DE2025791C2 - Dotriacontapeptides, processes for their preparation and pharmaceuticals containing these compounds - Google Patents

Description

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H, R-C- or R'O —C -

R '

Alkyl with 1 to 3 carbon atoms, tert-butyl or one optionally with a halogen atom, a lower alkoxy group or phenyl or benzyl radical substituted by the nitro group,

Ethyl, propyl, allyl, benzyl, p-nitrobenzyl, p-chlorobenzyl, p-bromobenzyl, p-phenylazobenzyl or p-methoxyphenylazobenzyl

mean

and in the

in position 15

the L-glutamyl residue against an L-glutaminyl,

an L-asparaginyl or an L-aspartyl

rest,
in position 19

the L-Leucy residue against an L-tyrosyl residue and in position 22

the L-Tyrosy! rest against an L-Phenylalani-

nylrest can be exchanged,
as well as their therapeutically effective acid addition salts and heavy metal complexes.

2. H -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro- Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-PrO-NH ₂ .

3. A method for producing the Dotriacontapeptide according to claim 1, characterized in that that these compounds are prepared by methods known per se from peptide chemistry and, if appropriate, then in a manner known per se in their therapeutically effective manner Acid addition salts with organic or inorganic acids or in their heavy metal complexes convicted.

4. Medicaments containing compounds of the formula given in claims 1 and 2.

The invention relates to dotriacontapeptides of the general formula

X-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg- Thr-Asn-Thr-Gly-Ser-Glv-Thr-PrO-NH ₂ ,

X H, R — C— or R'O —C -

Alkyl having 1 to 3 carbon atoms, tert-butyl or a phenyl or benzyl radical optionally substituted by a halogen atom, a lower alkoxy group or the nitro group, R 'ethyl, propyl, allyl, benzyl, p-nitrobenzyl. p-chlorobenzyl, p-bromobenzyl, p-phenylazobenzyl or p-methoxyphenylazobenzyl
mean
and in the
in position 15

the L-glutamyl residue against an L-glutaminyl-. an L-asparaginyl or an L-aspartyl resl,
in position 19

the L-leucyl residue against an L-tyrosyl residue and
in position 22

the L-Tyrosylresl against an L-Phenylalaninylrest can be exchanged,

as well as their therapeutically effective acid addition salts and heavy metal complexes.

The polypeptides or polypeptide derivatives of the general formula can be used for the synthesis of Compounds of this type are prepared using well-known methods, the amino acids in the in of the above formula individually or after the formation of smaller peptide units linked together. The amino acid and / or peptide units are linked e.g. Am the way that an amino acid or a peptide with a protected at-amino group and activated terminal carboxyl group with an amino acid or a Reacts peptide with free (X-amino group and free or protected terminal carboxyl group or that you have an amino acid or a peptide with activated Λ-amino group and protected terminal Carboxyl group with an amino acid or a peptide with a free terminal carboxyl group and protected «amino group implemented.

The carboxyl group can, for example, be converted into an acid azide, anhydride, imidazolide, -isoxazolide or an activated ester or by reaction by means of a carbodiimide or N, N'-carbonyl-diimidazole to be activated. Preferably, the condensation method is the carbodiimide method, which Azide method, the activated ester method, the anhydride method and the Merrifield method are used.

The introduction of a possibly desired on the end product on the terminal L-hemicystine molecule Protecting group can be at any stage before

last stage.

In the last stage of the condensation, however, methods are to be used in which racemization does not occur or can be kept low, preferably by using the azides or the activated ester, the activation preferably being carried out with N-hydroxysuccinimide.

Free, functional groups not involved in the reaction can be used in the construction of the inventive Peptides can be protected as follows:

For blocking the guanido group of the arginine residue in the partial sequence B described below the nitro group was used, but other suitable protective groups such as the tosyl group, the p-nitrocarbobenzoxy group or the 2- (isopropyloxycarbonyl) -3,4,5,6-tetrachlorobenzoyl group be used. One can also use the protective effect of protonation of the guanido group during synthesis use.

When building the polypeptides or polypeptide derivatives, blocking the ^ -carboxyl group, for example, in the partial sequence D described below, the tert-butyloxy group has proven successful, but can also use other protective groups, such as methoxy, ethoxy, tert-amyloxy, amide or Benzyloxy group can be used.

For blocking the ω-amino group of the lysine residue, for example in the one described below Partial sequence C, a carbo-tert-alkoxy group, preferably the carbo-tert-butoxy group, can be used will.

The mercapto protecting group used in the partial sequence EF described below is preferably used the benzyl or trityl group. Usually those are used to protect the SH groups Benzyl or trityl residues at the end of the synthesis by treatment with sodium in liquid ammonia cleaved. It has now been found that the elimination of the benzyl or trityl protective groups and the Establishing the S — S bond before the last stage leads to particularly good yields of the end product.

The conversion of a protected mercapto or amino group into a free group and the conversion a functionally modified carboxyl group into a free carboxyl group in the course of the process for the production of the polypeptides takes place according to methods known per se by treatment with hydrolyzing or reducing agents.

The starting products for the production of the polypeptides or polypeptide derivatives can, if they were previously were not known, can be obtained by the methods known for peptide chemistry, the Amino acids are linked individually or after prior formation of smaller peptide units.

The polypeptides or polypeptide derivatives can also be obtained or used in the form of their salts. as Salts come those with organic acids, such as acetic acid, lactic acid, succinic acid, Benzoic acid, salicylic acid, methanesulfonic acid, toluenesulfonic acid and polymeric acids such as tannic acid or carboxymethyl cellulose and salts with inorganic acids such as hydrohalic acids, e.g. B. Hydrochloric acid or sulfuric acid and phosphoric acid in question. As a heavy metal complex z. B. the one from zinc * ® in question.

The compounds prepared according to the invention represent an important therapeutic principle and can be used as remedies. So they lower z. B. the calcium plasma level and act as antagonists of the parathyroid hormone a positive calcium balance in the bones. The biological effect of the new compounds was tested on rats, resulting in a value of Yielded 3000-5000 MRC units / mg peptide.

The dose to be administered depends on the desired effect and the type of application

ίο off. In general, however, with a once too administering daily dose of 1 to 10 units per kg of animal weight achieved satisfactory results. For For larger mammals, the daily dose is about 70 to 700 units, taken all at once or in multiple portions can be administered. For intramuscular administration contains a suitable form of administration about 70 to 700 units of the active ingredient mixed with a liquid carrier.

The compounds produced according to the invention are thus indicated in all conditions in which lowering the plasma calcium level or influencing bone metabolism is desired, e.g. B. Hypercalcaemia as a result of a deficiency in endogenous thyreocalcitonin due to the loss of thyroid tissue or hyperfunction of the parathyroid glands. They are also indicated for all bone affections that lead to based on increased degradation or in which a calciurtifixation in the bone is desired, z. B. Osteoporosis of various origins (e.g. post-climacteric, post-traumatic, caused by corticosteroid therapy or inactivity, etc.), fractures, osteomalacia, rickets and renal osteodystrophy, as well especially for combination therapy with calcium or phosphate.

An advantage of the polypeptides or polypeptide derivatives according to the invention of the general formula wherein X is R-CO- or R'-O-CO- is their resistance to the degrading action of aminopeptidases.

The compounds prepared according to the invention can be used as medicaments, e.g. B. in the form of pharmaceutical preparations, use. These contain the compounds mentioned in a mixture with an organic or inorganic carrier material suitable for parenteral administration. They can also be administered in the form of a depot preparation.
The following abbreviations are used:

Z = carbobenzoxy (benzyloxycarbonyl)

BzI = benzyl

BOC = tert-butyloxycarbonyl

Trt = trityl = triphenylmethyl

OTB = tert-butyloxy

ONP = p-nitrophenyl ester

OCP = 2,4,5-trichlorophenoxy

OMe = methoxy

OEt = ethoxy

NO ₂ = nitro

Ser = L-seryl

bo Asn = L-asparaginyl

Leu = L-leucyl

Thr = L-threonyl

VaI = L-valyl

Tyr = L-tyrosyl

h5 Arg = L-arginyl

GIn = L-glutaminyl

GIu = L-glutamyl

His = L-histidyl

Per L-prolyl GIy Glycyl Lys = L-lysyl Cys L-cysteinyl Piv = Pivaloyl OSu N-oxysuccinimide EOC Ethoxycarbonyl DCHA = Dicyclohexylamine

In the following examples, which explain how the process is carried out, all temperatures are given in degrees Celsius. The value c for the optical rotation is 1. The amino acid analysis in the following examples shows that the individual amino acids are present in the expected ratios.

Partial sequence A:

H-Ser-Gly-Thr-Pro-NH ₂
a) H-Thr-Pro-NH ₂ · HCl

134 g of Z-Thr-NH-NH _{2 are dissolved} in 21 1 N hydrochloric acid at -5 °, and 0.55 1 N sodium nitrite is added. After 5 minutes, potassium carbonate is added to pH 9, the azide formed is extracted with ethyl acetate and a solution of 80 g of H-PrO-NH ₂ · hydrochloride in 100 ml of water, 500 ml of dimethylformamide and 77 ml of triethylamine is added. The ethyl acetate is evaporated at 20 ° in vacuo and left to stand at 25 ° overnight. The remaining solution is evaporated in vacuo, the residue is dissolved in ethyl acetate, the solution is washed with water, dilute hydrochloric acid and an aqueous calcium carbonate solution and dried over sodium sulfate. It is evaporated in vacuo, dissolved in warm ethyl acetate and cooled. Z-Thr-Pro-NHj is obtained; Mp. 148 °, [<x] 'S = -72 ° in 95% acetic acid. 90 g of Z-Thr-Pro-NH _{2 are then dissolved} in 2 l of dioxane and 260 ml of 1 N hydrochloric acid and hydrogenated at 20 ° and normal pressure in the presence of a palladium catalyst. It is filtered, the solution is evaporated in vacuo, the crystalline residue is washed with ethyl acetate and H-Thr-Pro-NH ₂ · HCl is obtained; Mp. 216 °, [«] 'S = -64 ° in 95% acetic acid.

b) Z-Ser-Gly-NHNH ₂

14.5 g of Z-Ser-OH are dissolved in 100 ml of chloroform and 6.1 g of N-methylmorpholine are added; then 8.2 g of isobutyl chloroformate are added dropwise. After 10 minutes, a solution of 6.6 g of Glydn-ethyl ester in 50 ml of chloroform is added and the mixture is stirred at room temperature for 1 hour. The reaction mixture is washed with dilute ammonia and then with hydrochloric acid solution, dried over sodium sulfate and the organic phase is evaporated. The Z-Ser-Gly-OEt obtained is recrystallized from ethyl acetate. Mp. 103 °, [λ] ϊ = + 3 ° in dimethylformamide. 19.4 g of Z-Ser-Gly-OEt are dissolved in 270 ml of ethyl alcohol, 27 ml of hydrazine hydrate are added and the mixture is left to stand for 2 days at room temperature. The crystalline mass is filtered off, washed with methanol and dried. Z-Ser-Gly-NHNH ₂ , melting point 180 °, [*] 'S = + 2 ° in dimethylformamide is obtained.

c) H-Ser-Gly-Thr-Pro-NH ₂

25 ml of a 4 N hydrogen chloride solution in ethyl ether are added to 200 ml of dimethylformamide and 11 g of Z-Ser-Gly-NHNH _{2 are} dissolved therein. 5.4 ml of tert.-butyl nitrite are then added dropwise at -10.degree. C., and 18 ml of triethylamine are then added to the solution while being filtered. At the same time, one dissolves 12 g of H-TTIr-PrO-NH ₂ -HCl in 100 ml of dimethylformamide, gives 6,? ml of triethylamine, the precipitated triethylamine hydrochloride is filtered off and the dipeptide azide solution obtained above is added to the filtrate. The mixture is left to stand for 2 hours at 0 °, evaporated to dryness, the residue is dissolved in a mixture of 500 ml of methanol and 100 ml of water and the resulting solution is passed through a 100 ml

ίο Dowex 50 column (H + form). It is evaporated and the residue is crystallized from methanol / ethyl acetate. Z-Ser-Gly-Thr-Pro-NH _{2 is obtained} with a melting point of 132 ° (with decomposition), [«] J ⁰ = -22 ° in dimethylformamide. Dissolve 22 g of Z-Ser-Gly-Thr-Pro-NH ₂ in a mixture of

440 ml of methanol and 88 ml of water and hydrogenated at 20 ° and normal pressure in the presence of palladium carbon. It is then filtered, evaporated to dryness, the residue washed with ether and dried. H-Ser-Gly-Thr-PrO-NH _{2 is obtained} with a melting point of 95 ° (with decomposition), [α] c? = - 27 ° in dimethylformamide.

Partial sequence B:

BOC-Arg-Thr-Asn-Thr-Gly-NHNH ₂
a) Z-Asn-Thr-Gly-OEt

43 g of Z-Thr-Gly-OEt are dissolved in 500 ml of dimethylformamide and hydrogenated at normal pressure and room temperature in the presence of palladium carbon. It is filtered, 60 g of Z-Asn-OCP are added, left to stand for 16 hours at 25 °, concentrated to about 250 ml, ethyl acetate is added, washed with dilute hydrochloric acid, dried over sodium sulfate and evaporated to dryness. The residue is washed with ethyl acetate and dried. Z-Asn-Thr-Gly-OEt is obtained. Mp. 225 °, [tu] 'S = -4 ° in dimethylformamide.

b) Z-Thr-Asn-Thr-Gly-OEt

29 g of Z-Asn-Thr-Gly-OEt are dissolved in 700 ml of dimethylformamide, hydrogenated at normal pressure and room temperature in the presence of palladium-carbon, filtered and the filtrate is treated with Z-Thr-N ₃ , which is obtained from 30 g of Z-Thr-NHNH _{2 was} produced (see partial sequence A a)). After 12 hours at 0 °, it is evaporated to dryness and the residue is washed with water, ether and ethanol. Z-Thr-Asn-Thr-Gly-OEt, m.p. 230 =, [«] 'S = -8 ° in dimethylformamide is obtained.

c) BOC-Arg (NO ₂ ) -Thr-Asn-Thr-Gly-NHNH ₂

22 g of Z-Thr-Asn-Thr-Gly-OEt are dissolved in 700 ml of dimethylformamide, hydrogenated at normal pressure and room temperature in the presence of palladium-on-carbon and filtered. 23 g of BOC-Arg (NO ₂ ) -OH are dissolved in 500 ml of dimethylacetamide, 12 ml of triethylamine are added, the mixture is cooled to -10 °, 9.4 ml of isobutyl chloroformate is added, allowed to react for 10 minutes and the tetrapeptide solution obtained above is added. After 30 minutes at 25 °, 100 ml of water are added, the solution obtained is treated with Amberlite-IRA-410 (OH form) until the chlorine reaction is negative, filtered and evaporated to dryness. The residue is washed with chloroform, ethyl acetate and ether and dried. BOC-Arg (NO ₂ ) -Thr-Asn-Thr-Gly-OEt is obtained. Mp -100 °, [ _Å ] jp = -4 ° in dimethylformamide.

12 g of BÖC-Arg (NO ₂ ) -Thr-Asn-Thr-Gly-OEt are dissolved in a mixture of 200 ml of acetic acid and 40 ml of water, hydrogenated in the presence of palladium carbon at 20 ° and normal pressure, filtered and evaporated in the

Vacuum at 20 ° to dryness. The residue is dissolved in dimethylformamide and evaporated. This treatment is repeated until acetic acid can no longer be detected. The residue is dissolved in 120 ml of dimethylformamide, 6 ml of hydrazine hydrate are added, the mixture is left to stand for 16 λ hours at 25 ", evaporated, the residue is washed with ether, methanol / ether (1: 1) and then again with ether and dried obtained BOC-Arg-Thr-Asn-Thr-Gly-NHNHj. Mp. 175 ° (with decomposition), [α], = -42 ° in dimethylformamide / HCl in 1 N (I: 1).

Partial sequence AB:

H-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂
2 CH ₃ COOH

13.5 g of BOC-Arg-Thr-Asn-Thr-Gly-NHNH ₂ (partial sequence B) are dissolved in 270 ml of dimethylformamide / water (8: 2), cooled to -15 °, and 40 ml of a 4 N solution of Hydrochloric acid in dioxane and 2.5 ml of tert-butyl nitrite. The mixture is stirred for 10 minutes at -15 °, 28 ml of triethylamine are added, the mixture is filtered off, 10.5 g of H-Ser-Gly-Thr-Pro-NH ₂ (partial sequence A) are added, and the mixture is stirred for 16 hours at 0 °. It is then evaporated to dryness, the residue is washed with ether and dried. The residue is dissolved in methanol, a tenth volume of water and seven times the amount of chloroform are added and the resulting solution is passed through a column of 500 g of silica gel. After elution with an increasing amount of methanol, after evaporation, BOC-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH _{2 is obtained} . This is suspended in 200 ml of a 4N solution of hydrochloric acid in dioxane and stirred for 2 hours at 25 °. It is evaporated to dryness, the residue is dissolved in 0.2 N acetic acid, treated with Amberlit-IRA-410 (acetate form) and the aqueous solution is lyophilized. The residue is then washed with ether, ethyl acetate and again with ether and dried over sodium hydroxide shavings. H-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · 2 CH ₃ COOH is obtained. Mp. 195 ° (with decomposition), [α] f = -64 ° in dimethylformamide / HCl 1 N (I: 1).

Partial sequence C:

Z-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-OH
a) H-Thr-Tyr-Pro-OH

176 g of Z-Tyr (Z) -Pro-OMe are dissolved in 1000 ml of methanol / HCl 1N, hydrogenated in the presence of palladium-carbon at 20 ° and 1 atm. and evaporates. The residue is dissolved in 400 ml of dimethylformamide, 100 in! Triethylamine is added, the triethylamine hydrochloride which has crystallized out is filtered off and the filtrate is treated with Z-Thr-N ₃ (prepared from 87 g of Z-Thr-NHNH ₂ , see partial sequence A a)). The mixture is then left to stand at 0 ° for 16 hours, evaporated to dryness, the residue is dissolved in ethyl acetate, washed successively with dilute hydrochloric acid and dilute ammonia, dried and evaporated. The residue is pulverized in heptane, washed with petroleum ether and dried. Z-Thr-Tyr-Pro-OMe, melting point 80 ° (decomposition), [a] ⁷ S = -23 ° in dimethylformamide is obtained

53 g of Z-Thr-Tyr-Pro-OMe are dissolved in 530 ml of methanol, add 100 ml of 2N sodium hydroxide solution, leave for 1 hour Stand at 25 °, add 50 ml of 2N hydrochloric acid, concentrate on approx. 200 mL add 100 ml more water, adjust the pH 10, the aqueous solution washes twice with ethyl acetate, then made up with 4N hydrochloric acid pH 1, extracted the precipitated tripeptide Z-Thr-Typ-Pro-OH with ethyl acetate, dried and evaporated a. M.p. 80 °, [λ]; = -23 ° in dimethylformamide. The residue is dissolved in a mixture of 500 ml Dioxane and 100 ml of water and hydrogenated at 20 ° and normal pressure in the presence of a palladium catalyst. It is filtered, evaporated to dryness, the residue is washed with ether and dried. You get H-Thr-Tyr-Pro-OH, m.p. 180 ° (with decomposition), [«] / = -31 ° in acetic acid (95%).

b) Z-Leu-Gln-OMe

70 g of Z-GIn-OH are dissolved in 1.51 of dioxane and left as it is a lot of essential diazomethane solution until the

π solution remains yellow in color. Then the Solution evaporated and the white residue processed with ether. One obtains Z-GIn-OMe, m.p. 136 °, [λ]; ' = -21 ° in dimethylformamide. 71 g of ester are dissolved in 1.7 l of methanol. Then 5 g of 10% palladium are applied

jo activated charcoal mixed with 59 ml of a 4 N hydrochloric acid and added to the solution. The whole thing is a 3 hour hydrogenation at room temperature and Subject to normal pressure. Approx. 83% of the theoretical amount of hydrogen is consumed here. Of the

2 ') Catalyst is filtered off and the filtrate is complete evaporated to give H-GIn-OMe · HCl as a viscous oil.

64 g of Z-Leu-OH are dissolved in 200 ml of acetonitrile, 27.7 g of N-hydroxysuccinimide are added and the mixture is cooled

in - 10 ° C. A solution of 50 g is poured into it Dicyclohexylcarbodiimide in 100 ml acetonitrile. The dicyclohexylurea is filtered off in about 30 minutes. The filtrate is mixed with 47.3 g of H-GIn-OMe HCl, dissolved in dimethylformamide, with simultaneous addition

st be from an equivalent N-methylmorpholine. It is left to stand for 4 hours and evaporated. The residue is dissolved in ethyl acetate and washed with 5% sodium bicarbonate solution, 1 N hydrochloric acid and water. The organic phase is dried over sodium sulfate and evaporated in vacuo. After recrystallization from methanol and ether, Z-Leu-Gln-OMe, melting point 179 °, [α] ΐ - - 16 ^C in dimethylformamide is obtained.

c) Z-Lys (BOC) -Leu-Gln-OMe

42 g of Z-Leu-Gln-OMe are dissolved in 1500 ml of methanol. Then 16 g of 10% palladium are applied Activated charcoal was mixed with 25.8 ml of 4 N hydrochloric acid and added to the solution. The whole becomes one 8 hours of hydrogenation at room temperature and

so subjected to normal pressure. Here 95% of the theoretical amount of hydrogen consumed. The catalyst is filtered off and the filtrate is evaporated. H-Leu-Gln-OMe ■ HCl is obtained in the form of a Foam. H-Leu-Gln-OMe · HCl is then dissolved in 300 ml of dimethylformamide, and 34 g of Z-Lys (BOC) -OCP are added and 8.4 ml of triethylamine. After shaking, the mixture is left to stand for 16 hours. Then that will The reaction mixture was evaporated in a vacuum. The residue is treated with water and removed from the water filtered off. Then it is dissolved in a little methanol and precipitated with ether. Obtained after filtration and drying man Z-Lys (BOC) -Leu-Gln-OMe, [a] F = -21 ° in Dimethylformamide

d) Z-His-Lys (BOC) -Leu-Gln-NH-NH2

38 g of Z-Lys (BOC) -Leu-Gln-OMe are dissolved in 300 ml Dimethylformamide, 15 g of 10% palladium carbon are added, hydrogenated at normal pressure and room temperature

and filtered off from the catalyst. Z-His-Nj, prepared from 25.2 g of Z-HiS-NHNH ₂ , is then added, the mixture is left to stand at 20 ° for 5 hours, evaporated, the residue is dissolved in ethyl acetate, washed with water, dried over sodium sulfate, evaporated and washes the residue with ether. Z-His-Lys (BOC) -LeU-GIn-OMe, melting point 120 ° (with decomposition), [λ] '= -19 ° in dimethylformamide is obtained. 33 g of Z-His-Lys (BOC) -LeU-GIn-OMe are dissolved in 330 ml of methanol, 7.5 ml of hydrazine hydrate are added, the mixture is left to stand for two days at 25 ° and then evaporated to dryness. It is then dissolved in dimethylformamide, evaporated and this treatment is repeated until traces of hydrazine are no longer detectable. The residue is washed with ether, water and ether, dried and obtained Z-His-Lys (BOC) -LeU-GIn-NH-NH ₂ , melting point 199 °, [&] ί - -22 ° in methanol.

e) Z-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-OH

40 g of Z-His-Lys (BOC) -Leu-Gln-NH-NH _{3 are dissolved} in 400 ml of dimethylformamide, the mixture is cooled to -20 °, 75 ml of dioxane / HCl 2 N and then 6 ml of tert-butyl nitrite are added, and 10 is stirred Min. At -20 °, add 30 ml of triethylamine and 20 g of H-Thr-Tyr-Pro-OH and leave to react for 16 hours at 25 °. It is then evaporated to dryness, the residue is washed with ether, dilute acetic acid, ether and finally with hot ethyl acetate. It is then dried in a high vacuum and Z-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-OH, m.p. 215 ° (decomp.), [Λ]? = -23 ° in dimethylformamide.

Partial sequence Cl:

Z-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-OH

a) H-Thr-Tyr-Pro-OH

See partial sequence C a).

b) Z-Gln-Thr-Tyr-Pro-OH

40.1 g of Z-GIn-ONP and 38.4 g of H-Thr-Tyr-Pro-OH are dissolved in 500 ml of dimethylformamide, and 12.2 ml of N-methylmorpholine are added. The mixture is left to stand for 16 hours at room temperature and the solution is then completely evaporated in vacuo. The evaporated residue is taken up in ethyl acetate / n-butanol (10: 1) and washed with 1N sulfuric acid / 30% sodium chloride solution (1: 1) and then with 15% sodium chloride solution. After drying over sodium sulfate ■■ the organic phase in vacuo is completely evaporated. The residue is dissolved in chloroform / methanol (1: 1) and filtered through a silica gel column (20 times the amount). Z-Gln-Thr- Tyr-Pro-OH is obtained in the form of a foam. [«]? = -19 ° in dimethylformamide.

c) Z-Lys (BOC) -Tyr-OMe

1433 g of Z-Lys (BOC) -OH · DCHA are dissolved in 600 ml of warm dimethylformamide and, after cooling to 35 °, a solution of 65.1 g of H-Tyr-OMe - HQ in 200 ml of dimethylformamide is added, with DCHA - HQ Quantitatively crystallized out After filtration, 30 g of N-hydroxysuccinimide are added to the filtrate, the mixture is cooled to -15 ° and a solution of 53.6 g of dicyclohexylcarbodiimide in 200 ml of acetonitrile is added. The mixture is shaken for 15 hours at 0 °, the dicyclohexylurea is filtered off and evaporated the filtrate in a vacuum. The residue is dissolved in ethyl acetate, washed with 5% sodium bicarbonate solution, water, 1 N sulfuric acid and water, dried over sodium sulfate and evaporated to a foam. This gives Z-Lys (BOC) -Tyr-OMe, [at]; ' = -6 ° in methanol.

d) Z-His-Lys (BOC) -Tyr-NHNH ₂

113.5 g of Z-Lys (BOC) -Tyr-OMe are in:> Dissolved 1 methanol, with a suspension of 10 g of 10% palladium on activated carbon in 50 ml of 4N hydrochloric acid added and hydrogenated at room temperature and normal pressure. After 3 hours of hydrogenation, 90% the theoretical amount of hydrogen consumed. The catalyst is filtered off and the filtrate on Vacuum completely evaporated. The foam-like H-Lys (BOC) -Tyr-OMe HCI is thin-layer chromato-

Jd graphically uniform and becomes with Z-H1S-N3 as follows coupled: Dissolve 88.5 g of Z-His-NHNHj in 1800 ml of dimethylformamide, cool to -20 °, give 405 ml of one 3.7 N solution of HCl in ether and, dropwise, 36.6 ml of tert-butyl nitrite. After stirring for 10 minutes at

j-, -20 ° are added 270 ml of triethylamine and a solution of H-Lys (BOC) -Tyr-OMe HCI in 600 ml of dimethylformamide, which already contains 27.3 ml of triethylamine, added. After 5 hours, the crystallized material is filtered off Triethylamine ■ HCI from. The filtrate is in

completely evaporated in a high vacuum. One solves the Residue in ethyl acetate / water (2: 1), wash the organic phase four times with 15% sodium chloride solution, dries over sodium sulfate and evaporates. After dissolving the residue in chloroform / metha-

j-, nol (98: 2) and filtration through a silica gel column (25-fold amount) gives Z-His-Lys (BOC) -Tyr-OMe in amorphous form, [λ] ⁷ S = -11 ° in dimethylformamide. 90.4 g of Z-His-Lys (BOC) -Tyr-OMe are dissolved in 300 ml of methanol, and 20 ml of hydrazine hydrate are added. To

The hydrazide formed crystallizes out if left to stand for 4 2 days. There are 300 m! Aether closed, sucks off, washes with ether and dries in a vacuum. This gives Z-His-Lys (BOC) -Tyr-NHNH ₂ , m.p. 187 °, [«] ² S = -24 ° in dimethylformamide.

e) Z-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-OH

36.7 g of Z-Gln-Thr-Tyr-Pro-OH are dissolved in 500 ml of dimethylformamide, 15 g of 10% palladium on activated carbon are added, the mixture is hydrogenated at room temperature and normal pressure, and the catalyst is then filtered off. The filtrate contains H-Gln-Thr-Tyr-Pro-OH. which is coupled with Z-His-Lys (BOC) -Tyr-N3 as follows: 40.4 g of Z-His-Lys (BOC) -Tyr-NHNH2 are dissolved in 400 ml of dimethylformamide, cooled to -20 °, with 88 ml of dioxane / HCl 2 N and then 73 ml of tert-butyl nitrite are added. After stirring for 10 minutes at -20 °, 37 ml of triethylamine and the dimethylformamide solution containing H-Gln-Thr-Tyr-Pro-OH are added . After 5 hours at 20 °, it is completely evaporated in vacuo, the residue is dissolved in ethyl acetate / n-butanol (5: 1), washed with dilute acetic acid with the addition of sodium chloride solution, dried over sodium sulfate and concentrated. The not quite evaporated oil

ether is added, processed well and suction filtered. After drying in a high vacuum, Z-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-OH, m.p. 193 °, [λ] ο ³ = -33 ° is obtained in dimethylformamide.

Partial sequence ABC:

H-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ • 3CHjCO ₂ H

10 g of heptapeptide (partial sequence C) are dissolved in 100 ml of dimethylformamide, evaporated, the residue is dissolved in 100 ml of dimethylformamide, 15 g of N-hydroxysuccinimide are added, the mixture is cooled to 0 °, 5 g of dicyclohexylcarbodiimide are added, allowed to react for 3 hours, and filtered the excreted dicyclohexylurea is concentrated to 30 ml and the resulting heptapeptide oxysuccinimide ester Z-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-OSu is precipitated by adding ether. After washing with ether, the residue is dissolved in 100 ml of dimethylformamide, mixed with 10 g of nonapeptide amide (partial sequence AB) and allowed to react for 16 hours. 500 ml of ethyl acetate are added and the mixture is filtered. The residue is dissolved in dimethylformamide, 10 ml of acetic acid are added and it is again precipitated with ethyl acetate. It is filtered, washed with ethyl acetate and ether and dried. You get

Z-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-Arg-

Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · 2 AcOH, M '"= -19 ° in dimethylformamide, which is dissolved directly in 80% acetic acid. Then 5 g of palladium-carbon are added, at Hydrogenated at normal pressure and room temperature, filtered, evaporated to dryness at 20 ° in a high vacuum and washed with ether. Drying over KOH chips gives the partial sequence ABC, mp 170 ° (with decomposition), [>] f 53 ° in 1 N acetic acid.

Partial sequence ABCl:

H-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ 3CHjCO ₂ H

Using the same method as described for the production of the ABC part- sequence, 10 g of heptapeptide (part-sequence CI) and 10 g of nonapeptide (part-sequence AB) give the part-sequence ABCl, melting point 166 ° (with decomposition), [α] 2 = - 50 ° in 1 N acetic acid.

Partial sequence D:

TrI-GIy-LyS (BOC) -LeU-SCr-GIn-GIu (OTB) -LeU- NHNH ₂

a) Z-Glu (OTB) -Leu-OMe

Dissolve 151 g of Z-Glu (OTB) -OH, 54 g N-Hydroxysuc- cinimid in 700 ml of acetonitrile and cooled to - from 20 °. Then 96.5 g of dicyclohexylcarbodiimide, dissolved in 350 ml of acetonitrile, are added. After about 30 minutes of standing, the resulting dicyclohexylurea is filtered off. 71.2 g of H-Leu-OMe are added to the filtrate, then left to stand for 4 hours and evaporated in vacuo. The solid residue is mixed with ethyl acetate / water (2: 1). The organic phase is then washed with 5% sodium bicarbonate solution, water and with dilute sulfuric acid (pH 3), after washing neutral, dried over sodium sulfate and evaporated. The residue is dissolved with chloroform, to which 1% methanol has been added, and filtered through a silica gel column ( ten times the amount). Z-GIu (OTB) -LeU-OMe, melting point 31 ", [a] S" 12 ° in dimethylformamide is obtained.

b) H-Gln-Glu (OTB) -Leu-OMe

Dissolve 118 g of Z-GIu (OTB) LeU-OMe in 2000 ml Methanol. Then 15 g of 10% palladium on activated charcoal are stirred in 63 ml of 4N hydrochloric acid and poured into the Solution given. The whole thing is a two-hour hydrogenation at room temperature and normal pressure subject. Approx. 85% of the theoretical amount of hydrogen is consumed here. The catalyst will filtered off and the filtrate evaporated completely, with H-Glu (OTB) -Leu-OMe ■ HCI in the form of a amorphous foam is obtained.

91.7 g of Z-GIn-ONP and 93.8 g of H-GIu (OTB) -LeU-OMe · HCl are dissolved in 500 ml of dimethylformamide, and 51 ml of N-methylmorpholine are added. The mixture is left to stand for 15 hours at room temperature and the solution is then completely evaporated in vacuo. The residue thus obtained is m ^; t water well processed and filtered off from the water. It is then recrystallized from methanol. Z-GIn-GIu (OTB) -Leu-OMe, melting point 210 ° (decomp.), [Λ] = -40 ^e in dimethylformamide is obtained.

44.4 g of Z-GIn-GIu (OTB) -Leu-OMc are dissolved in 12CO ml Methanol / dimethylformamide (1: 1). After that will be

15 g of 10% palladium on activated charcoal are added to 50 ml of water and added to the solution. After a hydrogenation time of about 2 hours, almost the quantitative amount of hydrogen has been consumed. The catalyst is filtered off and the filtrate is evaporated in vacuo. The residue is dissolved in ethyl acetate and washed three times with saturated sodium chloride solution. The organic phase is dried over sodium sulfate and the filtrate is evaporated to about 1/3 of the original volume. After adding petroleum ether, the solid tripeptide can be filtered off. H-Gln-Glu (OTB) -Leu-OMe, melting point 123 °, [λ] "^;=; - 14 ° in dimethylformamide is obtained.

c) Z-Lys (BOC) -Leu-Ser-OMe

43 g of H-Leu-Ser-OMe · HCl and 80 g of Z-Lys (BOC) -OCP are dissolved in 400 ml of dimethylformamide 22 ml of triethylamine are added, left to stand for 25 hours at 25 °, the precipitated triethylamine is filtered off and evaporated the filtrate to dryness. The residue is dissolved in ethyl acetate, washed with dilute hydrochloric acid and dilute potassium bicarbonate solution, dry over sodium sulfate, filtered and evaporated. One crystallizes the residue from ethyl acetate / ether and receives Z-LysiBOCJ-Leu-Ser-OMe, melting point 110 °, [λ]? = -14 ° in Dimethylformamide.

d) Trt-Gly-Lys (BOC) - Leu-Ser-N HNH ₂

158 g of Trt-Gly-OH are dissolved in 2000 ml of dichloromethane and 72 ml of triethylamine, the mixture is cooled to -5 °, 50 ml of ethyl chloroformate are added and the mixture is stirred at -5 ° for 10 minutes. At the same time, 300 g of Z-LysiBOCJ-Leu-Ser-OMe are dissolved in 3000 ml of dioxane / water (8: 2 hydrogenated in the presence of palladium-carbon at room temperature and normal pressure, filtered and evaporated. The residue is dissolved in chloroform and mixed with the The mixed anhydride obtained above is left to stand for 1 hour at 0 °, the chloroform solution is washed with dilute acetic acid and then with dilute potassium bicarbonate solution, dried and evaporated to dryness. The residue is dissolved in 1250 ml of methanol, 175 ml of hydrazine hydrate are added and left

Stand at 25 ° for 16 hours. Concentrate to 500 mL mixed with water, filtered, the residue was washed with water and dried over phosphorus pentoxide. Man

obtained Tit-Gly-Lys (BOC) -Leu-Ser-NHNH ₂ , m.p. 204 °, [&] ν = -6 ° in dimethylformamide.

e) Trt-Gly-Lys (BOC) -Leu-Ser-Gln-GIu (OTB) -LeU-NHNH ₂

76 g of Trt-Gly-Lys (BOC) -Leu-Ser-NHNH ₂ m 300 ml of dimethylformamide are dissolved, the mixture is cooled to -20 °, 75 ml of dioxane / HCl 4 N and then 11.6 ml of tert-butyl nitrite are added, and the mixture is stirred 10 minutes at -15 °, treated with 70 ml of triethylamine and with 49.5 g of H-Gln-Glu (OTB) -Leu-OMe. This is followed by stirring at 0 ° for 16 hours. The precipitated triethylamine hydrochloride is filtered off, the filtrate is concentrated to about 100 ml and the residue is treated with water. The precipitated residue is filtered off, which is then washed through with water and finally dried in a high vacuum at 50 °. The residue is boiled with ethyl acetate, washed with ether and dried. The heptapeptide ester obtained Tn-GIy-Lys (BOC) -Leu-Ser-Gln-Glu (OTB) -Leu-OMe (melting point 234 °, [α] f = -20 ° in dimethylformamide) is dissolved in 200 ml of dimethylformamide, mixed with 4 ml of hydrazine and left to stand at 25 ° for 24 hours. After concentrating to about 100 ml, 500 ml of water are added, the precipitated material is filtered off, washed with water until the reaction is neutral and dried over phosphorus pentoxide at 50 °. Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Glu (OTB) -Leu-NHNH ₂ , m.p. 265 °, [α] ■ - ■ '= -18 ° in dimethylformamide / water (8: 2).

Partial sequence ABCD:

H-GIy-LyS (BOC) -LeU-SCr-GIn-GIu (OTB) -LeU-HiS-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser -Gly-Thr-Pro-NH ₂ • 3 AcOH • 3 H ₂ O

3 g of heptapeptide hydrazide (partial sequence D) are dissolved in 30 ml of dimethylformamide, cooled to -20 °, 2 ml of dioxane / HCl 4 N and then 0.3 ml of tert-butyl nitrite are added. The mixture is stirred for 10 minutes at -20 °, 2 ml of triethylamine and 3 g of hexadecapeptide amide (partial sequence ABC) are added, the mixture is stirred for 16 hours at 0 ° and evaporated to dryness. The residue is washed at 0 ° with dilute acetic acid and then with water, filtered, the residue is dissolved in methanol / water (9: 1), filtered through silica gel, the residue is evaporated, washed with ethyl acetate, dissolved in 200 ml of 80% acetic acid on, left to stand for 4 hours at 25 °, evaporated to dryness at 20 °, the residue was washed with ether, ethyl acetate and ether, filtered and dried. The partial sequence ABCD, melting point 240 ° (with decomposition), [α] Ϊ ^Λ = - jo in L ~ ssigSaürc is obtained

Partial sequence Dl:

Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Gln-Leu-NHNH ₂ a) Z-Gln-Leu-OMe

40.1 g of Z-GIn-ONP and 19.1 g of H-Leu-OMe.HCl are dissolved in 300 ml of dimethylformamide, and 34.1 ml are added N-methylmorpholine to and left for 15 hours with occasional shaking at 20 °. The reaction solution is then completely evaporated in a high vacuum. The residue is with Water well processed and filtered off. After crystallization from methanol, Z-Gln-Leu-OMe, mp. 168 °, [a] F = -11 ° in dimethylformamide.

b) H-Gln-Gln-Leu-OMe

Dissolve 27.8 g of Z-Gln-Leu-OMe in 130 ml of glacial acetic acid and mixed with 220 ml of 40% hydrogen bromide in ^r) of glacial acetic acid. The whole is left to stand for 1 hour with occasional shaking. After complete evaporation in vacuo, the residue is pulverized in anhydrous ether. After filtration, the strongly hygroscopic H-Gln-Leu-OMe · HBr in

in an ether-moist state in a drying cabinet at room temperature dried.

77.5 g of Z-GIn-ONP and 81.4 g of H-Gln-Leu-OMe ■ HBr are dissolved in 750 ml of dimethylformamide and mixed with 66.5 ml of N-methylmorpholine. Let 15

Stand for> hours at room temperature and steam thereupon the solution in a vacuum completely. The residue obtained in this way is processed well with water and filtered off from the water. The suction filter residue is processed with acetone and using a press

: ii separated from the acetone phase. After drying in vacuo, Z-Gln-Gln-Leu-OMe, m.p. 238 ", [«] · ■ = -16 ^Γ in dimethylformamide is obtained ml of dimethylformamide, then a suspension of 10 g

r, 10% palladium on activated carbon in 70 ml of water. After a hydrogenation time of about 1 hour at room temperature and normal pressure is the quantitative amount of hydrogen consumed. The catalyst is filtered off and the filtrate was completely evaporated in vacuo.

in The residue is recrystallized from methanol / ether. H-Gln-Gln-Leu-OMe, melting point 151 ^{° C.} , [λ] Jf = -10 ° in dimethylformamide is obtained.

c) Z-Lys (BOC) -Leu-Ser-OMe
" See partial sequence Dc).

d) Trt-GIy-Lys (BOC) -Leu-Ser-NHNH ₂
See partial sequence D d).

e) Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Gln-Leu-NHNH ₂

Using the same procedure as described for partial sequence D e), where H-GIn-GIu (OTB) -Leu-OMe is replaced by the same amount of H-Gln-Gln-Leu-OMe, one obtains Trt-Gly-Lys ( BOC) -Leu-Ser-Gln-Gln-Leu-NHNH ₂ , m.p. 270 °, [α]? = - 12 ° in dimethylformamide / water (8: 2).

Partial sequence ABCDl:

H-Gly-Lys (BOC) -Leu-Ser-Gln-Gln-Leu-His-Lys (BOQ-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-

Gly-Thr-Pro-NH ₂ - 3 AcOH - 3 H ₂ O

When using the same method as for the production of the partial sequence ABCD and replacing the partial sequence D with the same amount of the partial sequence Dl, the partial sequence ABCD1, melting point 245 ° (with decomposition), [a] I ⁰ = -40 ° is obtained in acetic acid.

Partial sequence ABClD:

H-GIy-LyS (BOC) -Leu-Ser-Gln-Glu (OTB) -Leu-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser -Gly-Thr-Pro-NH ₂ • 3 AcOH - 3 H ₂ O

When using the same procedure as for the production of the partial sequence ABCD and under replacement

the partial sequence ABC by the same amount of the partial sequence ABCl, one obtains the partial sequence ABClD, melting point 230 ° (with decomposition), [λ] f = -40 ° in acetic acid.

Partial sequence ABCl Dl:

H-Gly-Lys (BOC) -Leu-Ser-Gln-Gln-Leu-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-

Ser-Gly-Thr-Pro-NH ₂ • 3 AcOH • 3 H ₂ O

When using the same method as for the production of the partial sequence ABCD and replacing the partial sequence ABC with the same amount of the partial sequence ABCl and replacing the partial sequence D with the same amount of the partial sequence Dl, the partial sequence ABClDl is obtained. Mp. 255 ° (dec.), [Tx] S "= -41 ° in acetic acid.

Partial sequence E:
H-Thr-Cys (Bzl) -Val-Leu-OH

a) H-Cys (Bzl) -Val-Leu-OMe · HBr

Dissolve 21.0 g of Z-Cys (Bzl) -OCP and 12.3 g of H-VaI-Leu-OMe-HCl in 120 ml of dimethylformamide. Then 5.9 ml of triethylamine are added, the mixture is left to stand for 16 hours at 25 °, ethyl acetate is added, washed with dilute hydrochloric acid, dried over sodium sulfate, evaporated to dryness and the residue crystallized from ethyl acetate / diethyl ether. Z-Cys (Bzl) -Val-Leu-OMe, melting point 160 °, [α] Ό ⁰ = -28 °, is obtained in dimethylformamide, which is dissolved in 210 ml of a 40% strength solution of hydrogen bromide in glacial acetic acid. The mixture is left to stand at 25 ° for 1 hour, evaporated to dryness and the residue is recrystallized from isopropanol / diethyl ether. H-Cys (Bzl) -Val-Leu-OMe · HBr, melting point 168 °, [tx] l ^c = + 14 ° in dimethylformamide is obtained.

b) H-Thr-Cys (Bzl) -Val-Leu-OMe ■ 1.3 HBr

20 g of Z-Thr-NHNH _{2 are dissolved} in 350 ml of dimethylformamide, the mixture is cooled to -20 °, 100 ml of a solution of 2N hydrochloric acid in dioxane and then 10 ml of tert-butyl nitrite are added. After 10 minutes at -20 °, 45 ml of triethylamine and 25.5 g of H-Cys (Bzl) -Val-Leu-OMe ■ HBr are added and the resulting mixture is shaken at 0 ° for 16 hours. It is evaporated to dryness, the residue is dissolved in a mixture of ethyl acetate / water, the organic phase is washed with dilute hydrochloric acid, dried over sodium sulfate and evaporated, and the residue is recrystallized from ethyl acetate. Z-Thr-Cys (Bzl) -Val-Leu-OMe is obtained; Mp. 208 °, [tx] O ⁰ = -27 ° in dimethylformamide.

20 g of the tetrapeptide obtained above are dissolved in 200 ml of a mixture of trifluoroacetic acid / ethyl acetate (1: 1), passes in a stream of gaseous hydrogen bromide for 1 hour at 0 °, evaporates then one and the residue crystallizes out Methanol / diethyl ether. H-Thr-Cys (Bzl) -Val-Leu-OMe · 1.3 HBr, melting point 202 °, [λ] Jf = -10 ° in Dimethylformamide.

c) H-Thr-Cys (Bzl) -Val-Leu-OH

372 g of H-Thr-Cys (Bzl) -Val-Leu-OMe 1.3 HBr are dissolved in 1800 ml of methanol, and 900 ml are added Add 2 N sodium hydroxide solution, leave to stand at 25 ° for 1 hour, add 240 ml of glacial acetic acid and leave to stand at 0 ° for 2 hours. Man the precipitated crystalline mass is filtered off, washed first with 1N acetic acid, then with Water and dried at 50 ° in a high vacuum. H-Thr-Cys (Bzl) -Val-Leu-OH, melting point 219 °, [«] S" = is obtained - 53 ° in 1 N ammonia.

Partial sequence F:
BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-NHNH ₂

a) H-Asn-Leu-Ser-OMe - HCl

43 g of H-Leu-Ser-OMe · HCl and 53 g of BOC-Asn-ONP are dissolved in 400 ml of dimethylformamide, 22 ml of triethylamine are added, the mixture is left to stand at 25 ° for 16 hours, evaporated to dryness and the crystallized Residue from methanol. BOC-Asn-Leu-Ser-OMe, melting point 190 °, [λ] I ⁰ = -24 ° is obtained in dimethylformamide, which is dissolved in 500 ml of a 4N solution of hydrochloric acid in methanol. The mixture is left to stand at 25 ° for 1 hour, evaporated to dryness, the residue is dissolved in methanol and precipitated with diethyl ether. H-Asn-Leu-Ser-OMe · HCl, melting point 180 °, [λ] I ⁰ = -23 ° in dimethylformamide is obtained.

b) H-Ser-Asn-Leu-Ser-OMe · HCl

39.5 g of BOC-Ser-NHNH _{2 are} dissolved in 500 ml of dimethylformamide, the mixture is cooled to -20 °, 200 ml of a 2 N solution of hydrochloric acid α-dioxane and then 20 ml of tert-butyl nitrite are added. After 10 minutes at -20 °, 90 ml of triethylamine and 38.0 g of H-Asn-Leu-Ser-OMe · HCl are added, then the mixture is stirred for 16 hours at 0 °, evaporated to dryness and the residue is recrystallized from chloroform / diethyl ether. BOC-Ser-Asn-Leu-Ser-OMe, melting point 135 °, [tx] i "= -22 °, is obtained in dimethylformamide, which is dissolved in 420 ml of a 4N hydrochloric acid solution in methanol ° stand, evaporate to dryness and

j5 recrystallizes from methanol / ethyl acetate. H-Ser-Asn-Leu-Ser-OMe · HCl, melting point 155 ° (decomp.), [A] ¹ S = -15 ° in dimethylformamide is obtained.

c) BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-NHNH ₂

Dissolve 18.5 g of H-Ser-Asn-Leu-Ser-OMe · HCl and 18 g of BOC-Cys (Bzl) -ONP in 100 ml of dimethylformamide, add 10 ml of water, 3.5 ml of acetic acid and 5.6 ml of triethylamine, left to stand for 16 hours at 25 °, evaporated to dryness and recrystallized from methanol. Is obtained BOC-Cys (Bzl) -Ser-Asn-Leu-Ser OMe, mp. 182 °, [ei] 'd = -17 ° in dimethylformamide, which is dissolved with gentle heating in 200 ml of dimethylformamide. 200 ml of methanol and 20 ml of hydrazine hydrate are added, the mixture is left to stand for 16 hours at 30 °, it is precipitated with ethyl ether, the precipitate is washed with diethyl ether / methanol (1: 1) and the BOC-Cys (Bzl) -Ser-Asn thus obtained is dried -Leu-Ser-NHNH ₂ , m.p. 224 °, [tx] ? ' = -13 ° in dimethylformamide.

Partial sequence EF:

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH

Dissolve 18.4 g of BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-NHNH ₂ (partial sequence F) in 150 ml of dimethylformamide, cool to -20 °, give 40 ml of a 2N solution of hydrochloric acid in dioxane and 15 ml of tert-butyl nitrite. After 10 minutes at -20 °, 28 ml of triethylamine and 16.2 g of H-Thr-Cys (Bz!) - Val-Leu-OH (partial sequence E) are added and the mixture is stirred at 25 ° for 16 hours. It is then filtered, the solution is evaporated and the residue is washed through with 1N acetic acid. BOC-

230 222/34

CysiBzO-Ser-Asn-Leu-Ser-Thr-CysiBzO-Val-Leu-OH, m.p. 217 °, [λ] i ° = -17 ° in dimethylformamide. The product obtained is dissolved in 5000 ml of dried ammonia, and sodium metal is added with stirring and boiling of the ammonia until it turns deep blue. Ammonium chloride is added for the purpose of decolorization. The mixture is evaporated to dryness and the residue is washed through with 1N acetic acid and acetone. After drying, BOC-Cys-Ser-Asn-Leu-Ser-Thr-

Cys-Val-Leu-OH, m.p. 248 ° (dec.), [<X] 'S 41 ° in

Dimethylformamide / water (3: 1). This is how you solve it obtained nonapeptide in 5000 ml of 0.01 N ammonia, are 1 N hydrogen peroxide with stirring up to negative nitroprussiate reaction, then another 200 ml of glacial acetic acid, filtered and lyophilized

Partial sequence EFl:

Piv-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH

(Partial sequence EF) is dissolved in 300 ml of trifluoroacetic acid, left to stand at 25 ° for 30 minutes, evaporated, the residue is washed with ethyl acetate and dissolved in 300 ml of dimethylformamide. 30 g of pivalic acid p-nitrophenyl ester - which is prepared as described below - and 14 ml of triethylamine are added to this solution, the mixture is left to stand for 16 hours at 25 °, evaporated and the residue is washed with chloroform. It is dissolved in 300 ml of dimethylformamide, 50 ml of water and 50 g of Dowex-50 are added, the mixture is stirred for 15 minutes, filtered, the resin is washed with dimethylformamide and the filtrate is evaporated to dryness. The residue is washed first with chloroform, then with ethyl acetate and dried. You get the _JS

Piv-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH,

Mp. 243 °, \ a \ ² S = -17 ° in dimethylformamide / water (3: 1).

Pivalic acid p-nitrophenyl ester

95 g of pivalic acid and 131 g of p-nitrophenol are dissolved in 1000 ml of ethyl acetate gives 197 g of dicyclohexylcarbodiimide to, stirred for 2 hours at 25 °, filtered, the filtrate evaporated and the residue crystallized from a mixture of ethyl acetate-petroleum ether. Pivalic acid p-nitrophenyl ester is obtained, M.p. 104 °.

60

Partial sequence EF2:

EOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH

Using the same method as for the production of the partial sequence EF1, starting from the partial sequence EF and carbonic acid p-nitrophenyl ethyl ester, which is produced as described below, the partial sequence EF2 is kept e. Mp. 263 °, [«]? = -21 ° in dimethylformamide / water (3: X).

Carbonic acid p-nitrophenyl ethyl ester

200 g of p-nitrophenol and 113 ml of pyridine are dissolved in 1200 ml of ethyl acetate cools to 0 °, 156 ml of ethyl chloroformate are added with stirring, and a further 30 is stirred Minutes at 0 °, washed with water, dried over Na2SC> 4 and evaporated to dryness. One solves the Residue in diethyl ether and the carbonic acid p-nitrophenyl-ethyl ester crystallized by addition of petroleum ether, m.p. 65 °.

example 1

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH.

Mp. 238 ° (dec.), [A \ i '= -18 ° in dimethylformamide / water (3: 1).

a) BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys (BOC) -Leu-Ser-Gln-Glu (OTB) -

Leu-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-

Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-

Pr ₀ -NH ₂ • 2 CH ₃ CO ₂ H • 3 H ₂ O

1.0 g of nonapeptide (partial sequence EF) is dissolved in 10 ml of dimethylformamide, and 1.5 g of N-hydroxysuccinimide are added and 0.52 g of dicyclohexylcarbodiimide, stirred for 6 hours at 25 °, filtered and the filtrate evaporated to dryness. The residue is washed with ethyl acetate and diethyl ether and dried. You get

30 BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OSu,

Mp. 242 °, which is dissolved in 10 ml of dimethylformamide. 3.1 g of tricosapeptide diacetate (partial sequence ABCD) and 1.2 g of N-hydroxysuccinimide are added to this solution and the mixture is stirred at 25 ° for 16 hours. It is evaporated to dryness and the residue is washed with diethyl ether, chloroform and acetone. This gives the crude, protected Dotriacontapeptide, which is dissolved in 50 ml of a mixture of chloroform / methanol / water (70: 30: 5): The solution obtained is layered on a silica gel column (5 × 100 cm) with the above Mixture has been equilibrated. The elution is carried out with an increasing concentration of methanol. The combined fractions, which contain the pure peptide, are evaporated, then washed with diethyl ether and dried over potassium hydroxide in a high vacuum. The title compound is obtained, mp 230 ° (decomp.), [A] ' _D ° = -45 ° in 50% acetic acid.

b) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-

Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-

Gly-Thr-Pro-NH ₂ · hexaacetate · decahydrate

3.3 g of protected diacontapeptide diacetate trihydrate from stage a) are dissolved in 100 ml of trifluoroacetic acid under a nitrogen atmosphere, left to stand for 15 minutes at 20 ° and evaporated to dryness. It is dissolved in 300 ml of 0.2 N acetic acid, treated with 20 ml of Amberlite IRA-410 (acetate), lyophilized, washed with diethyl ether and dried over potassium hydroxide in a high vacuum. The title compound is obtained, m.p. 220 ° (decomp.), [A] ^! D = -46 ° in 50% acetic acid.

Using the same procedure as in Example 1 a)

and b), the following were also described Connections made:

Example 2 Example 5

I I

a) Piv-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys (BOC) -Leu-Ser-Gln-Glu (OTB) - Leu-His-Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-

Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-

PrO-NH ₂ • diacetate • trihydrate

Starting product: partial sequences EfI AND ABCD method according to example 1 a

Mp. 220 ° (decomp.), La] _B ° = -45 ° in 50% acetic acid.

b) Piv-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-GIy-Ser-GIy-Thr-Pro-NH ₂ · Hexaacetate ■ Octahydrate

Method according to Example 1 b

Mp. 216 ° Ia] ² B = -49 ° in 50% acetic acid.

Example 3

a) EOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys (BOC) -Leu-Ser-Gln-Glu (OTB) - Leu-His-Lys (BOC) -Leu-GIn-Thr-Tyr-

Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NHj · Diacetate · trihydrate

Starting product: partial sequences EF2 and ABCD process according to example 1 a

Mp. 230 ° (decomp.), La] _B ° = -45 ° in 50% acetic acid.

b) EOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ Hexaacetate octahydrate

Method according to Example 1 b

Mp. 240 ° (decomp.), La] ² _B = -52 ° in 50% acetic acid.

Method according to Example 1 b

Mp. 220 ° (decomp.), Ia ]% = -60 ° in 50% acetic acid.

Example 4

a) BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys (BOC) -Leu-Ser-Gln-Gln-Leu-His-

Lys (BOC) -Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · 2 CH ₃ CO ₂ H · 3 H ₂ O

Starting products: partial sequences EF and ABCDl method according to example 1 a

Mp. 235 ° (decomp.), La] ² _B ° = -46 ° in 50% acetic acid.

I I

b) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Gln-Leu-His-Lys-

Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · 6 CH ₃ CO ₂ H · 10 H ₂ O

Method according to Example 1 b

M.p. 230 ° (decomp.), La] ² _B ° = -48 ° in 50% acetic acid.

a) BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys (BOC) -Leu-Ser-Gin-Glu (OTB) -Leu-His-Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser -Gly-Thr-Pro-NH ₂ · 2 CH ₃ CO ₂ H · 3 H ₂ O

Starting products: partial sequences EF and ABClD method according to Example 1 a

Mp. 230 ° C (dec.), La] _D ² ° = -45 ° in 50% acetic acid.

b) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-

Ser-Gly-Thr-Pro-NH ₂ · hexaacetate · decahydrate

Method according to Example 1 b
M.p. 216 ° (decomp.), Ia] ² J = -43 °

in 50% acetic acid.

Example 6

a) BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-GIy-LystBOQ-Leu-Ser-Gln-Gln-Leu-His-

Lys (BOC) -Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · diacetate · trihydrate

Starting products: partial sequence EF and ABClDl method according to example 1 a

M.p. 235 ° (decomp.), La] ^! _D ° = -43 ° in 1 N acetic acid / methanol (2: 1).

b) H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Gln-Leu-His-Lys-

Tyr-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · hexaacetate · decahydrate

Method according to Example 1 b

Mp. 216 ° (decomp.), La] '" = -43 ° in 1N acetic acid.

Example 7

H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-

Leu-Gly-Lys-Leu-Ser-Gln-Asp-Leu-His-Lys-Leu-Gln-Thr-Phe-Pro-Arg-Thr-Asn-Thr-Gly-

Ser-Gly-Thr-Pro-NH ₂ · hexaacetate · decahydrate

Partial sequence AB:

H-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · 2 CH ₃ COOH

13.5 g of BOC-Arg-Thr-Asn-Thr-Gly-NHNH ₂ (partial sequence B) are dissolved in 270 ml of dimethylformamide / water (8: 2), cooled to -15 °, and 40 ml of a 4N solution of Hydrochloric acid in dioxane and 2.5 ml of tert-butyl nitrite. The mixture is stirred for 10 minutes at -15 °, 28 ml of triethylamine are added, filtered off, 10.5 g of H-Ser-Gly-Thr-Pro-NH ₂ (partial sequence A) are added, the mixture is stirred for 16 hours at 0 °, then evaporated it is dried to dryness, the residue is washed with ether and dried. The residue is dissolved in methanol, a tenth volume of water and seven times the amount of chloroform are added and the resulting solution is passed through a column of 300 g of silica gel. After elution with an increasing amount of methanol, one obtains

after evaporation BOC-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ . This is suspended in 200 ml of a 4N solution of hydrochloric acid in dioxane and stirred for 2 hours at 25 °. It is evaporated to dryness, the residue is dissolved in 0.2 N acetic acid, treated with Amberlit-IRA-410 (acetate form) and the aqueous solution is lyophilized. The residue is then washed with ether, ethyl acetate and again with ether and dried over sodium hydroxide shavings. H-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · 2CH ₃ COOII is obtained. Mp. 195 ° (with decomposition), [a] f = -64 ° in dimethylformamide / HCIIN (l: 1).

Partial sequence C2:
Z-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro-NHNH ₂ ''

a) Z-Gln-Thr-Phe-Pro-OMe

84.8 g of Z-Thr (t.But.) - OMe are dissolved in 1500 ml of methanol and mixed with 125 ml of 2N hydrochloric acid and 10 g of palladium (10% on charcoal). After 4 hours of hydrogenation, the catalyst is filtered off and complete evaporated.

61.2 g of Z-GIn-OH and 25.6 g of N-hydroxysuccinimide are dissolved in 1000 ml of acetonitrile / dimethylformamide and cooled to -15 °. A solution of 45.7 g of dicyclohexylcarbodiimide in 200 ml of acetonitrile is rinsed in, as is the HCl · H-Thr (t.But.) - OMe obtained above. dissolved in dimethylformamide, with the addition of 30.4 ml of triethylamine. With occasional shaking μ is allowed to stand for 16 hours approximately at room temperature. The crystallized triethylamine hydrochloride or the dicyclohexylurea are filtered off and the filtrate is completely evaporated. The residue is taken up in ethyl acetate and washed with 1 N hydrochloric acid and sodium bicarbonate 5%. After drying over sodium sulfate, the organic phase is concentrated to a volume of approx. 200 ml and petroleum ether is added. The precipitated oil is converted into a powder and filtered off. Z-GIn-Thr (t.But.) - OMe, melting point 106 °, [λ] ¹ S = + 11 ° in dimethylformamide is obtained.

78.0 g of Z-Gln-Thr (t.But.) - OMe are dissolved in 800 ml of methanol, and 65 ml of hydrazine hydrate are added. After 48 hours of standing at room temperature, it is completely evaporated. The residue is treated with water and then recrystallized from methanol / water. Z-Gln-Thr (t.But.) - NHNH ₂ , melting point 202 °, [α] '"= -13 ° in methanol is obtained.

45.3 g of Z-Phe-Pro-OMe are dissolved in 750 ml of methanol dissolved and mixed with 55 ml of 2N hydrochloric acid and 12 g of palladium (10% on charcoal). After hydrogenation for 2 hours the calculated amount of hydrogen is almost quantitatively consumed. The catalyst is filtered off and that The filtrate was completely evaporated.

49.5 g of Z-Gln-Thr (t.But.) - NHNH ₂ are dissolved in 500 ml of dimethylformamide and cooled ^{to - 20 ° C.} After adding 77 ml of 5N hydrochloric acid in ether and 13.4 ml of tert-butyl nitrite, stirring is continued for 10 minutes and the mixture is neutralized with 54 ml of triethylamine. The HCl · H-Phe-Pro-OMe obtained above bo is dissolved in 150 ml of dimethylformamide, mixed with 15.4 ml of triethylamine and rinsed to the dipeptide azide. After stirring for 4 hours at room temperature, the triethylamine hydrochloride which has crystallized out is filtered off and the filtrate is evaporated completely. The residue is filtered through a silica gel column using chloroform / methanol. After crystallization from ethyl acetate / petroleum ether, Z-Gln-Thri.tButO-Phe- ^ ro-OMe, melting point 110 °, [α] ί? = -41 ° in methanol.

The tetrapeptide is dissolved in 250 ml of trifluoroacetic acid. After standing for 20 minutes at room temperature, the solution is concentrated to half its volume and the product is precipitated with ether. By filtration and sufficient washing with ether to 154 after drying, Z-Gln-Thr-Phe-Pro-OMe, mp. ^R, [ «] f = -59 ° in methanol.

b) Z-His-Lys (BOC) -LeU-NHNH ₂

114 g of Z-Lys (BOC) -OH, 48 g of H-Leu-OMe and 69 g of N-hydroxysuccinimide are dissolved in 600 ml of ethyl acetate and cooled to -20 °. A solution of 65 g of dicyclohexylcarbodiimide in 300 ml of ethyl acetate is rinsed in. The mixture is left to stand at room temperature for 16 hours with occasional shaking. Dicyclohexylurea which has crystallized out is filtered off and the filtrate is completely evaporated. The solid residue is treated with water After recrystallization from Essigesfer / petroleum ether obtained Z-Lys (BOC) -Leu-OMe, mp. 113 °, [α] ί ° = -18 ° in methanol.

101.5 g of Z-Lys (BOC) -Leu-OMe are in 1000 ml Dissolve ethyl acetate and add a pre-cooled solution of 40 ml of 5 N hydrochloric acid in ether in 300 ml of ethyl acetate offset. After adding 5 g of palladium (10% on charcoal) it is slurried in 50 ml of dimethylformamide Hydrogenated for 3½ hours. The calculated amount of hydrogen is used almost quantitatively. Afterward the catalyst is filtered off and the filtrate is evaporated.

78.7 g of Z-HiS-NHNH ₂ are dissolved in 400 ml of dimethylformamide, 182 ml of 5N hydrochloric acid in ether are added and the mixture is cooled to -10 °. After adding 32.5 ml of tert-butyl nitrite, stirring is continued for 10 minutes and the mixture is neutralized with 140 ml of triethylamine.

The HCl · H-Lys (BOC) -Leu-OMe obtained above is dissolved in 300 ml of dimethylformamide, mixed with 36.4 ml of triethylamine and rinsed to the azide solution. After stirring for 4 hours at room temperature, the triethylamine hydrochloride which has crystallized out is filtered off and the filtrate is completely evaporated. An Oi is obtained which is dissolved in ethyl acetate. After washing with water, the organic phase is dried over sodium sulfate, filtered and concentrated. Then it is precipitated with petroleum ether and filtered off. The crude product is purified by chromatography on silica gel and elution with chloroform / methanol. Crystallization from ether / petroleum ether gives Z-His-Lys (BOC) -Leu-OMe, mp 146 ", [(X] S ⁰ 28 ° in methanol.

64.5 g of Z-His-Lys (BOC) -Leu-OMe are dissolved in 300 ml of methanol, and 15 ml of hydrazine hydrate are added. After standing for 3 days at room temperature, the solution is completely evaporated. The residue is mixed with ethyl acetate / n-butanol and washed several times with saturated sodium chloride solution. The organic phase is evaporated to dryness and the residue is filtered through a silica gel column with chloroform / methanol. Z-His-Lys (BOC) -LeU-NHNH ₂ , melting point 181 °, [α] = -26 ° in methanol is obtained.

c) Z-His-Lys (BOC) -Leu-Gin-Thr-Phe-Pro-NHNH ₂

38.4 g of Z-Gln-Thr-Phe-Pro-OMe are dissolved in 1500 ml of methanol / dimethylformamide. 10 g palladium (10% on charcoal) are slurried in 50 ml of water and rinsed in. After about 1/2 hour the calculated amount of hydrogen practically consumed.

The catalyst is filtered off and the filtrate is evaporated.

38.6 g of Z-His-Lys (BOC) -Leu-NHNH ₂ are dissolved in 250 ml of dimethylformamide. After cooling to -15 °, 42 ml of 5N hydrochloric acid in ether and 7.3 ml of tert-butyl nitrite are added. It is stirred for 10 minutes and neutralized with 29.5 ml of triethylamine. The H-Gln-Thr-Phe-Pro-OMe obtained above is dissolved in 250 ml of dimethylformamide and rinsed to the peptide azide solution. After stirring for 4 hours at room temperature, the precipitated triethylamine hydrochloride is filtered off and the filtrate is completely evaporated. The residue is taken up in ethyl acetate / n-butanol and washed with 1N ammonia, 1N sulfuric acid and several times with water. The organic phase is completely evaporated and the residue is purified on a silica gel column with chloroform / methanol / water. Z-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro-OMe, m.p. 17Γ, [λ]? = -54 ° in methanol.

43.6 g of Z-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro-OMe are dissolved in 1000 ml of methanol / dimethylformamide, and 44 ml of hydrazine hydrate are added. After standing for 4 days at room temperature, the solution is completely evaporated. The residue is boiled in acetonitrile. After cooling to approx. 20 °, it is filtered off. Z-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro-NHNH ₂ , m.p. 175 °, [α]? = -58 ° in methanol.

Partial sequence D2:

Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Asp (OTB) -LeU-NHNH ₂

a) Z-ASp (OTB) - Leu-OMe

151 g of Z-Asp (OTB) -OH and 54 g of N-hydroxysuccinimide are dissolved in 700 ml of acetonitrile and cools to -20 °. Then 96.5 g of dicyclohexylcarbodiimide are added, dissolved in 350 ml of acetonitrile. After about 30 minutes of standing, the resulting dicyclohexylurea becomes discharged. 71.2 g of H-Leu-OMe are added to the filtrate, then left to stand for 4 hours and evaporated in a vacuum. The solid residue is mixed with ethyl acetate / water. The organic Phase is then added with 5% of the sodium bicarbonate solution. Water and with dilute sulfuric acid (pH 3) washed, dried over sodium sulfate after washing neutral and evaporated Residue with chloroform to which 1% methanol was added and filtered on a silica gel column (ten times the amount). Z-Asp (OTB) -Leu-OMe is obtained. M.p. 58 °, [α]? = - 16 ° in dimethylformamide.

b) Z-Gln-Asp (OTB) -Leu-OMe

118 g of Z-Asp (OTB) -Leu-OMe are dissolved in 2000 ml Methanol. Then 15 g of 10% palladium on activated charcoal are stirred in 63 ml of 4N hydrochloric acid and poured into the Solution given. The whole thing is a two-hour hydrogenation at room temperature and normal pressure subject. Approx. 85% of the theoretical amount of hydrogen is consumed here. The catalyst is filtered off and the filtrate evaporated completely where H-Asp (OTB) -Leu-OMe · HCl in the form of a amorphous foam is obtained.

91.7 g of Z-GIn-ONP and 93.8 g of H-Asp (OTB) -Leu-OMe-HCl are dissolved in 500 ml of dimethylformamide and mixed with 51 ml of N-methylmorpholine. The mixture is left to stand for 15 hours at room temperature and the solution is then evaporated on the vacuum completely. The residue obtained in this way is processed well with water and filtered off from the water. It is then recrystallized from methanol. Z-Gln-Asp (OTB) -Leu-OMe, melting point 189 ° (decomp.), [Α] 'S = -30 ° in dimethylformamide is obtained.

c) Z-Ser-Gln-Asp (OTB) -Leu-OMe

44.4 g of Z-Gln-Asp (OTB) -Leu-OMe are dissolved in 1200 ml of methanol / dimethylformamide. Then 15 g of 10% palladium on activated charcoal are in 50 ml of water added and added to the solution. After about 2 hours of hydrogenation is the calculated amount of hydrogen almost used up. The catalyst is filtered off and the filtrate is evaporated in vacuo.

21.4 g of Z-Ser-NHNH ₂ dissolved in 100 ml of dimethylformamide are cooled to -20 °, and 50.6 ml of 5N hydrochloric acid in ether and 10.3 ml of tert-butyl nitrite are added. After 10 minutes of stirring, the pH is adjusted to 7.5 with 35.6 ml of triethylamine and 34.2 g of H-GIn-Asp (OTB) -Leu-OMe, dissolved in 100 ml of dimethylformamide, are added. The mixture is stirred for 4 hours at 20 °, the precipitated triethylamine hydrochloride is filtered off and the filtrate is completely evaporated. After treatment of the residue with water and subsequent crystallization from water / methanol, the crude product is purified by filtration through a silica gel column with chloroform / methanol. Z-Ser-Gln-Asp (OTB) -Leu-OMe, melting point 172 °, [<x] ^: - '= -24 ° in dimethylformamide is obtained.

d) Trt-Gly-Lys (BOC) -Leu-NHNH ₂

50.8 g of Z-Lys (BOC) -Leu-OMe are in 600 ml Dissolved ethyl acetate and after adding 25 ml of 4N hydrochloric acid in ether and 3 g of palladium (10% on carbon) catalytically hydrogenated. After approx. 4 hours, the calculated amount of hydrogen is almost completely consumed. The catalyst is filtered off and the filtrate is concentrated to half its volume.

33, OgTn-GIy-OH and 12.0 g of N-hydroxysuccinimide are dissolved in 150 ml of dimethylformamide and cooled to -15 °. A solution of 21.6 g of dicyclohexylcarbodiimide in 100 ml of acetonitrile is rinsed in and the mixture is left to stand for 3 hours at room temperature with occasional shaking. The dicyclohexylurea which has crystallized out is filtered off and the dipeptide solution obtained from the above hydrogenation is added to the filtrate with the addition of 25 ml of N-methylmorpholine. After 16 hours of standing, it is completely evaporated. The residue is taken up in ethyl acetate and treated at 0 ° with 1 N sulfuric acid and 5% sodium bicarbonate solution. After drying over sodium sulfate, the organic phase is completely evaporated. Filtration of the resulting foam through a silica gel column with chloroform / methanol gives Trt-Gly-Lys (BOC) -Leu-OMe in the form of an amorphous foam, [«] ² S = -10 ° in methanol.

The residue is dissolved in 300 ml of methanol, 17 ml of hydrazine hydrate are added and the mixture is left to stand at room temperature for 16 hours. After complete evaporation and subsequent drying in a high vacuum, the mixture is filtered through a silica gel column using chloroform / methanol. Crystallization from chloroform / petroleum ether gives Tn-GIy-LyS (BOC) -LeU-NHNH ₂ . M.p. 175 °, [«]? = -12 ° inMethanoL

e) Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Asp (OTB) -Leu-NHNH ₂

26.6 g of Z-Ser-Gln-Asp (OTB) -Leu-OMe are in 1 i.v. Dissolved methanol / dimethylformamide, with 5 g of palladium (10% on carbon), slurried in 100 ml of water, added and subjected to catalytic hydrogenation. After 1 hour is the calculated amount of hydrogen consumed. The catalyst is filtered off and the filtrate is concentrated to remove methanol.

29.6 g of Trt-Gly-Lys (BOC) -Leu-NHNH ₂ are dissolved in 150 ml of dimethylformamide. It is cooled to -20 °, and 26.2 ml of 5N hydrochloric acid in ether and 5.3 ml of tert-butyl nitrite are added. After stirring for 10 minutes, 18.5 ml of triethylamine and the concentrated solution of H-Ser GIr. Asp (OTB) -Leu-OMe added. The mixture is stirred for 4 hours at room temperature, then the precipitated triethylamine hydrochloride is filtered off and the filtrate is completely evaporated. The residue is taken up in ether, processed into a powder and suction filtered. This crude product is purified by filtration through a silica gel column with chloroform / methanol. Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Asp (OTB) -Leu-OMe, melting point 217 °, [α]; is obtained. = -30 ° in methanol.

The heptapeptide is dissolved in 400 ml of methanol, 1.4 ml of hydrazine hydrate are added and the mixture is carried out for 48 hours stirred at room temperature. The precipitated hydrazide is filtered off, washed with ether and until

Dried constant weight. Mp 218 °, [α] ΐ 16 °

in dimethylformamide.

Partial sequence ABC2:

Z-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂

5.4 ml of a 5 are added to a solution, precooled to -18 °, of 1.58 g of Z-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro-NHNH ₂ (partial sequence C2) in 60 ml of dimethylformamide , 65 η solution of hydrochloric acid in dioxane and 0.8 ml of butyl nitrite to. The temperature is kept at -15 to -20 °, 4.5 ml of triethylamine are added and the mixture is filtered to give a cold solution of H-Arg-Thr-Asn-Thr-Gly-Val-Gly-Ala-PrO-NH ₂ in dimethylformamide. After two hours of stirring at 0 °, the solution is concentrated in vacuo. mixed with ethyl acetate and filtered off. The crude product obtained in this way is dissolved in a mixture of methanol / chloroform / water and applied to a silica gel column. It is eluted, the pure fractions are combined and evaporated to dryness in vacuo. This gives Z-His-Lys (Boc) Gln-Thr--Lea-Phe-Pro-Arg-Thr-Asp-Thr-Gly-Ser-Gly-Thr-Pro-NH _2. M.p. approx. 150 °; [λ]? = -52.2 ° in methanol, -27.0 ° in dimethylformamide

Partial sequence ABC2D2: Trt-Gly-Lys (BOC) -Leu-Ser-Gln-Asp (OTB) - Leu-His-Lys (BOC) -Leu-Gln-Thr-Phe-Pro- Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2

4.6 g of hexadecapeptide (partial sequence ABC2) are in 250 ml of methanol / water dissolved, with 2.1 g of palladium (10% on charcoal) slurried in a little water, added and subjected to catalytic hydrogenation. After the hydrogenation has ended, the catalyst is filtered off and the filtrate is completely evaporated.

3.04 g of heptapeptide (partial sequence D2) are dissolved in 20 ml of dimethylformamide. After cooling to -20 °

1.53 ml of 5.87 η hydrochloric acid in ether and 0.31 ml of tert-butyl nitrite are added. It is allowed to continue for 5 Stir for minutes and then neutralize with 1.26 ml of triethylamine. The one obtained in the above hydrogenation

^r > Hexadecapeptide is cooled to -30 ° and the heptaazide solution is filtered in. The reaction mixture is left to stand for a few hours and then evaporated completely. The residue is precipitated by adding ethyl acetate. The raw product is sent to a

ίο silica gel chromatography and with chloroform / Methanol / water eluted. The title compound is obtained with a melting point of 170 ° (decomp.); [α] f = -11.6 ° in Dimethylformamide.

ij partial sequence EF:

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH

Dissolve 18.4 g of BOC-Cys (Bzl) -Ser-Asn-Leu-Ser-NHNH ₂ (partial sequence F) in 150 ml of dimethylformamide, cool to -20 °, give 40 ml of a 2N solution of hydrochloric acid in dioxane and 15 ml of tert-butyl nitrite. After 10 minutes at -20 °, 28 ml of triethylamine and 16.2 g of H-Thr-Cys (Bzl) -Val-Leu-OH (partial sequence E) are added and the mixture is stirred for 16 hours at 25 °. It is then filtered, the solution is evaporated and the residue is washed through with 1N acetic acid. BOC-CysiBzlJ-Ser-Asn-Leu-Ser-Thr-CysiBzlJ-Val-Leu-OH is obtained,

in m.p. 217 °, [α] l ^c = -17 ° in dimethylformamide. The product obtained is dissolved in 5000 ml of dried ammonia, and sodium metal is added with stirring and boiling of the ammonia until it turns deep blue. Ammonium chloride is added for the purpose of discoloration.

j-, joined. It is evaporated to dryness and the residue is washed through with 1N acetic acid and acetone. After drying, BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH, m.p. 248 ° (decomp.), [Α] ί ° = -41 ° in dimethylformamide / water (3 :1). The nonapeptide obtained in this way is dissolved in 5000 ml of 0.01 N ammonia, 1 N hydrogen peroxide is added with stirring until the nitroprussiate reaction is negative, then another 200 ml of glacial acetic acid, filtered and lyophilized. You get

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OH.

Mp. 238 ° (dec.), [A \) " = -18 ° in dimethylformamide / water (3: 1).

example

H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Asp-Leu-His-Lys-

Leu-Gln-Thr-Phe-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂ · hexaacetate ■ decahydrate

900 mg tricosapeptide (part sequence ABC2D2) dissolved in 70 ml of 60% acetic acid and during 20

Left to stand for hours at room temperature. To Adding water is several times with ether

washed. The water phase is eluted by a

AmberIite IRA 410 acetate column and evaporate on it

Solution completely.

1.0 mg of nonapeptide (partial sequence EF) is dissolved in 10 ml of dimethylformamide, 169 mg of N-hydroxysuccinimide and 0.52 g of dicyclohexylcarbodiimide are added, and the mixture is stirred. 6

Hours at 25 °, filtered and the filtrate evaporated to dryness. The residue is washed with ethyl acetate and diethyl ether and dries. You get

BOC-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-OSn,

Mp. 242 °, which is dissolved in 10 ml of dimethylformamide. That obtained above by splitting off the protecting group Tricosapeptide is dissolved in 5 ml of dimethylformamide and rinsed into the nonapeptide solution. After 48 Hours standing at room temperature, the product is precipitated by adding ethyl acetate. It will centrifuged, decanted and the resulting

Triturated residue in ether, centrifuged and decanted off. After vacuuming and drying are 145 mg of the protected crude Dotriacontapeptide in 3 ml of a methylene chloride / trifluoroacetic acid / water solved. After standing for 1 hour, the solution is precipitated with ether, centrifuged and dried with ether. Of the The residue is taken up in 18 ml of 0.2 η acetic acid and layered on a biogel column Pb (1 300 cm). the Elution is carried out with 0.2 η acetic acid. The combined pure fractions are evaporated and then lyophilized.

The title compound is obtained, [α] % = -72.9 ° in 1 η acetic acid.

Claims (1)

Patent claims: 1. Dotriacontapeptides of the general formula X-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg- Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH,