glacial acetic acid, formic acid (82.8%) and water (7: 1: 2). The methyl ester can be converted into the hydrazide with hydrazine hydrate. The methyl or ethyl ester group is hydrolyzed with dilute sodium hydroxide solution. The tert-butyl ester is z. B.



  split with trifluoroacetic acid, as well as the tert-butyl ether.



   The tert-butyl ester group is preferably used to protect the carboxyl group of the side chain and, if appropriate, the terminal carboxyl group, the tert-butyloxycarbonyl group to protect the amino group of the side chain, and the hydroxyl groups of the serine, threonine and tyrosine residues, if these are protected at all, the tert-butyl ether group and, if desired, the 2,2,2-trifluoro-1-tert-butyloxycarbonylaminoethyl group to protect the imino group of the histidine. All of these protecting groups can, if desired, in one step by acid hydrolysis, e.g. B.



  be split off by means of trifluoroacetic acid, hydrochloric acid or hydrogen fluoride.



   The mercapto groups are preferably protected by benzyl, trityl or by acylamidomethyl. 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. The S-acylamidomethyl groups can be obtained from the protected peptide by stirring the peptide, preferably at room temperature, in an aqueous solution of a water-soluble heavy metal salt such as an acetate or nitrate of mercury, silver, cadmium, tin etc. or an organometallic salt of a heavy metal.



  In addition to water, other solvents such as methanol, dimethylformamide or mixtures of these solvents can also be used. In all cases the protected peptide is obtained with free mercapto groups. This can lead to the protected disulfide, e.g. B. be oxidized with iodine in glacial acetic acid, with diiodoethane in organic solvents or with atmospheric oxygen in liquid ammonia. It is particularly advantageous to protect the mercapto groups by trityl or acylamidomethyl groups and remove them from the protected peptide with simultaneous formation of the disulfide bridge with iodine z. B. to remove in methanol or acetic acid. The formation of the disulfide ring can take place at the stage of one of the two part-sequence containing cysteine residues, e.g. B. the decapeptide 1-10, or at the stage of the Dotriacontapetidamids.

 

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



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



   The formation of acid addition salts is carried out in a known manner.



   The formation of complexes also takes place according to known or equivalent methods.



   Complexes with inorganic substances such as poorly soluble
Metal, e.g. B aluminum or tin compounds are preferably used in a manner analogous to that known for ACTH, e.g. B.



   by reaction with a soluble salt of the relevant
Metal, e.g. B. zinc chloride or zinc sulfate, and precipitation with an alkali metal phosphate and / or hydroxide produced.



   Complexes with organic compounds such as polyoxygelatine,
Carboxymethyl cellulose, polyvinylpyrrolidone, polyphloretin phosphate, polyglutamic acid, etc. are made by mixing these
Obtain substances with the peptide in aqueous solution. In the same way, insoluble compounds can also be used
Alkali metal polyphosphates are produced.



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



   The linkage of the amino acid and / or peptide units takes place, for. B. in such a way that an amino acid or a peptide with a protected a-amino group and activated terminal carboxyl group with an amino acid or a peptide with a free a-amino group and free or protected, z. B. esterified or amidated terminal
Carboxyl group, or that an amino acid or a peptide with activated a-amino group and protected terminal carboxyl group with an amino acid or a
Peptide with free terminal carboxyl group and protected a
Reacts amino group.

  The carboxyl group can, for example, be converted into an acid azide, anhydride, imidazolide, isoxazolide or an activated ester such as cyanomethyl ester,
Carboxymethyl ester, p-nitrophenyl thioester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, N
Hydroxysuccinimide ester, N-hydroxyphthalimide ester, 8-hydroxyquinoline ester, N-hydroxypiperidine ester or by
Reaction using a carbodiimide (optionally under
Addition of N-hydroxysuccinimide or an unsubstituted or z. B. by halogen, methyl or methoxy substituted 1-hydroxybenzotriazole) or N, N'-carbonyl-diimidazole, the
Amino group can be activated, for example, by reaction with a phosphite amide.

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



   It has been found that it is advantageous to start from a sequence which comprises the first 10 N-terminal amino acids and to condense the entire remaining sequence with this N-terminus, preferably according to Wegand Wünsch.



   But you can also use the N-terminal sequence mentioned e.g.



  link with the fragment up to the 28th amino acid (glycine) with a free terminal carboxyl group and condense the octacosapeptide with the tetrapeptide of amino acids 29-32, for example according to Weygand-Wünsch.



   In the following, as an example of a preferred embodiment, the preparation of the peptide of the formula I, in which R is NH2 and X is L-methionine and in which the 26th



     Amino Acid 'B.



   The N-terminal decapeptide (1-10) can e.g. B. after Helv.



  53, 556 (1970).



   The sequence to be connected with the N-terminal sequence 1-10 comprising the 11th to 32nd amino acid is, for example, from the sequences 11-16, 17-20, 21-23, 24-28 L-asparagine and the 27th amino acid L-threonine is, and composed of 29-32.



   The sequence 2428 can e.g. be prepared in such a way that the 27th amino acid (Thr) is linked with the 28th amino acid (Gly), this sequence of 2 is linked to the 26th amino acid (Asn), the resulting 3- he sequence connects with the 25th amino acid (Thr) and finally with the 24th amino acid (Gln). The hydroxyl groups of the threonine residues can e.g. be protected by the tert-butyl ether group.



   The sequence 24-28 with the protected amino acid of the glutamine residue and free carboxyl group of the glycine residue is combined with the sequence 29-32 H-Val-Gly-Ala-Pro-NH2 [Helv.



     53, 2135 (1970)], preferably by the carbodiimide-N-hydroxysuccinimide method.



   After the amino protective group of the glutamine residue of sequence 2432 has been split off, the sequence 21-23 Z-Thr (tBu) -Phe-Pro-OH [Helv.53, 2135 1970)] is attached to sequence 24, preferably by the carbodiimide method in the presence of N-hydroxysuccinimide -32 coupled.



   The junction of the sequence 17-20 Z-Asn-Lys (Boc) -Phe His-NH-NH2 [HeIv. 53, 2135 (1970)] with the sequence 21-32 freed from the amino protective group Z takes place, for. B. by means of the azide method.



   After splitting off the amino protective group Z of the asparagine residue of the sequence 17-32, the sequence 11-16 Z-Thr (tBu) Tyr (tBu) - Thr- (tBu) -Gln-Asp (OtBu) - is used to complete the required fragment 11-32 Phe-NHNH2 [Helv. 53, 2135 (1970)] coupled to the sequence 17-32, preferably by means of the azide method.



   The linkage of the sequence 11-32 just described
EMI3.1
 is preferably carried out by the carbodiimide-N-hydroxysuccinimide method.



   All protective groups that are still present are preferably removed in trifluoroacetic acid or concentrated hydrochloric acid and leads to Asn26-Thr27-calcitonin-M.



   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, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, hydroxymaleic acid, dihydroxymaleic acid, benzoic acid, phenylacetic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, anthranic acid, cinnamic acid, mandelic acid,
4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
Naphthalenesulfonic acid, or sulfanilic 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 43C: tert-amyl alcohol-isopropanol-water (51:21:28) System 45: sec. Butanol- 3% aqueous ammonia (70:30) System 52: nButanol-glacial acetic acid - Water (75: 7.5: 21) System 52A: n-butanol-glacial acetic acid-water (67:10:23) System 70: ethyl acetate-pyridine-water (40:20:40),
Upper phase system 89: ethyl acetate-acetone-water (72: 24: 4) system 96: sec-butanol-glacial acetic acid-water (67:10:23) system 100: ethyl acetate-pyridine-glacial acetic acid-water (62: 21: 6 : 11) System 101 A: n-butanol-pyridine-glacial acetic acid-water (42: 24: 4: 30) System 107: ethyl acetate-pyridine-water (49:24:27) System 112E: n-butanol-pyridine-formic acid - Water (44: 24: 2: 20) Rf data for thin layer chromatography:
DS:

  On silica gel prefabricated plates SL 254 from Antec, Birsfelden.



   TLC: On prefabricated cellulose plates from Merck, Darmstadt.



   DA: On Alox plates (45 g Al203 from Camag, Muttenz, + 3.5 g plaster of paris, thickness 0.3 mm).



   The following abbreviations are used in the examples: Boc tert-butyloxycarbonyl Z carbobenzoxy OtBu tert-butyl ester ONp p-nitrophenyl ester OMe methyl ester OSu hydroxysuccinimide ester tBu tert-butyl ether Bmp ss-mercaptopropionyl DMF dimethylformamide
example 1
EMI4.1
 at 0 in 1.2 ml purest 12-n. Dissolved hydrochloric acid (approx. 1-2 minutes), briefly flushed with N2 and left to stand at 0 for 10 minutes. It is then cooled with dry ice, evacuated in a high vacuum and concentrated to a syrup while slowly increasing the temperature. This is dissolved in 2 ml of water, lyophilized, the residue is again lyophilized from 2 ml of water and finally equilibrated with the humidity.

  The product thus obtained (hydrochloride of Asn26-Thr27-calcitonin M) is an amorphous, water-soluble powder.



     TLC: Rf (45) = 0.45; Rf (101A) = 0.53; Rf (112E) = 0.39. The connection runs in thin-layer electrophoresis (on cellulose plate) at pH 1.9 and 16 volts / cm in 12 hours for approx.



  3.7 cm to the cathode.



   The protected Dotriacontapeptidamid is z. B. prepared in the following way: a) Z-Thr (tBu) -Gly-OMe
22.3 g of Z-Thr (tBu) -OSu and 8.29 g of HCI - H-Gly-OMe are dissolved or suspended in 100 ml of dimethylformamide, mixed with 7.49 ml of N-methylmorpholine, for 1 day at 22 " stirred and then concentrated to dryness in a high vacuum.



  The residue is taken up in ethyl acetate, washed with citric acid (5%), KHCO3 (5%) and water, dried over sodium sulfate and concentrated to dryness. The oily residue crystallizes on standing overnight at 0, m.p.



  The dipeptide is chromatographically uniform and is processed further directly.



   DS: Rf (45) = 0.77; Rf (chloroform-methanol = 9: 1) = 0.70 b) H-Thr (tBu) -Gly-OMe-HCl
5.0 g of Z-Thr (tBu) -Gly-OMe are dissolved in 200 ml of methanol and, after adding 13.2 ml of 1-n. HCI and 0.5 g palladium carbon (10% Pd) hydrogenated to saturation with CO2 absorption. The catalyst is then filtered off and evaporated to dryness, a white foam being obtained.



   DS: Rf (45) = 0.64; Rf (chloroform-methanol = 9: 1) = 0.41; Rf (52) = 0.42 c) Z-Asn-Thr (tBu) -Gly-OMe
18.9 g of Z-Asn-ONp and 12.5 g of H-Thr (tBu) -Gly-OMe HCI are dissolved in 95 ml of DMF, 4.6 ml of N-methylmorpholine are added and the mixture is left to stand at room temperature for 15 hours . The solvent is removed in a high vacuum, the residue is pulverized by trituration with ethyl acetate and filtered off and crystallized by crystallization from methanol-chloroform-petroleum ether and from ethanol-water, mp = 1650.



   DS: Rf (45) = 0.68; Rf (chloroform-methanol = 9: 1) = 0.38, Rf (52) = 0.70 d) H-Asn-Thr (tBu) -Gly-OMe
8.5 g of Z-Asn-Thr (tBu) -Gly-OMe are hydrogenated as usual in 210 ml of methanol. The product is an amorphous foam, m.p. approx. 600.



     DS: Rf (45) = 0.42; Rf (52) = 0.27 e) Z-Thr (tBu) -Asn-Thr (tBu) -Gly-OMe
A solution of 7.51 g of Z-Thr (tBu) -OSu and 5.55 g of H Asn-Thr (tBu) -Gly-OMe in 50 ml of DMF is left at 22 "overnight and then concentrated to dryness in a high vacuum.



  The residue is dissolved in ethyl acetate-n-butanol (1: 1), washed with citric acid (5%), KHCO3 (5%) and water, evaporated to dryness and the solid residue is crystallized from methanol-ether; M.p. 145-146 ".



     DS: Rf (45) = 0.73; Rf (52) = 0.75 f) H-Thr (tBu) -Asn-Thr (tBu) -Gly-OMe
2.0 g of Z-Thr (tBu) -Asn-Thr (tBu) -Gly-OMe are hydrogenated to saturation as usual in 20 ml of methanol, an amorphous (foamy) product being obtained.



      DS: Rf (45) = 0.41; Rf (52) = 0.34 g) Z-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-OMe
A solution of 5.33 g of H-Thr (tBu) -Asn-Thr (tBu) -Gly OMe and 4.95 g of Z-Gln-ONp in 80 ml of DMF is left to stand overnight at 22, whereby the pentapeptide that forms starts to crystallize after a short time. Finally, 500 ml of water are added, the mixture is filtered off at 0, and the residue is dried and purified by slurrying in acetone-acetonitrile 1: 1.



  The crystalline product has a melting point of 220.



   DS: Rf (100) = 0.65; Rf (chloroform-methanol 8: 2) = 0.35; Rf (52) = 0.58 h) Z-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-OH
6.42 g of pentapeptide methyl ester (of g) are dissolved in 58 ml of trifluoroethanol with heating, at room temperature with 25 ml of 1-n. Sodium hydroxide solution was added and the mixture was stirred at 22 for 6 hours. After adding 25 ml of 1-n. HCI begins to crystallize out the saponified pentapeptide. It is concentrated to 50 ml, filtered off and washed free of chloride with water.



     DS: Rf (52) = 0.46; Rf (100) = 0.29 i) Z-Gin-Thr (tBu) -Asn-Thr (tBu) -G (y-Val-Gly-AIa-Pro-NH2
500 mg Z-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-OH, 335 mg H-Val-Gly-Ala-Pro-NH2 (Helv. 53,2135 (1970)) and 105 mg N- Hydroxysuccinimide are dissolved in 4 ml of DMF with heating. After the addition of 202 mg of dicyclohexyl-carbodiimide, the mixture is stirred for 4 hours at 40, whereupon the reaction product is precipitated as a jelly by adding to a mixture of 100 ml each of ether and petroleum ether and is filtered off. The crude product is purified by dissolving it twice in methanol-water (85:15) and precipitating with ether, mp.



     198-199.



   DS: Rf (96) = 0.41; Rf (107) = 0.50, Rf (chloroform methanol 7: 3) = 0.35 j) H-Gln-Thr (tBu) -Asn-Thr (tBu) - Gly-Val-Gly-Ala-Pro-NH2
3.1 g of the above-mentioned Z-derivative are hydrogenated to saturation in 120 ml of 80% strength trifluoroethanol with 310 mg of palladium carbon (10% Pd) with CO2 absorption.

  The filtrate is concentrated to approx. 5 ml and, after the addition of 40 ml of water, is left to stand overnight at 0 °, the nonapeptide crystallizing out in pure form, mp approx. 2400 (decomp.)
DS: Rf (96) = 0.20; Rf (107) = 0.32; Rf (70) = 0.18 k) Z-Thr (tBu) -Phe-Pro-Gln-Thr (tssu) -Asn-Thr (tBu) -Gly Val-Giy-Aia-Pro -NH2
517 mg of Z-Thr (tBu) -Phe-Pro-OH (Helv. 53, 2135 (1970)), 410 mg of the above-mentioned nonapeptide and 74 mg of N-hydroxy-succinimide are dissolved in 4 ml of trifluoroethanol-chloroform (6 : 4) dissolved or suspended, mixed with 177 mg of dicyclohexyl-carbodiimide at 22 and stirred for 20 hours.

  The reaction mixture is then concentrated to dryness and the residue is reprecipitated once from chloroform-petroleum ether. The crude product thus obtained is purified in a Craig distribution in the solvent system methanol buffer-chloroform-carbon tetrachloride 10: 10: 3: 5: 4 (buffer: 28.6 ml glacial acetic acid + 19.25 g ammonium acetate + 960 ml water) with phase volumes of 5 ml each over 420 levels.

  The pure dodecapeptide is obtained by concentrating the contents of the distribution elements No. 135-184 (rmax = 160; K = 0.61) to dryness and subliming away the ammonium acetate in a high vacuum at 45 as an amorphous powder with a melting point of about 195 (decomp.)
DS: Rf (96) = 0.44; Rf (70) = 0.51; Rf (52A) = 0.38 I) H-Thr (tBu) -Phe-Pro-Gin-Thr (tBu) -Asn-Thr (tBu) - Gly Val-Gly-Ala-Pro-NH2
504 mg of the above-mentioned Z-dodecapeptide are dissolved in 30 ml of methanol with heating and, after the addition of 100 mg of palladium-carbon, hydrogenated as usual. The product obtained when the filtrate is concentrated to dryness is an amorphous, white powder.



   DS: Rf (43C) = 0.25; Rf (70) = 0.35; Rf (52A) = 0.20 m) Z-Asn-Lys (Boc) Phe-His-Thr (tBu) - Phe-Pro-Gln Thr (tBu) - Asn-Thr (tBu) - Gly- Val-Gly- Ala-Pro-NH2
To a solution of 479 mg of Z-Asn-Lys (Boc) -Phe-His NHNH2 (Helv. 53, 2135 (1970)) in 4.5 ml of DMF is added at -20, eel 3, 2-a. HCl in dioxane and 90 l tert. -Butyl nitrite too.



  After stirring for 15 minutes at -10z, 410 mg of H Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-Val Gly-Ala-Pro-NH2, dissolved in 6 ml of DMF , and then 260 l of ethyl diisopropylamine were added. It is left to stand at 0 overnight and the crude product is then precipitated by dropping it into 100 ml of ether. For purification, it is subjected to a Craig partition in the system methanol-buffer-chloroform-carbon tetrachloride 10: 5: 10: 5 [buffer as for k] over 200 hours with phase volumes of 3 ml each. The pure hexadecapeptide is obtained from distribution elements No. 18-37 (rman = 27; K = 0.16) by concentrating to dryness and subliming away the buffer in a high vacuum at 40 as an amorphous powder.



   DS: Rf (43C) = 0.40; Rf (52) = 0.20; Rf (70) = 0.58 n) H-Asn-Lys (Boc) -Phe-His- Thr (tBu) -Phe-Pro- Gln- Thr (tBu) - Asn-Thr (tBu) -Gly- Val- Gly-Ala-Pro-NH2, acetate
270 mg of the aforementioned Z-hexadecapeptides are hydrogenated in 27 ml of 80% acetic acid with 50 mg of Pd-charcoal for 2 hours, the catalyst is filtered off, the filtrate is reduced to approx.



  3 ml concentrated and lyophilized.



   DS: Rf (52) = 0.07; Rf (96) = 0.28; Rf (l07) = 0.65 o) Z-Thr (tBu) -Tyr (tBu) -Thr (tBu-Gln-Asp (OtBu) - Phe-Asn Lys (Boc) - Phe-His- Thr (tBu) - Phe-Pro-Gin-Thr (tBu) -Asn Thr (tBu) - Gly-Val-Gly-Ala-Pro-NH2
To a solution of 165 mg of Z-Thr (tBu) -Tyr (tBu) Thr (tBu) -Gln-Asp (OtBu) Phe-NHNH2 (Helv. 53, 2135 (1970)) in 2 ml of DMF is added at -200 117 HCl 3.24-n. in dioxane and 21 l of tert-butyl nitrite.

  The mixture is stirred for 15 minutes at -100, a solution of 198 mg H-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Thr is added, which has been pre-cooled to 0 ° (tBu) -Gly-Val-Gly-Ala-Pro-NH2 (acetate) in 4 ml DMF and 80 M 1 ethyl-diisopropylamine and let stand at 0 overnight. The crude product is precipitated by dripping the reaction solution into a mixture of 40 ml of ether and 20 ml of petroleum ether and then a Craig distribution in the system methanol-buffer-chloroform-carbon tetrachloride 20: 5: 10: 10 [buffer as for k] over 300 Subjected to stages with phase volumes of 3 ml each.

  The chromatographically pure docosapeptide is obtained as an amorphous powder from distribution elements No. 98-117 (rmax = 107, K = 0.55) by concentrating to dryness and subliming away the buffer at 40 in a high vacuum.



   DS: Rf (43C) = 0.39; Rf (52) = 0.30; Rf (96) = 0.48 p) H- Thr (tBu) - Tyr (tBu) - Thr (tBu) -Gitt-Asp (OtBu) -Phe-Asn Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBIl) -Asn- Thr (tBu) - Gly-Val-Gly-Ala-Pro-NH2
260 mg of Z-docosapeptide are hydrogenated in 26 ml of 80% acetic acid with 50 mg of Pd-charcoal for 15 hours, the catalyst is filtered off, the filtrate is concentrated to about 5 ml and lyophilized. To remove the acetic acid, the residue is reprecipitated twice from methanol-sodium bicarbonate solution.

 

     DS: Rf (43C) = 0.31; Rf (100) = 0.29
EMI5.1
 170 mng of the hydrogenated docosapepti described under p)
EMI5.2
 1.2 ml of DMF dissolved with heating, mixed with 15.8 mg of N-hydroxysuccinimide and 21.3 mg of dicyclohexylcarbodiimide and stirred under nitrogen at 45 for 3+ hours. The crude product is then precipitated by dropwise addition of the reaction mixture in 20 ml of peroxide-free ether and to purify a Craig distribution in the system methanol-buffer chloroform-carbon tetrachloride 11: 3: 6: 7 [buffer as in k] over 450 stages with phase volumes of 3 each ml subjected. The pure, protected dotriacontapeptide derivative is isolated from elements no. 190-210 (rmax = 200; K = 0.8) when the buffer is concentrated and sublimed away as an amorphous powder.



   DS: Rf (70) = 0.53; Rf (100) = 0.35; Rf (52A) = 0.29.



   Example 2
EMI6.1
 Acidolysis with 12-n. HCI at 0 ", as described in Example 1, converted into the above peptide.



      TLC: Rf (45) = 0.50; Rf (101A) = 0.59; Rf (112E) = 0.43
Thin-layer electrophoresis: pH 1.9, 16 volts / cm, 11 hours: distance about 2.4 cm to the cathode.



   The protected Dotriacontapeptidamid is z. B. manufactured in the following way:
EMI6.2
 Ala-Pro-NH2 (Example Ip) and 10.5 mg of N-hydroxy-succinimide are dissolved in 0.9 ml of DMF. After adding 14 mg of dicyclohexylcarbodiimide, the reaction vessel is flushed with N2, closed and stirred at 45 for 30 hours.



  The crude product is precipitated by adding 15 ml of peroxide-free ether and purified by means of Craig partitioning in the system methanol-buffer-chloroform-carbon tetrachloride 11: 3: 6: 7 [buffer in Example I k) j over 240 stages with phase volumes of each 3 ml. The pure product is isolated from the distribution elements No. 108-129 (rmax = 118; K = 0.97) when the buffer is concentrated and sublimated as an amorphous powder.



   DS: Rf (52) = 0.29; Rf (70) = 0.65; Rf (100) = 0.38.



   Example 3
EMI6.3
 Acidolysis with 12-n. HCI at 0 as described in Example 1, converted into the above peptide.



      TLC: Rf (101A) = 0.51; Rf (112E) = 0.45
Thin-layer electrophoresis: pH 1.9, 16 volts / cm 1 hour: Distance about 3.7 cm to the cathode.



   The protected Dotriacontapeptidamid can e.g. B. be prepared in the following way: a) Z-Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-OMe
4.15 g of H-Thr (tBu) -Gln-Asp (OtBu) -Phe-OMe (Helv. 53, 2135 (1970)) are dissolved in 40 ml of DMF, mixed with 3.26 g of Z-Leu ONp and over Left at 22 night. The resulting pentapeptide is crystallized out by adding water and purified by crystallization from methanol-chloroform-petroleum ether and from methanol-water; M.p.



  204.



   DS: Rf (chloroform-methanol 9: 1) = 0.39; Rf (89) = 0.52 b) H-Leu-Thr (tBu) -Gin-Asp (OtBu) -Phe-OMe
4.8 g of Z-pentapeptide are hydrogenated to saturation as usual in 100 ml of methanol and the solution filtered off from the catalyst is concentrated to dryness, giving an amorphous, white foam.



   DS: Rf (chloroform-methanol 9: 1) = 0.14; Rf (89) = 0.29 c) Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-OMe
4 g of H-Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-OMe and 3.5 g of Z-Lys (Boc) -ONp are dissolved in 25 ml of DMF and left at 22 overnight. The resulting hexapeptide is precipitated as a jelly by adding ether and purified by crystallization from methanol-ethyl acetate-petroleum ether; M.p. 211-212.



   DS: Rf (chloroform-methanol 9: 1) = 0.50; Rf (89) = 0.55 d) Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-NHNH2
5 g of the above-mentioned methyl ester are dissolved in 50 ml of DMF with heating, 5 ml of hydrazine hydrate are added and the mixture is left to stand at 22 for 5 hours. It is precipitated by adding 250 ml of water, the finely powdered precipitate is filtered off and washed with water until the film is negative. M.p. about 220 (decomp.).



   DS: R <(chloroform-methanol 9: 1) = 0.24; Rf (89) = 0.27 e) Z- Thr (tBu) - Tyr (tBu) -Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
393 mg of 2-Thr (tBu) -Tyr (tBu) -Pro-OH (DOS 2 050 434) and 66 l of N-methylmorpholine are dissolved in 5 ml of tetrahydrofuran, cooled to -20 and in the course of 1 minute with 82 l of isobutyl chlorocarbonate offset. The mixture is stirred for a further 10 minutes, then a solution, precooled to 0, of 300 mg H-Gln-Thr (tBu) -Asn-Thr (tBU7-Gly-Val-Gly-Ala Pro-NH2 [analogous to example lj)] in 6 mltrifluoroethanol-chloro form (6: 4), stirred for 1 hour at 0 and left to stand at 22 overnight.

  The reaction mixture is concentrated until a gelatinous mass is formed and this is reprecipitated from carbon tetrachloride petroleum ether. For purification, the crude product is subjected to a Craig distribution in the methanol-buffer-chloroform-carbon tetrachloride system 10: 3: 5: 4 [buffer as in Example 1 k)] over 400 stages with phase volumes of 3 ml each. The chromatographically pure dodecapeptide is isolated as an amorphous powder from the distribution elements nos. 118-143 (rmnX = 130; K = 0.48) when the buffer is concentrated to dryness and the buffer is sublimated in a high vacuum at 40.



     DS: Rf (43C) = 0.42; Rf (52A) = 0.37; Rf (70) = 0.65 f) H-Thr (tB) -Tyr (tBu) -Pro-Gln-Thr (tBu) -Asn-Thr (tBu) Gly-Val-Giy-Ala-Pro-NH2
1.56 g of dodecapeptide from e) are dissolved in 80 ml of methanol with heating and hydrogenated as usual. When the hydrogenation solution is concentrated to dryness, the product is obtained as an amorphous residue with a melting point of approx.

 

     DS: R <(43C) = 0.30; Rf (52A) = 0.21; R <(70) = 0.56 g) Z-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Tyr (tBu) -Pro-Gln Thr (tBu) -Asn-Thr (tBu) -Gly - Val-Gly-Ala-Pro -NH2
445 mg of Z-Asn-Lys (Boc) -Phe-His-NHNH2 (Helv. 53, 2135 (1970)) are dissolved with warming in 4 ml of DMF and at -20 in succession with 350 .mu.l HCl 3.2-n. in dioxane and mixed with 83 <1 tert-butyl nitrite.

  The mixture is stirred for a further 15 minutes at -100, then a solution of 400 mg of the above-mentioned hydrogenated dodecapeptide in 5 ml of DMF, and 192 HlAethyldiisopropylamin and stirred for 2 hours at 00. This is another 48 l of ethyl diisopropylamine and leaves at 0 0 overnight. The crude product is precipitated by dripping the reaction solution into 100 ml of ether and purified in a Craig distribution in the system methanol buffer-chloroform-carbon tetrachloride 10: 3: 7: 4 [buffer as in example lk) j with phase volumes of 3 ml over 650 hours . As usual, the pure hexadecapeptide is isolated as an amorphous powder from distribution elements no.230-275 (rmax = 255; K = 0.65).



   DS: Rf (43C) = 0.37; Rf (52A) = 0.27; R6 (70) = 0.63 h) H-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Tyr (tBu) -Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly - Val-Gly- Ala-Pro -NH2, acetate
250 mg of hexadecapeptide from g) are hydrogenated in 30 ml of 80% acetic acid as usual. After filtering off the catalyst, the filtrate is concentrated to approx. 4 ml and lyophilized.



   DS: Rf (43C) = 0.24; Rf (52) = 0.14; Rf (96) = 0.34 i) Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Tyr (tBu) -Pro-Gln- (Thr (tBu) - Asn-Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
274 mg of Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe NHNH2 are dissolved in 2 ml of DMF while warming and, at -20, successively with 154 l HCl 3.2-n. in dioxane and treated with 37 Hl of tert-butyl nitrite.

  The mixture is stirred for 15 minutes at -100, a solution of 275 mg of the above-mentioned hexadecapeptide acetate in 2 ml of DMF (precooled to 0) and 85 Cll of ethyl diisopropylamine are added and the mixture is stirred for 2 hours at 0 ". After a further addition of 21 l of ethyldiisopropylamine are left to stand at 0 for 15 hours and the crude product is precipitated as a powdery precipitate with ether-petroleum ether (7: 3) .For purification, it is given a Craig distribution in the system methanol-buffer-chloroform carbon tetrachloride 10: 3: 5 : 5 [buffer in example lk)] with phase volumes of 3 ml each over 345 steps.

  The chromatographically pure docosapeptide is isolated as usual from the distribution elements nos. 98-132 (rmaX = 115; K = 0.5).



     DS: R <(43C) = 0.39; Rf (52) = 0.29; Rf (96) = 0.48 j) H-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn Lys (Boc) -Phe-His-Thr (tBu) -Tyr ( tBu) -Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
173 mg docosapeptide from k) are hydrogenated in 30 ml 80% acetic acid with 50 mg Pd-charcoal for 15 hours, the filtrate is concentrated to about 3-4 ml and lyophilized. To remove the acetic acid, the lyophilizate from trifluoroethanol-sodium bicarbonate solution (5%) is reprecipitated.



   DS: Rf (52) = 0.21; Rf (70) = 0.63; Rf (100) = 0.17
EMI7.1
 127 mg of the docosapeptide described under j), 65 mg
EMI7.2
 (DOS 2 050 434), 12 mg N-hydroxy-succinimide and 17.5 mg dicyclohexyl-carbodiimide are dissolved in 0.5 ml DMF or



  suspended and stirred at 45 for 3¸ hours.



   The crude product is precipitated by adding 50 ml of ether and purified by means of Craig distribution in the system methanol buffer-chloroform-carbon tetrachloride 10: 3: 5: 4 [buffer as in example lk) j over 250 stages with phase volumes of 3 m each. From the distribution elements No. 88-110 (rmax = 100; K = 0.67), the chromatographically uniform, protected dot triacontapeptide is isolated as an amorphous powder as usual.



   DS: Rf (52) = 0.28; Rf (70) = 0.65; Rf (100) = 0.27.



   Example 4
EMI7.3

Gly-Val-Gly-Ala-Pro-NH2 are obtained by treating with 12-n.



   HCI at 0, as described in Example 1, cleaved to the free peptide.



     TLC: Rf (45) = 0.47; Rf (101A) = 0.50; R <(1 12E) = 0.42
Thin-layer electrophoresis: pH 1.9, 16 volts / cm, 1¸
Hours: Running distance approx. 3.7 cm to the cathode.



   The protected Dotriacontapeptidamid can e.g. B. be prepared in the following way: a) H-Leu-Thr (tBu) -Gln-Asp (OtBil) -Leu-OMe
3.6 g Z-Leu-Thr (tBu) -Gln-Asp (OtBu) -Leu-OMe (DOS
2 050 434), are hydrogenated to saturation in 90 ml of methanol with 360 mg of Pd carbon with CO2 absorption, filtered off from the catalyst and evaporated to dryness.



   DS: R <(chloroform-methanol) 9: 1) = 0.15; Rf (89) = 0.32 b) Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Leu-OMe
840 mg of H-Leu-Thr (tBu) - Gln-Asp (OtBu) -Leu-OMe are dissolved in 5 ml of DMF, 765 mg of Z-Lys (Boc) -ONp are added and the mixture is left to stand at 22 overnight. It is then concentrated to a syrup in a high vacuum and the crude product is precipitated as a jelly by adding 30 ml of ether. The
Purification is carried out by reprecipitation from methanol-ethyl acetate
Petroleum ether, the pure hexapeptide derivative, has a melting point of
218-220 on.



   DS: R <(chloroform-methanol 9: 1) = 0.41; Rf (89) = 0.61 c) Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Leu-NHNH2
720 mg of the hexapeptide methyl ester described under b) are dissolved in 40 ml of absolute methanol and after
Addition of 3.6 ml of hydrazine hydrate and left to stand at 22 for 5 hours. The resulting hexapeptide hydrazide is precipitated by adding 75 ml of water, filtered off with
Water washed well and dried, m.p. 2152180.



   DS: R <(chloroform-methanol 8: 2) = 0.55; Rf (89) = 0.29 el) Z-Asn-Lys (Boc) -Leu-His-Thr (tBu) -Tyr (tBu) -Pro-Gln
Thr (tBu) -Asn-Thr (tBu) - Giy-Val-Giy-Ala-Pro-NH2
636 mg Z-Asn-Lys (Boc) -Leu-His-NHNH2 (DOS
2 050 434) are dissolved in 10 ml of DMF while warming, cooled to -20 and successively with 525 ul HCl 3.2-n. in dioxane and mixed with 119 l of tert-butyl nitrite. The mixture is stirred for 15 minutes at -15 and then a pre-cooled to 0 solution of 600 mg H-Thr (tBu) -Tyr (tBu)
Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Tly-Val-Gly-Ala-Pro-NH2 [Example 3f)] in 4.5 ml of DMF, and 230M1 of N-methylmorpholine.

  The mixture is stirred for 2 hours at 0, and another 46 l are added
N-methylmorpholine and let stand at 0 overnight. The
Crude product is precipitated with 100 ml of ether and a Craig partition in the acetonitrile-buffer-chloroform system
Methanol 1: 1: 1: 1 [buffer as in Example 1 k) j subjected to over 500 stages with phase volumes of 3 ml each. The pure hexa decapeptide is obtained from distribution elements no. 38-62 (rmax = 50; K = 0.11) by concentrating to dryness and subliming off the buffer at 40 in a high vacuum as an amorphous powder.



     DS: Rf (43C) = 0.38; Rf (52A) = 0.28; Rf (70) = 0.51 e) H-Asn-Lys (Boc) -Leu-His-Thr (tBu) -Tyr (tBu) -Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly - Val -Gly-Ala-Pro-NH2, acetate
380 mg of hexadecapaptide from d) are hydrogenated in 45 ml of 80% acetic acid with 50 mg of Pd-charcoal until the starting product disappears (thin-layer chromatographic control). After the catalyst has been filtered off, it is concentrated to approx. 4 ml and lyophilized.



     DS: Rf (43C) = 0.29; Rf (70) = 0.40 f) Z-Lys (Boc) -Leu-Thr (tBu) - Gln-Asp (OtBu) -Leu-Asn-Lys (Boc) -Leu -His-Thr (tBu) - Tyr (tBu) -Pro-Gln-Thr (tBu) -Asn Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
230 mg of Z-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Leu NHNH2 are dissolved in 2 ml of DMF and at 200 mg in succession with 135 μl of HCl 2,3-n. in dioxane and mixed with 32 l of tert-butyl nitrite. The solution is stirred for 15 minutes at -10, after which a solution of 306 mg of the hexadecapeptide acetate described under e) in 3 ml of DMF and 63 μl N-methylmorpholine is added.

  The mixture is stirred for 2 hours at 0 ", another 15 pol N-methylmorpholine is added, the mixture is left to stand at 0 overnight and the crude product is precipitated by adding 60 ml of ether and 30 ml of petroleum ether Hours in the system methanol-buffer-chloroform-carbon tetrachloride 10: 3: 5: 5 [buffer as in example lk)] Isolation is carried out as usual from the distribution elements No. 83-107 (rmaX = 95; K = 0.65) the pure docosapeptide as an amorphous powder.



   DS: Rf (52) = 0.27; Rf (70) = 0.59; Rf (100) = 0.41 g) H-Lys (Boc) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Leu-Asn Lys (Boc) - Leu-His- Thr (tBu) - Tyr ( tBu) - Pro-Gln- Thr (tBu) Asn- Thr (tBu) - Gly-Val-Gly-Ala-Pro -NH2
100 mg of docosapeptide from f) are hydrogenated in 16 ml of 80% acetic acid with 15 mg of Pd-charcoal overnight, the catalyst is filtered off, the filtrate is concentrated to about 2 ml and lyophilized. To remove the acetic acid, the residue is dissolved in 0.6 ml of trifluoroethanol and reprecipitated by dropping it into 5 ml of sodium hydrogen carbonate solution (5%).



   DS: Rf (52) = 0.18; Rf (70) = 0.50; Rf (100) = 0.20
EMI8.1
   Leu-Gly-0H (DOS 2 050 454), 80 mg of the hydrogenated docosapeptide mentioned above, 7.5 mg of N-hydroxysuccinimide and 10 mg of dicyclohexylcarbodiimide are dissolved in 0.6 ml of DMF, and the mixture is added in an N2 atmosphere for 39 hours 45 stirred. The crude product is then precipitated by adding 15 ml of ether and by means of Craig distribution in the system methanol-buffer-chloroform-carbon tetrachloride 11: 3: 6: 7 [buffer as in example lk)] over 200 stages with phase volumes of 3 each ml cleaned.

  The pure substance is isolated as usual from the distribution elements No. 90-115 (rmax = 100; = 1.0) as an amorphous powder.



   DS: Rf (52) = 0.27; R <(70) = 0.66; Rf (100) = 0.31.



   Example 5
EMI8.2

EMI8.3
 described in Example 1 by treating with 12-n. HCI cleaved at 0 to the free peptide.



     TLC: Rf (45) = 0.43; Rf (10IA) = 0.43; Rd (112E) = 0.38
Thin-layer electrophoresis: pH 1.9; 16 volts / cm, 1¸ hours, distance approx. 4.9 cm to the cathode.



   The protected dotriacontapeptide amide can be prepared as follows:
EMI8.4
 Gly-Val-Gly-Ala-Pro-NH2 [Example 4g)] and 17.2 mg of N-hydroxysuccinimide are dissolved in 1.5 ml of DMF while warming, mixed with 23 mg of dicyclohexylcarbodiimide and stirred at 45 under N2 for 3+ hours.

  The crude product is then precipitated by adding 30 ml of peroxide-free ether and purified in a Craig distribution over 350 stages in the system methanol-buffer-chloroform-carbon tetrachloride 11: 3: 6: 7 [buffer as in example lk)]. The chromatographically pure protected Dotriacontapeptide is isolated from the distribution elements No. 140-179 (rmox = 159; K = 0.83) by concentrating to dryness and subliming off the buffer in a high vacuum at 450,
DS: Rf (52A) = 0.28; Rf (70) = 0.54; R <(100) = 0.29.



   Example 6
EMI8.5
   in 6 ml of 90% trifluoroacetic acid, degassed with nitrogen and left to stand at 23 for 90 minutes. Then it is poured into 50 ml of ice-cold, peroxide-free ether, the precipitate is sucked off, washed with ether and dried in vacuo at 400. 185 mg of Thr2'-calcitonin M are obtained as trifluoroacetate. This is converted into the acetate by elution through a column of Merck ion exchanger No. II (weakly basic, acetate form); the yield is 158 mg of lyophilized acetate.



   To purify, 80 mg of the acetate are dissolved in 5 ml of water and, while stirring, a total of 1.2 ml of Merck ion exchanger No. II (weakly basic, free base form) is established, a pH of about 6.5 and a fine precipitation fails. To complete the precipitation, the mixture is stirred for 2 hours, the pH of the solution increasing to 7.1.

 

   The precipitate and ion exchanger are then sucked off together, washed thoroughly with water and the precipitated peptide is removed from the ion exchanger by adding warm 90% acetic acid. The ion exchanger is filtered off with suction, washed with 90% strength acetic acid and the acetic acid solution is lyophilized. This gives 74 mg of Thr2, -calcitonin M as an acetic acid salt, which, according to thin-layer chromatography and electrophoresis, has a high degree of purity.



      DA: Rf (52) = 49 (calcitonin M: Rf (52) = 0.55);
TLC: Rf (101A) = 0.52 (calcitonin M: Rf (101A) = 0.57).



   In thin-layer electrophoresis (cellulose plates, pH 1.9, 2 hours, 280 V), the distance traveled is 4.5 cm against the cathode (calcitonin M: distance traveled 4.5 cm).



   In the amino acid analysis after total hydrolysis (6 N HCl, 110 ", 24 hours), the product shows the calculated amino acid ratio.



   The starting material can be prepared as follows: a) Z-Ala-Thr (tBu) -Gly-OMe
A mixture of 18.9 g of Z-Ala-ONp, 13.4 g of H Thr (tBu) -Gly-OMe is stirred. HCl, 100 ml of DMF and 6.2 ml of N-methylmorpholine for 17 hours at room temperature, the solvent is removed in a high vacuum, the residue is taken up in ethyl acetate, washed and dried as described in Example 1 under a), the solution evaporates and crystallizes the residue from ether-petroleum ether. M.p. 104-106 "; 16 g.



  b) H-Ala-Thr (tBu) -Gly-OMe
13.3 g of the product obtained under a) are hydrogenated in methanol in the presence of 1.5 g of palladium carbon (10% Pd).



  9.4 g of colorless resin are obtained.



  c) Z-Thr (tBu) -Ala-Thr (tBu) -Gly-OMe
9.4 g of the product obtained under b) and 13.2 g of Z-Thr (tBu) -OSu are dissolved in 100 ml of DMF and left to stand for 17 hours at room temperature. The product is then precipitated by pouring into 500 ml of ice water, suction filtered, dried and crystallized from acetone-petroleum ether with the addition of a little water. M.p. 193-195 ": 13.6 g.



  d) H-Thr (tBu) -Ala-Thr (tBu) -Gly-OMe
13.1 g of the product from c) are hydrogenated in 750 ml of methanol with 2.6 g of palladium carbon. 9.95 g of colorless oil are obtained.



  e) Z-Gln-T7zr (tBu) -Ala-Thr (tBu) -Gly-OMe
This compound is prepared analogously to the pentapeptide derivative written in Example 1 under g. It crystallizes from methanol. 198-202 "; 10.7 g.



     D H-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-OMe
10 g of the product from e) are hydrogenated in 1 liter of methanol with 2 g of palladium carbon. The product (8.2 g) is processed further immediately.



  g) Z-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-OMe
8.2 g of the product from f) are mixed with 8.3 g of Z Thr (tBu) -Phe-Pro-OH (Helv. Chim. Acta 53, 2135 (1970)), and 100 ml of DMF, 4.0 g are added N-hydroxysuccinimide and 4.2 g of dicyclohexylcarbodiimide and the mixture is stirred for 17 hours at room temperature. The dicyclohexylurea is then filtered off with suction, the filtrate is poured into 1.3 l of ice water, the precipitate is suction filtered after 2 hours, dried and stirred with 120 ml of ethyl acetate. The insoluble powder is sucked off, washed with ethyl acetate and dried.

  8.5 g of a product with the following Rr values are obtained: DS: Rt (121A) = 0.78; Rf (102A) = 0.85; Rf (43C) = 0.71 h) Z-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-OH
5.15 g of the product from g) are dissolved in 110 ml of 90% methanol with heating, cooled to 230 and 13.5 ml of 1.0-n are added. aqueous sodium hydroxide solution too. After 10 minutes, 45 ml and, after a further 5 minutes, a further 25 ml of water are added.



  After 20 minutes, the solution is poured into 270 ml of ice-cold 0.05N aqueous hydrochloric acid, the precipitate is filtered off with suction, washed with ice water and with water-acetonitrile (4: 1) and dried. The product shows in the DS: Rf (45) = 0.27; Rf (100) = 0.33; Rf (43C) = 0.28; 4.1 g.



  i) H-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-OH
3.65 g of the product from h) are suspended in 125 ml of DMF and hydrogenated in the presence of 0.5 g of palladium-carbon (10% Pd). The product (2.62 g) is triturated with water, suction filtered and dried.



     DS: Rf (100) = 0.12; Rf (52) = 0.23 j) Z-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-OH
7.2 g of Z-Asn-Lys (Boc) -Phe-His-NH-NH2 (Helv.



  Chim. Acta 53, 2135 (1970)) with heating to 80 in 30 ml of DMF, cools to 300 and gives 7.74 ml of 3.7-n. Add hydrogen chloride in dioxane. Then add 1.42 ml of tert-butyl nitrite at - 300, let react for 10 minutes at -10 ", cool again to 300 and then add dropwise a solution of 6.45 g of H-Thr (tBu) -Phe-Pro- Gln-Thr (tBu) -Ala-Thr (tBu) -Gly OH and 7.8 ml of N-ethyl-diisopropylamine in 70 ml of DMF.



  The mixture is left to stand at 0 for 18 hours and the solution is then poured into 400 ml of ice-cold 1% acetic acid. The precipitate is filtered off with suction, washed with water and dried over calcium chloride in vacuo. The product is digested with acetonitrile, suction filtered and dried.



     DS: Rf (70) = 0.33; Rf (100) = 0.21 Ar) Z-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBul-Ala-Thr (tBu) -Gly-Val-Gly -Ala-Pro-NH2
A mixture of 7.5 g of Z-Asn-Lys (Boc) -Phe His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-OH, 0, 99 g N-hydroxysuccinimide, 1.84 g H-Val-Gly-Ala-Pro-NH2 (Helv. Chim. Acta 1.c.) and 70 ml DMF with 1.11 g dicyclohexylcarboddiimide and stir for 5 hours at 45 ". The mixture is then poured into 1.2 liters of ice-cold ether, the precipitate is suction filtered and dried.The crude product is stirred twice with 200 ml of acetonitrile / methanol (9: 1) each time, suction filtered and dried.



   DS: Rf (45) = 0.50; Rf (96) = 0.46; Rf (100) = 0.29; R, (121) = 0.80; Yield 8.5g.



      1) H-Asn-Lys (Boc) -Phe-His-Thr (tBu) - Phe-Pro-Gln-Thr (tBu) - Aia-TIir (tBu) - Gly-Val-Gly-Ala-Pro-NH2, acetate
7.05 g of the product described under k) are hydrogenated in 400 ml of 80% strength acetic acid in the presence of 1 g of palladium carbon (10% Pd). The product (6.6 g) shows in the DS:
Rf (96) = 0.35; Rf (121) = 0.72; Rf (45) = 0.27 m) Z- Thr (tBu) - Tyr (tBu) - Thr (tBu) - Gln-Asp (Otu) -Pe-Asn-Lys (Boc) - Phe-His-Thr (tBu ) - Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
565 mg of Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln Asp (OtBu) -Phe-NH-NH2 (Helv. Chim. Acta 1. c.) Are dissolved in FIG. 4 with heating to 80 " ml DMF, cools to -20 ,, mixed with 0.35 ml 3.7-n.

  Hydrogen chloride in dioxane and 0.067 ml of tert. Butyl nitrite and stir for 10 minutes at -150 and then a solution, cooled to 0, of 600 mg H-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr is added dropwise (tBu) -Gly-Val- Gly-Ala-Pro-NH2-acetate in 5 ml DMF and 0.33 ml N-ethyldiisopropylamine in addition. The mixture is stirred for 5 minutes at -15 ", allowed to warm to 23 and stirred for 6 hours. Then it is poured into 100 ml of ice water, the precipitate is suction filtered, washed with water and dried in vacuo over calcium chloride. For purification, it is precipitated from methanol. Recirculating water and triturating the precipitated product after drying with acetone, giving 650 mg of product.

 

     DS: Rf (43C) = 0.33; Rf (70) = 0.60 n) H- Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn Lys (Boc) - Phe-His- Thr (tBu) - Phe-Pro-Gln-Thr (tBu) -Ala Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
610 mg of the product obtained under m) are hydrogenated in 25 ml of 80% acetic acid in the presence of palladium carbon.



  The product is dissolved in 50 ml of water-saturated n-butanol, and the solution is washed twice with dilute soda solution to remove the acetic acid, then twice with half-saturated sodium chloride solution and finally with water.



  The butanol phase is then evaporated and 605 mg of the product is obtained as a colorless foam.



     DS: Rf (100) = 0.20; Rf (96) = 0.40
EMI10.1

A mixture of 605 mg of H-Thr (tBu) Tyr (tBu) -Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe- Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2.



   Example 7
EMI10.2
 ice-cold 90% ie trifluoroacetic acid entered, degassed with nitrogen and warmed to room temperature. The mixture is left to stand for 90 minutes at room temperature and then cooled again to 0, the solution is poured into 150 ml of ice-cold, peroxide-free ether, left to stand at 0 for 2 hours, the fine precipitate is filtered off with suction, washed with ether and dried. It is then dissolved in 15 ml of 5% strength acetic acid and filtered through a column of Merck ion exchanger No. II, weakly basic, acetate form, to remove the trifluoroacetic acid. The column is washed 3 times with 10 ml of 5% acetic acid each time, the eluates are combined and lyophilized. 432 mg of Leul2-Thr27-calcitonin macetate are obtained as a colorless, light powder.



   TLC: R (101A) = 0.50;
DA: Rf (52) = 0.45.



   With thin-layer electrophoresis on cellulose plates (pH = 1.9.280 V, 2 hours), the distance from the cathode is 4.5 cm.



   The starting material can be prepared as follows: a) Z-Thr (tBu) -Leu-Thr (tBu) -Gln-Asp (OtBu) -P / le-NH-NH2
12.7 g of H-Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe OMe are dissolved in 100 ml of DMF, 9.0 g of Z-Thr (tBu) -OSu are added and left at 25 for 17 hours Then it is precipitated with water, suction filtered, dried and crystallized from ethyl acetate, yield 14.2 g; for further purification, it is reprecipitated from methanol-water, mp.



     197-199. To convert into the hydrazide, 13.9 g of the Z-Thr (tBu) -Leu-Thr (tBu) -Gln-Asp (OtBu) Phe-OMe obtained are dissolved in 700 ml of methanol, 70 ml of hydrazine hydrate are added and the mixture is left for 45 minutes stand at room temperature. Then it is concentrated in vacuo to a volume of 300 ml and precipitated by adding 500 ml of water. It is filtered off with suction, washed neutral with water and dried. 13.5 of the crude hydrazide are obtained. For purification, it is reprecipitated once from DMF-water and crystallized from methanol. 11 g of crystalline product are obtained, mp 225-227.



  b) Z- Thr (tBu) -Leu- Tltr (tBu) -Gln-Asp (OtBu) - Phe-Asn Lys (Boc) -Phe-His- Thr (tBu) -Phe-Pro-Gln- Thr (tBu) -Ala-Thr (tBu) - Gly-Val- Gly-Ala-Pro -NH2
A suspension of 4.00 g of Z-Thr (tBu) -Leu Thr (tBu) -Gln-Asp (OtBu) -Phe-NHNH2 and 25 ml of DMF is heated to 70 with stirring until everything is dissolved, and the solution is cooled down to 200 and gives 2.50 ml of 3.70-n. Hydrogen chloride in dioxane. 0.48 ml of tert-butyl nitrite is then added dropwise with stirring and cooling to -200, allowed to warm to -10 and stirred at this temperature for a further 15 minutes.

  A solution of 4.2 g of H-Asn-Lys (Boc) -Phe His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Ala-Thr (tBu) -Gly is added dropwise to this Val Gly-Ala-Pro-NH2, acetate in 50 ml of DMF and 1.6 ml of N-ethyldiisopropylamine, allow to warm to 0 and stir for a further 60 minutes, slowly adding another 8.0 ml of N-ethyldiisopropylamine dropwise. The mixture is left to react for 17 hours at 23, during which a gelatinous precipitation occurs. 500 ml of ice-cold, peroxide-free ether are then added, the precipitate is sucked off, washed with ether and dried. The product is triturated with 200 ml of ice water, suction filtered again, dried and precipitated three times from DMF water and twice from DMF ethyl acetate. 4.98 g of product are obtained in this way.



      DS: Rf (70) = 0.61; Rf (100) = 0.38 c) H-Thr (tBu) -Leu-Thu (tBIL) -Gln-Asp (OtBu) -Phe-Asn Lys (Boc) -Phe-His- Vir (tBu) -Phe- Pro-Gln-Thr (tBu) -Ala Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2
3.0 g of the product obtained under b) are dissolved in 200 ml of 90% strength acetic acid and hydrogenated as usual in the presence of 0.5 g of palladium carbon (10% Pd). The acetic acid salt obtained is dissolved in 200 ml of water-saturated n-butanol to remove the acetic acid, and the solution is washed several times with dilute soda solution, dilute sodium chloride solution and finally with water. The strong emulsions that occur when washing the butanol solution are separated through
Centrifuges.

  The butane solution is then evaporated to dryness and 2.15 g of H-Thr (tBu) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (Boc) -Phe-His-Thr ( tBu) -Phe-Pro- Gln- Thr (tBu) -Ala-Thr (tBu) -Gly-Val-Gly-Ala-Pro-NH2 as a colorless resin.



      DS: Rf (96) = 0.37; Rf (100) = 0.19
EMI10.3
   Met-Leu-Gly-OH, 250 mg of N-hydroxysuccininnide and 30 ml of DMF are mixed with 400 mg of dicyclohexylcarbodiimide while stirring and the mixture is stirred for 6 hours at 43 ". The mixture is then cooled to 0 for 3 hours and the precipitated material is sucked off Dicyclohexylurea and the filtrate is poured into 250 ml of ice-cold, peroxide-free ether. The precipitate is filtered off with suction, washed with ether and dried. For purification, the product is reprecipitated three times from DMF water and then three times from DMF acetone receives 1.72 g of purified, protected Leus2-Thr27-Cylcitonin M.



   DS: Rf (52A) = 0.33; R <(100) = 0.30; R (107) = 0.58.



   Example 8
EMI10.4
  
EMI11.1
   90% trifluoroacetic acid, leave to stand for 90 minutes at 25 ", the peptide trifluoroacetate precipitates with peroxide-free ether, sucks it off, washes it with ether, dries it and dissolves it in 1% acetic acid. Then it is filtered through a column from Merck ion exchanger (weakly basic, acetate form), eluted with 1% acetic acid and lyophilized the eluate (116 mg) For purification, the acetate obtained is dissolved in 5 ml of water (pH of the solution = 4.2) and added in portions Stir a total of 2.8 ml of Merck ion exchanger No. II (weakly basic, base form) in. The pH of the solution increases slowly and when the value reaches about 6.7, the free peptide begins to precipitate.

  The mixture is stirred for a total of 2 hours, a pH of 7.1 being reached. Then the precipitate and the ion exchanger are sucked off together, washed with water and the peptide is then dissolved from the ion exchanger by mixing with 90% acetic acid heated to 600. It is suction filtered, rewashed with 90% acetic acid and lyophilized. 98 mg of Leul2-Asn26-Thr27-calcitonin M are obtained as an acetate of high purity. TLC: Rf (101A) = 0.33; DA: Rf (52) = 0.47.



   The inventive purification process described here by precipitation of the peptide in the isoelectric range is the subject of Swiss application Case 4-9157.



   The protected peptide used as starting material can be prepared as follows: a) Z-Thr (tBu) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn Lys (Boc) -Phe-His-Thr (tBu ) -Phe-Pro-Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-Vai-Giy.Aia-Pro-NH2
380 mg of Z-Thr (tBu) -Leu-Thr (tBu) -Gln-Asp (OtBu) -Phe-NHNH2 (cf. Example 7) are dissolved in 5 ml of DMF with stirring and heating to 70 ", and the solution is cooled - 20 "from, gives 0.265 ml 3.71-n. Hydrogen chloride in dioxane and then 0.051 ml of tert-butyl nitrite and allowed to react at 100 for 10 minutes. Then a solution of H-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) Asn-Thr (tBu) -Gly-Val-Gly- Ala-Pro-NH2, acetate (cf.



  Example 1, sub. N) in 5 ml of DMF and 0.20 ml of N-ethyldiisopropylamine are added and the mixture is stirred for 1 hour at 0 ". During this time, a further 0.07 ml of N-ethyldiisopropylamine are added and the mixture is then left warm to room temperature and leave to stand for 1 hour at 25 and 20 hours at 5, gelatinous precipitation now occurring by adding 100 ml of ice water, the precipitate is filtered off with suction after 1 hour at 0, washed with water and dried The powder is triturated with ether, suction filtered and dried again. For purification, the crude product (colorless powder) is reprecipitated once from DMF-water and twice from DMF-acetone. 440 mg of product are obtained.



      DS: Rf (70) = 0.52; Rf (100) = 0.31; Rf (96) = 0.40 b) H- Thr (tBu) -Leu-Thr (tBu) -Gln-Asp (OtBu) - Phe-Asn Lys (Boc) -Phe-His-Thr (tBu) -Phe- Pro -Gln-Thr (tBu) -Asn-Thr (tBu) -Gly-Vai-Gly-Aia-Pro-NH2
260 mg of the product obtained under a) are dissolved in 25 ml of 80% strength acetic acid and hydrogenated as usual in the presence of 50 mg of palladium carbon (10% Pd). The product (266 mg of resin) is dissolved in 20 ml of water-saturated n-butanol to remove acetic acid. It is washed several times with soda solution, dilute sodium chloride solution and water and then the butanol phase is evaporated. Yield: 199 mg.



     DS: Rf (100) = 0.13; Rf (96) = 0.36
EMI11.2

Add to a mixture of 10Z mg Boc- (: ys-tily-Asn-Leu-Ser (tBu) -Thr (tBu) -Cys-Met-Leu-Gly-OH, 199 mg H Thr (tBu) -Leu- Thr (tBu) -Gln-Asp (OtBu) -Phe-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Thr (tBu) Gly-Val -Gly-Ala-Pro-NH2.16 mg N-hydroxysuccinimide and 3 ml DMF with stirring 27 mg dicyclohexylcarbodiimide, stir for 6 hours at 45 ", pour the mixture into ice-cold ether and suck off the precipitate after 2 hours at 0 Washed with ether, dried, the powder dissolved in 5 ml of DMF with heating, cooled to room temperature and the product precipitated by adding 50 ml of ethyl acetate.

  It is filtered off with suction, washed with ethyl acetate, dried, the powder is then dissolved by heating in 10 ml of DMF-methanol (1: 1), cooled in ice and precipitated by adding ice water. After 17 hours at 0, the fine precipitate is filtered off with suction, washed with water and dried in vacuo over calcium chloride. 175 mg of the protected Dotriakontapeptide mentioned in the title are obtained.



      DS: Rf (107) = 0.60; Rf (52A) = 0.23; Rf (100) = 0.22.



   Example 9
EMI11.3

115 mg, finely pulsed, are added to 2.5 ml of ice-cold, 90% strength trifluoroacetic acid, while stirring and degassing with nitrogen
EMI11.4
 Peptides, the solution is allowed to warm to 21 "and left to stand for 90 minutes. Then it is poured into 50 ml of ice-cold, peroxide-free ether, suction filtered and dried. 84 mg of trifluoroacetic acid salt of Asn26-calcitonin M are obtained. For conversion into the acetate it is dissolved in 1% acetic acid and filtered through an ion exchange column from Merck ion exchanger No. II (weakly basic, acetate form) .The eluate is lyophilized and 75 mg of Asn26-calcitonin M is obtained as acetate.



  After total hydrolysis (6 N HCl, 24 hours 110) the product shows the expected amino acid ratio.



   In thin-layer chromatography on cellulose plates, it shows the following Rf values (the comparative values for calcitonin M in brackets): Rf (101A) = 0.55 (0.60); Rf (112A) = 0.58 (0.62).

 

   The protected peptide used as starting material can be prepared as follows: a) Z-Asn-lle-Gly-OMe
A mixture of 9.1 g of H-Ile-Gly-OMe HCI, 16.3 g of Z-Asn-ONp, 50 ml of DMF and 4.1 ml of N-ethylmorpholine is allowed to stand at room temperature for 18 hours. The solution solidifies in a gelatinous state. Then 300 ml of ether are added, triturated, suction filtered, dried, the insoluble powder is rubbed with 200 ml of water, suction filtered, dried and crystallized from ethyl acetate, hexane. 15 g of Z-Asn-Ile-Gly-OMe of melting point are obtained.



     218-219.



  b) H-Asn-lle-Gly-OMe
14 g of Z-Asn-Ile-Gly-OMe are suspended in 500 ml of DMF and hydrogenated with 1 g of palladium carbon (10% Pd). The starting material goes into solution. When the hydrogenation is complete, the catalyst is suction filtered and evaporated to dryness. 9.7 g of H-Asn-Ile-Gly-OMe, which is free from starting material, are obtained.



  c) Z-Thr (tBu) -Asn-lle-Gly-OMe
A mixture of 9.5 g of H-Asn-Ile-Gly-OMe, 80 ml of DMF and 13.6 g of Z-Thr (tBu) -OSu is left to stand at 25 "for 18 hours, complete solidification occurring. 300 ml are added Aether is added, triturated, suction filtered and dried. Then it is triturated with 200 ml of water, suction filtered and dried. The residue is crystallized from ethanol; 12.4 g of 207-208 ".



  d) H-Thr (tBu) -Asn-lle-Gly-OMe
11.0 g of Z-Thr (tBu) -Asn-Ile-Gly-OMe are dissolved in 500 ml of DMF with heating and hydrogenated in the presence of 1 g of palladium-carbon (10% Pd). When the uptake of hydrogen has ended, the catalyst is suctioned off and evaporated to dryness. 8.0 g of H-Thr (tBu) -Asn-Ile-Gly-OMe are obtained; on thin layer chromatography on silica gel plates, Rf (43A) = 0.12; Rf = 0.28 in the chloroform-methanol system (70:30).



  e) Z-Gln-Thr (tBu) -Asn-lle-Gly-OMe
7.5 g of H-Thr (tBu) -Asn-Ile-Gly-OMe are dissolved in 100 ml of DMF and 7.7 g of Z-Gln-ONp are added. The reaction mixture slowly solidifies to a solid mass.



   After 18 hours at room temperature, 375 ml of ether are added, thoroughly ground, suction filtered, dried, triturated with 250 ml of water, suction filtered and dried again. Then it is precipitated from trifluoroethanol-water; 11 g are obtained; M.p.



     275-2770. The product is very sparingly soluble in the usual organic solvents.



     n H-Gln-Thr (tBu) -Asn-lle-Gly-OMe
8.5 g of Z-Gln-Thr (tBu) -Asn-Ile-Gly-OMe are suspended in 500 ml of DMF and hydrogenated in the presence of 1.5 g of palladium-on-carbon (10% Pd). The initially insoluble starting material goes into solution. When the hydrogenation is complete, the catalyst is filtered off with suction and evaporated; 6.5 g of H-Gln-Thr (tBu) - Asn-Ile-Gly-OMe are obtained as resin. In thin layer chromatography on silica gel plates, Rf (43C) = 0.20; Rf (101A) = 0.16.



  g) Z-Thr (tBu) -PIze-Pro-Gin-Thr (tBu) -Asn-lle-Gly-OMe
6.0 g of H-Gln-Thr (tBu) -Asn-Ile-Gly-OMe are dissolved in 500 ml of DMF and mixed with 5.8 g of Z-Thr (tBu) -Phe-Pro-OH, 2.3 g of N Hydroxysuccinimide and 3 g of dicyclohexylcarbodiimide were added.



  The reaction mixture is stirred for 16 hours at room temperature, the dicyclohexylurea is suctioned off, concentrated to a volume of 200 ml and poured into 1.5 liters of ether.



  The mixture is then left to stand at 0 for 3 hours and the fine precipitate is filtered off with suction (10 g of powder). The powder is rubbed with 200 ml of water, suction filtered and dried. 9.6 g of crude product are obtained, which for further purification is first triturated with ethyl acetate.



  It is suction filtered, dried and suspended in acetone, suction filtered again and dried. The insoluble product (9.3 g) is pure enough for further processing. When performed by thin layer chromatography on silica gel plates, Rf (102A) is 0.66.



  h) Z-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly-OH
3.1 g of Z-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly OMe are dissolved with heating in a mixture of 155 ml of methanol, 45 ml of dioxane and 55 ml of water. It is then cooled to room temperature, 8 ml of 1.0-n. NaOH and left to stand for 30 minutes at room temperature.



      The mixture is then cooled to 0 and 150 ml of ice water and 24 ml of 1.0 HCl are added. The precipitate is filtered off with suction, washed with ice water and dried. 2.56 g of powder are obtained which, according to thin-layer chromatography, turns out to be uniform; Rf (45) = 0.21; Rf (100) = 0.19.



  c) H-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-lle-Gly-OH
2.37 g of Z-Thr (tBu) -Phe-Pro-Glan-Thr (tBu) - Asn Ile-Gly-OH are dissolved in 60 ml of 80% acetic acid and hydrogenated in the presence of 200 mg of palladium carbon (10% Pd) . When the hydrogenation has ended, the product is filtered off with suction, evaporated and dried.



  2.28 g of resin are obtained which, on thin layer chromatography, was found to be free from unhydrogenated starting material; Rf (52) = 0.21; Rf (107) = 0.55.



  k) Z-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln Thr (tBu) -Asn-Ile-Gly-OH
1.41 g of Z-Asn-Lys (BOC) -Phe-His-NHNH2 are dissolved in 20 ml of DMF and the mixture is cooled. Then 1.5 ml of 3.2-n.



  HCI in dioxane and the 0.29 ml of tert-butyl nitrite. Then it is allowed to warm to - 120 and stirred for 15 minutes at - 120.



  A solution, cooled to 0, of 1.65 g of H Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly-OH and 1.2 ml of a 40% solution of trimethylbenzylammonium hydroxide is then added dropwise Methanol in 30 ml DMF and 6 ml water. The mixture is stirred while cooling with ice and a further 1.1 ml of N-ethyldiisopropylamine are added in the course of 30 minutes.



   It is left to stand at 0 for 5 hours, a gelatinous filling separating out. The reaction mixture is then poured into 300 ml of 1% acetic acid, stirred for 2 hours at 0 and the precipitate is filtered off with suction. It is washed with water, dried, treated with 50 ml of methanol and stirred for 2 hours at room temperature.



  Then the methanol-insoluble Z-Asn-Lys (Boc) Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly-OH is sucked off.



   399 mg are obtained; for thin layer chromatography on silica gel plates, Rf (100) = 0.18; Rf (52) = 0.24; Rf (70) = 0.33.



     i) Z-Asn-Lys (boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly-Val-Gly-Ala-Pro-NH2
A mixture of 420 mg of Z-Asn-Lys (Boc) is added
Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly-OH,
55 mg of N-hydroxysuccinimide and 7 ml of DMF with stirring
62 mg dicyclohexylcarbodiimide and 103 mg H-Val-Gly-Ala
Pro-NH2. The mixture is then heated to 45 "and stirred for 5 hours at this temperature. It is then cooled to 0 and poured into 80 ml of ice-cold ether.

  The precipitate is filtered off with suction, dried and, for further purification, reprecipitated once from DMF-acetone and then from DMF-acetonitrile; 420 mg of the hexadetrapeptide mentioned in the title are obtained; for thin layer chromatography on silica gel plates, Rf (100) = 0.25; Rf (107) = 0.60.



  m) H-Asn-Lys (Boc) -Phe-His-Thr (tBu) -Phe-Pro-Gln-Thr (tBu) -Asn-Ile-Gly-Vai-Giy-Ala.Pro-NH2
395 mg of the product obtained under 1) are dissolved in 25 ml of 80% strength acetic acid and hydrogenated in the presence of 100 mg of palladium carbon (10% Pd). When the hydrogenation has ended, the catalyst is filtered off with suction and evaporated to dryness. 380 mg of product are obtained which are free from the starting material according to thin layer chromatography on silica gel plates; Rf (107) = 0.30.

 

  n) Z- Thr (tBu) - Tyr (tBu) - Thr (tBu) - Gln-Asp (OtSu) -Phe-Asn- Lys (Boc) -Phe-His- Thr (tBu) -Phe-Pro-Gln- Thr (tBu) -Asn-Ile-
Gly-Val-Gly-Ala-Pro-NH2
Dissolve 358 mg of Z-Thr (tBu) -Tyr (tBu) -Thr (tBu) -Gln Asp (OtBu) - Phe-NHNH2 by heating in 3 ml of DMF, cool to -200 and add 0.26 ml of 3.2 -n. HCI in dioxane and then 0.045 ml of t-butyl nitrite. The mixture is stirred for 15 minutes at - 150.



      A solution, cooled to 0, of 380 mg of the product obtained under m) in a mixture of 10 ml of DMF, 2 ml of water, 1 ml of acetonitrile, 2 ml of dimethyl sulfoxide and 0.2 ml of N-ethyldiisopropylamine, is then added and the mixture is stirred for 18 hours 00. The precipitated product is suction filtered and reprecipitated twice from DMF / ethyl acetate for purification. The powder is then stirred with 20 ml of methanol, and the methanol-insoluble product is filtered off with suction and dried. 240 mg of the peptide mentioned in the title are obtained. It shows Rf (43C) = 0.40 by thin layer chromatography on silica gel plates; Rf (100) = 0.28; Rf (96) = 0.42.



  o) H-Thr (tBu) -Tyr (tBu) -Thr (tBu) - Gln-Asp (OtBu) -Phe-Asn Lys (Boc) - Phe-Hls-Thr (tBu) -Phe-Pro.Gln-Thr (tBu) - Asn-Ile Gly-Val-Gly-Ala-Pro-NH2
220 mg of the product obtained under n) are dissolved in 15 ml of 80% strength acetic acid and hydrogenated in the presence of 20 mg of palladium carbon (10% Pd). When the hydrogenation has ended, the catalyst is filtered off with suction and evaporated to dryness. 210 mg of residue are obtained. To remove the acetic acid, the residue is dissolved in 15 ml of water-saturated n-butanol and the solution is washed with dilute NaHCO3 solution and then several times with water. The butanol phase is then evaporated to dryness, 202 mg of the acetic acid-free peptide mentioned in the title being obtained. Thin layer chromatography on silica gel plates shows Rf (96) = 0.40; Rf (43C) = 0.32.
EMI13.1




  peptide obtained under (o), 16 mg of N-hydroxybenzotriazole and 3 ml of DMF with 25 mg of dicyclohexylcarbodiimide and stirred under nitrogen for 5 hours at 45 ". The reaction mixture is then poured into 50 ml of ice-cold peroxide-free ether and the fine precipitate is filtered off with suction. For cleaning it is reprecipitated twice from DMF-water and twice from DMF-ethyl acetate, giving 120 mg of the protected peptide mentioned in the title. Thin-layer chromatography on silica gel plates gives Rf (52A) = 0.31; Rf (100) = 0.17; Rf (107) = 0.65.



   PATENT CLAIM 1
Process for the preparation of new dotriacontapeptide amides of the formula I.
EMI13.2
 wherein R represents hydrogen or a free or acylated amino group and X represents the L-methionine, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine or La-aminobutyric acid radical and wherein L -Alanine26 is replaced by L-asparagine and / or L-isoleucine27 by L-threonine and in which one or more of the amino acids in positions 11, 12, 16, 19, 22 and 24 are replaced by another amino acid, namely L. -Threonine "against L-Lysine, L-Tyrosins2 against L-Leucine, L-Phenylalanini6 against L-Leucine, L-Phenylalanint9 against L-Leucine,

   L-phenylalanine22 against L-tyrosine and L-glutamine24 against L-arginine or its acid addition salts, characterized in that the corresponding amino acids, where proline32 is optionally present as an amino acid amide and cysteine optionally as deamino acid or N-acylated amino acid, or their derivatives with activated terminal carboxy group or



  activated a-amino group, with intermediate protection of the functional groups to be excluded from the reaction, with the formation of the peptide bonds correspondingly condensed with one another, the two mercapto groups in the first part of the sequence being combined by oxidation to form the disulfide bridge.



   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 ester group.



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



   3. Process according to claim I, characterized in that hydroxyl groups in the side chains to be excluded from the reaction are protected by the tert-butyl ether group.



   4. The method according to claim I, characterized in that a protected peptide of the formula I in which R is the NH2 group and X is L-methionine and in which L-alanine26 by L-asparagine and L-isoleucine27 by L threonine is replaced and where amino groups by the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids.



   5. The method according to claim I, characterized in that a protected peptide of the formula I in which R is hydrogen and X is L-methionine and in which L Alanini6 is replaced by L-asparagine and L-isoleucine27 by L-threonine and wherein amino groups through the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids.



   6. The method according to claim I, characterized in that desamino-Val8-Lystt-Leu12-Tyr22-Asn26-Thr27-calcitonin M, wherein amino groups through the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids.

 

   7. The method according to claim I, characterized in that desamino-Lyslt-Leut2st6jt9-Tyr22- Asn26 Thr27-calcitonin M, wherein amino groups through the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids.



   8. The method according to claim I, characterized in that Lyst-Leul2Xt6fi19-Tyr22-Asn26-Thr27 calcitonin M, in which amino groups are protected by the tert-butyloxycarbonyl group, carboxyl groups by the tert-butyloxy group and hydroxyl groups by the tert-butyl group are split off the protective groups by means of acids.



   PATENT CLAIM II
Use of according to the method of claim I.

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



   

 

Claims (1)

** WARNING ** Beginning of CLMS field could overlap end of DESC **. under m) product obtained in a mixture of 10 ml of DMF, 2 ml of water, 1 ml of acetonitrile, 2 ml of dimethyl sulfoxide and 0.2 ml of N-ethyldiisopropylamine and stirred for 18 hours at 00. The precipitated product is suction filtered and cleaned twice DMF ethyl acetate reprecipitated. The powder is then stirred with 20 ml of methanol, and the methanol-insoluble product is filtered off with suction and dried. 240 mg of the peptide mentioned in the title are obtained. It shows Rf (43C) = 0.40 by thin layer chromatography on silica gel plates; Rf (100) = 0.28; Rf (96) = 0.42. o) H-Thr (tBu) -Tyr (tBu) -Thr (tBu) - Gln-Asp (OtBu) -Phe-Asn Lys (Boc) - Phe-Hls-Thr (tBu) -Phe-Pro.Gln-Thr (tBu) - Asn-Ile Gly-Val-Gly-Ala-Pro-NH2 220 mg of the product obtained under n) are dissolved in 15 ml of 80% strength acetic acid and hydrogenated in the presence of 20 mg of palladium carbon (10% Pd). When the hydrogenation has ended, the catalyst is filtered off with suction and evaporated to dryness. 210 mg of residue are obtained. To remove the acetic acid, the residue is dissolved in 15 ml of water-saturated n-butanol and the solution is washed with dilute NaHCO3 solution and then several times with water. The butanol phase is then evaporated to dryness, 202 mg of the acetic acid-free peptide mentioned in the title being obtained. Thin layer chromatography on silica gel plates shows Rf (96) = 0.40; Rf (43C) = 0.32. EMI13.1 peptide obtained under (o), 16 mg of N-hydroxybenzotriazole and 3 ml of DMF with 25 mg of dicyclohexylcarbodiimide and stirred under nitrogen for 5 hours at 45 ". The reaction mixture is then poured into 50 ml of ice-cold peroxide-free ether and the fine precipitate is filtered off with suction. For cleaning it is reprecipitated twice from DMF-water and twice from DMF-ethyl acetate, giving 120 mg of the protected peptide mentioned in the title. Thin-layer chromatography on silica gel plates gives Rf (52A) = 0.31; Rf (100) = 0.17; Rf (107) = 0.65. PATENT CLAIM 1 Process for the preparation of new dotriacontapeptide amides of the formula I. EMI13.2 wherein R represents hydrogen or a free or acylated amino group and X represents the L-methionine, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine or La-aminobutyric acid radical and wherein L -Alanine26 is replaced by L-asparagine and / or L-isoleucine27 by L-threonine and in which one or more of the amino acids in positions 11, 12, 16, 19, 22 and 24 are replaced by another amino acid, namely L. -Threonine "against L-Lysine, L-Tyrosins2 against L-Leucine, L-Phenylalanini6 against L-Leucine, L-Phenylalanint9 against L-Leucine, L-phenylalanine22 against L-tyrosine and L-glutamine24 against L-arginine or its acid addition salts, characterized in that the corresponding amino acids, where proline32 is optionally present as an amino acid amide and cysteine optionally as deamino acid or N-acylated amino acid, or their derivatives with activated terminal carboxy group or activated a-amino group, with intermediate protection of the functional groups to be excluded from the reaction, with the formation of the peptide bonds correspondingly condensed with one another, the two mercapto groups in the first part of the sequence being combined by oxidation to form the disulfide bridge. 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 ester group. 2. The method according to claim I, characterized in that amino groups to be excluded from the reaction are protected by the tert-butyloxycarbonyl group. 3. Process according to claim I, characterized in that hydroxyl groups in the side chains to be excluded from the reaction are protected by the tert-butyl ether group. 4. The method according to claim I, characterized in that a protected peptide of the formula I in which R is the NH2 group and X is L-methionine and in which L-alanine26 by L-asparagine and L-isoleucine27 by L threonine is replaced and where amino groups by the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids. 5. The method according to claim I, characterized in that a protected peptide of the formula I in which R is hydrogen and X is L-methionine and in which L Alanini6 is replaced by L-asparagine and L-isoleucine27 by L-threonine and wherein amino groups through the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids. 6. The method according to claim I, characterized in that desamino-Val8-Lystt-Leu12-Tyr22-Asn26-Thr27-calcitonin M, wherein amino groups through the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids. 7. The method according to claim I, characterized in that desamino-Lyslt-Leut2st6jt9-Tyr22- Asn26 Thr27-calcitonin M, wherein amino groups through the tert. Butyloxycarbonyl group, carboxyl groups through the tert. Butyloxy group and hydroxyl groups are protected by the tert-butyl group, which splits off the protective groups by means of acids. 8. The method according to claim I, characterized in that Lyst-Leul2Xt6fi19-Tyr22-Asn26-Thr27 calcitonin M, in which amino groups are protected by the tert-butyloxycarbonyl group, carboxyl groups by the tert-butyloxy group and hydroxyl groups by the tert-butyl group are split off the protective groups by means of acids. PATENT CLAIM II Use of according to the method of claim I. obtained peptides for the production of sparingly soluble zinc complexes of these peptides, characterized in that the peptides are reacted with zinc phosphate, zinc pyrophosphate, zinc polyphosphate and / or zinc hydroxide.