DE1643274B2 - NEW PEPTIDES WITH ACTH EFFECT - Google Patents

Description

acid or aspartic acid.

The new compounds have a considerably stronger and / or longer adrenocorticotropic effect

The main application DT-AS 14 93 559.2 concerns al? the previously known peptides, as in-vivoiie ACTH-effective peptides with increased or, respectively, ver Tesits, z. B. by determining the plasma Corticoigerter Effect, which is characterized by showing steiron values in hypophysectomized rats.

For example, the D-SerMjly'-Lys ¹⁷ · ¹⁸ -ß ^l - ^Zi - group, or in particular the terL-Butyloxycarbonyl-

Corticotropin compared to the natural rest. To protect the amino group in the guanido

The nitro group is suitable for corticotropin at least 10 times increased and min- grouping of arginine;

at least 3 times longer effect. the said amino group of arginine must, however, be present

The new peptides are not necessarily protected for the reaction. the

formation of peptides with long chain lengths. The imino group of histidine can be

known methods. In doing so, the or trityl radical will be protected.

Amino acids, with the use of suitable protective The conversion of a protected amino or

groups, individually in the order mentioned or imino group in a free group as well as the

after previous formation of smaller peptide units, a functionally modified carboxyl

connected. At the end of the synthesis, the protective group is converted into a free carboxyl group in the course of the

groups in a single or, if appropriate, in multiple process takes place according to methods known per se

rer levels split off. by treatment with hydrolyzing or reducing

The method according to the invention is therefore decorative means.

characterized in that one of peptides which differ from 15 A preferred method is that one ACTH-effective peptides with at least the first 3 amino acids H-D-Ser-Tyr-Gly-OH N-terminus towards the complete sequence of the naturally-containing tripeptide (the abbreviated notation borrowed adrenocorticotropic hormones thereby under- refers to L-amino acids, when the D- does not separate that it is designated as the first amino acid D-serine, as special) or that also the third amino acid glycine and as 17 and 18 position 20 4-amino acid containing tetrapeptide with the hepta-amino acids L-lysine, and in which min- peptide or hexapeptide of the following amino acids up to at least the α-amino group and the side chain condensed to the amino acid 10 and then the decapepamino groups as well as the terminal carboxyl group tid with the entire remaining peptide sequence, e.g. at and optionally present side chain carboxyl production of the octadecapeptide amide with the octo groups are protected, the protective groups split off 25 peptiaamid of amino acids 11-18, in the case of Nonaund, if desired, the compounds obtained in decapeptide with the nonapeptide of amino acids their acid addition salts, derivatives or complexes 11-19, in the case of the eicosapeptide with the decapeptide of convicted. The linkage of the amino and / or amino acids 11-20, in the case of the docosapeptide Bait dem Peptide units are made in such a way that a dodecapeptide of amino acids 11-22, in the case of the tri-amino acid, is obtained or a peptide with a protected α-amino-30 kosapeptidamid with the tridecapeptide amide of group and activated terminal carboxyl group with amino acids 11-23, with tetrakosapeptide amide an amino acid or a peptide with a free tetradecapeptide amide of amino acids 11-24, α-amino group and free or protected, e.g. B. in the pentacosa peptide with the pentadecapeptide amide esterified or amidated terminal carboxyl group, amino acids 11-25, in the octacosa peptide with or that one amino acid or a peptide 35 of the octadecapeptide of amino acids 11-28, at with activated α-amino group and protected termi- Nonatriakontapeptid with the Nonakosa.peptid der nal carboxyl group condensed with an amino acid or amino acid l-39. With this condenser Peptide with a free terminal carboxyl group is preferably used as the coupling method and protected α-amino group. The carb-carbodiimide method or the activated method oxyl group can, for example, by converting 40 esters, especially by means of p-nitrophenyl ester, verin an acid halide, azide, anhydride, imidazolide, applies. In the latter case, the p-nitrophenyl -isoxazolid or an activated ester, such as cyano ester of the decapeptide nothing as such isolated methyl ester, carboxymethyl ester, p-nitirophenyl ester, but can also be obtained from the decapeptide with free carboxyl (optionally with the addition of N-hydroxysuccin group, p-nitrophenol and dicyclohexylcarbodiimide imid) or Ν, Ν'-carbonyl-diimidazoles are activated.

den, the amino group, for example, by reaction but one can use an N-terminal peptide fragment

be activated with a phosphate amide. As a £ e- instead of the decapeptide, any other peptide,

The most common methods are the Carbo- z. B. that containing the N-terminal amino acids diimide method, the azide method, the method of 50 tetrapeptide, pentapeptide, hexapeptide, heptapeptide,

activated ester and the anhydride methsde, prominent octapeptide, nonapeptide, with the whole remaining

Also to be emphasized is the so-called solid support peptide sequence condensing. If the N-iterminal

Synthesis in which the peptide from the carboxyl end, the fragment neither glycine nor proline as C-position

Is bonded like an ester to a polymer, has an amino acid structure, is chosen for the condensation is by adding the amino acids in sequence 55, preferably the C'arbodiimid-Hydroxysuccinimid-

condensed. method, (W e y g a η d, Z. Naturforschg. 21 b, 426—

Not involved in the reaction, free, functional ·; 428 [1966]) or the azide method.
Groups are expediently protected, especially the -amino group of the N-terminal fragment by means of hydrolysis or reduction and all side chain amino groups are removed from easily removable residues. B. with methanol, side chain carboxyl groups protected by the tert-butyl tert-butanol, benzyl alcohol, p-nitrobenzyl alcohol, ester group. The terminal carboxyl or amide formation, the amino group, for example, of the C-terminal fragment, if it is not amidated by the introduction of the tosyl, trityl, formyl, tri, is preferably as tert-butyl ester fluoroacetyl, o-nitrophenylsulfenyl -, phthalyl or 65 group. At the end of the synthesis, the tert-carbobenzoxy group or colored protective groups, butyloxycarbonyl groups and tert-butyl ester groups such as the p-phenylazo-benzyloxycarbonyl group can be split off at the same time by acid hydrolysis, for example with ρ - (p '- methoxyphenylazo) - benzyloxycarbonyl-trifluoroacetic acid will.

Depending on the method of operation, the new compounds of an organic base, such as triethylamine, procaine compounds, are obtained in the form of bases or their salts. From dibenzylamine, or other tertiary nitrogen bases, the salts can be obtained in a manner known per se. The polymers are known or can be obtained after basing. The latter, in turn, can be used to prepare processes, for example, by reaction with acids that can be used for the 5 according to the method described by M. I de 1 s et al., J. Am. Chem. Formation of therapeutically useful salts suitable Soc. 80, 4631 et seq. (1958) are to obtain salts, such as. B. those with anorga- So you can z. B. glutamic acid-a-carboxyanhydridic acids, such as hydrohalic acids, bei- y - benzyl esters or tert. - Butyl ester in dioxane with, for example, hydrochloric acid or hydrobromic acid, per-ammonia or an amine in a certain chloric acid, nitric acid or thiocyanic acid, sulfur io molar ratio, z. B. 100: react 1 (J ^{e NaCN} d ^em desired RDI or phosphoric acids, or organic acids, polymerization degree), and eliminate the protecting groups after bewie formic acid, acetic acid, propionic acid, glycolic consummate polymerization, acid, lactic acid, pyruvic acid, oxalic acid , e.g. B. the benzyloxy group with hydrogen bromide in ice-malonic acid, succinic acid, maleic acid, fumaric vinegar, the terL-butyloxy group with trifluoroacetic acid. acid, malic acid, tartaric acid, citric acid, ascorbic acid, 15 For the purpose of producing polymers with uniform, bic acid, hydroxymaleic acid, dihydroxymaleic acid, the polymers can also be benzoic acid, phenylacetic acid, 4-aminobenzoic acid, by synthesis according to the methods used in peptide chemistry. Hydroxybenzoic acid, anthranilic acid, cinnamic acid, known processes (carbodiimide method, azidme-mandelic acid, salicylic acid, 4-amino-salicylic acid, method, etc.).

2-phenoxybenzoic acid, 2-acetoxybenzoic acid, Me- ao The concentration of the polymer in the pharmathansulfonic acid, Ethanesulfonic acid, Hydroxyäthan- zeutischen preparations depends on the solubility of the sulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, the salt concerned and the viscosity. That Maphthalenesulfonic acid or sulfanilic acid. Polymers should be in the preparations in dissolved form

The peptides obtained according to the method can be present and injectable.
Form of pharmaceutical preparations Use 35 Find the concentration of the adrenocorticotropic effective. These contain the peptides in a mixture with peptides is, for example, 0.1 to 5 mg / ml,
one for enteral or parenteral administration. The invention is illustrated in the following examples

suitable pharmaceutical, organic or described. The temperatures are in degrees Celsius organic carrier material. For the same come specified.

substances that do not react with the polypeptides, such as gelatine, lactose, glucose, which are designated as follows:
Table salt, starch, magnesium stearate, talc, vegetable oils, benzyl alcohol, gum, polyalkylene glycols, petrolatum; Cholesterol Or Other Known System 40:

Drug carriers. The pharmaceutical preparations 35 n-butanol-ethanol-water (40:40:20)
can e.g. B. as lyophilisate or in liquid form Syytem 43 A:

as solutions, suspensions or emulsions pre-tert-amyl alcohol-isopropanol-water (67: 26: 7)

lie. If necessary, they are sterilized and / or System 45:

or contain auxiliaries such as preservatives, _s ek.-butanol-3% ammonia (70:30)

Stabilizing, wetting or emulsifying agents. You can- 40 <,, -,

NEN still other therapeutically valuable substances ^ ^s ". ', _. . . " _{/ Ιηη 1Λ} , _ηΛ
contain n-butanol-glacial acetic acid-water (100: 10: 30)

So you can also use them in the System 5 * .A:

ACTH therapy usual additives to extend n-butanol-glacial acetic acid-water (67:10: 23)

the effect, such as B. Gelatine, Polyphloretinphos- 45 System 54:

phate, carboxymethyl cellulose, or the above-mentioned sec-butanol-isopropanol-9% aqueous mono-

mentioned sparingly soluble metal compounds, ins- chloroacetic acid (58: 8: 34)

special phosphates, pyrophosphates and / or system 96:

Zinc, hydroxides combine. sec-butanol-glacial acetic acid-water (67:10: 23)

You can also extend the effective 50 System 100 "

wedge the new peptides into their complexes with polymer- ethyl acetate-pyridine-glacial acetic acid-water (62: 21: 6: 11)

seeds or copolymensates of amino acids, ms- _ft

special those that aystemiui a predominant part.

in acidic amino acids, such as glutamic acid or n-butanol-pyridine-E ^ acetic water (30: 20: 6: 24)

Aspartic acid of the L, D or D, L configuration, 55 System 101E:

have, transfer. The named polymers n-butanol-pyridine-euessig-water (42: 24: 2: 30)

and copolymers have free system 104 in the side chains:

Carboxyl groups, while the terminal carb- chloroform-methanol-17% ammonia (41:41:18)

oxyl group free or functionally modified, system 112A:

z. B. as an ester group or as an unsubstituted n-butanol-pyridine-formic acid-water

or by hydrocarbon residues, especially lower ones (42 ·. 24: 4: 20)

Alkyl groups, substituted amide groups are present System 112 E

can The molecular weight of the polymers can be n-butanol-pyridine-formic acid-water

between 10,000 and 100,000, preferably / 44 · 24 · 2 -20)

it is 20,000-80,000. One uses for the 6 _{5 Fo] dc} Abbreviations are used:
Preparation of the preparations expediently a water-soluble, physiologically acceptable salt, e.g. B. the BOC = tert-butyloxycarbonyl
Sodium or ammonium salt, or a salt with tBu = tert-butyl.

example 1

H-D-Ser-Tyr-Gly-Met-Glu-His-Phe-Arg-Try-

Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys-Lys-ProVal-

Lys-Val-Tyr-Pro-OHiD-Ser ^ GIy'-Lys ¹⁷ · ¹⁸ - !? ¹ - ²⁴ -

Corticotropin)

270 mg BOC-D-Ser-Ty r-GIy-Met-G Iu (OtBu) -His-Phe-Arg-Try-Gly-Lys (BOC) -Pro-Val-Gly-Lys- (BOC) -Lys ( BOC) -Lys (BOC) -Lys (BOC) -Val-Lys (BOC) -Val-Tyr-Pro-OtBu are dissolved in 13 ml of 90% trifluoroacetic acid and for 30 minutes at 22 ° ditched. The solution is then concentrated to about 2 ml, diluted with 10 ml of water and concentrated again to a volume of 3-4 ml and lyophilized. The trifluoroacetate of the free tetrakosapeptide obtained in this way is dissolved in 5 ml of water for conversion into the acetate and passed through a column (0 == 15 mm; / = 15 cm) of weakly basic ion exchanger (Merck No. II) in the acetate form. The eluate is concentrated to approx. 4 ml with the addition of a few ml of n-butanol and lyophilized. 230 mg are obtained Chromatographically and electrophoretically uniform acetate of D-Ser'-Giy'-Lys ^ - ^ - ^ '- ^ - corticotropin as an amorphous, water-soluble powder. It indicates aluminum oxide in the thin-layer chromatogram (DA) and on cellulose (DC) the following Rf values:

DA: Rf (101) = 0.42

Rf (52A) = 0.25

DC: Rf (112A) = 0.48

In the case of paper electrophoresis (pyridine acetate buffer, pH 6.1, 16 volts / cm), the compound migrates in 2 hours 8.6 cm against the cathode.

Compared to / ^ - "- the compound possesses corticotropin a greatly increased and prolonged corticotropic effect.

The tetracosa peptide derivative used as a starting material can be made as follows:

1. BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His-Phe-Arg-Try-Gly-OH

5.7 g (10 mmol) of BOC-D-Ser-Tyr-Gly-Met-NH-NH »(prepared as described in Belgian patent 6 68 250) are suspended in 50 ml of dimethylformamide and mixed with 12 with stirring at -15 ° , 5 ml of a 2-n. Solution of hydrogen chloride in ethyl acetate and then treated with 1.13 g (11 mmol) of tert-butyinitrite. The mixture is stirred for a further 10 minutes at -15 ° and then 3.5 ml (25 mmol) of triethylamine are added to the solution of the BOC tetrapeptide azide. The solution thus obtained is added dropwise to a solution, cooled to -10 °, of 7.1 g (8 mMoT) H-GIu (OtBu> -His-Phe-Arg-Try-GIy-OH in 150 ml of dimethylformamide and 7.5 ml of water The reaction mixture is stirred for 15 hours at 0 ° and then 500 ml of water are added. The precipitated amorphous product is filtered off with suction and washed with methyl formamide-water (1: 5) and acetonitrile. After purification by reprecipitation from aqueous acetonitrile and equilibration with atmospheric humidity, obtained 8, f2 g (73%) of pure BOC decapeptide tetrahydrate with a melting point of 230-240 =. [»}? = -15 ° ± 2 ° (c = 0.5 in pyridine-water 1 : 1) According to the thin-layer chromatogram on silica gel, the substance is uniform; Rf 52 = 0.3; Rf 101 = 0.6.

2. BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His-Phe-Arg-Try-Gly-Lys (BOC) -Pro-Val-Gly-Lys-
(BOC) -LyS (BOC) -LyS (BOC) -LyS (BOC) -PrO-Val-Lys (BOC) -Val-Tyr-Pro-OtBu

ίο 326 mg BOC-D-Ser-Tyr-Gly-Met-G Iu (OtBu) -HiS-Phe-Arg-Try-Gly-OH and 350 mg H-Lys (BOC) -Pro-Val-Gly-Lys ( BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -Pro-Val-Lys (BOC) -Val-Tyr-Pro-OtBu (see no. C40617 IVb / 12qu, Case 5805/5503/1 +2) in a mixture of 3.2 ml of pyridine, 0.1 ml of water and 0.25 ml of 1-n. Hydrochloric acid suspended. 112 mg of dicyclohexylcarbodiimide are added, the partially insoluble mixture is stirred for _3–2 hours at 50 °, then another equal amount of dicyclohexylcarbodiimide is added and the mixture is stirred for a further 4 hours at 50 °. Finally, it is concentrated to a volume of approx. 2 ml and the peptides are precipitated by adding 40 ml of water. The gelatinous, flaky precipitate is filtered off and dried, and then dissolved in hot methanol and precipitated with a benzene-petroleum ether mixture to separate dicyclohexylurea and other lipophilic impurities. The crude product obtained in this way is for final purification in the solvent system methanol-buffer-chloroform-carbon tetrachloride (10: 3: 5: 4) [buffer = 28.5 ml glacial acetic acid + 19.25 g ammonium acetate in 960 ml water] in a Craig distribution Subjected to 290 stages with phase volumes of 10 ml each. The distribution elements No. 74-97 (r _man = 85; K = 0.42) are converted to 295 mg of protected tetracosapeptide acetate in chromatographically uniform form as a white, amorphous powder with an indistinct decomposition point by concentrating to dryness and submerging the ammonium acetate at 45 ° in a high vacuum gained approx. 205 °. It has the following Rf values on thin-layer silica gel plates:

Rf (100) = 0.61

Rf (chloroform-methanol = 75:25) = 0.40

Example 2

HD-Ser-Tyr-Gly-Met-Glu-His-Phe-Arg-Try-
Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys-Lys · NH ₁

Lys ¹⁸ amide)

270 mg BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His-Phe-Arg-Try-Gly-LysiBOQ-Pro-Val-Gly-LvsiBOC) -LyS (BOC) -LyS (BOC) - LyS (BOC) -NH ₁ are in 13 ml 90% trifluoroacetic acid dissolved, left to stand for 30 minutes at 22 °, and then concentrated to about 2 ml. It is diluted with 10 ml of water, concentrated again to a volume of 3–4 ml of lyophilized: ie t. That thus obtained trifluoroacetate of the free Okiadekapepti des is converted to the acetate in 5 ml of water dissolved and passed through a column (0 = 15 mm; / = 15 cm) of weakly basic ion exchanger (Merck No. H) filtered in the acetate form. The eluate is concentrated to approx. 4 ml with the addition of a few ml of n-butanol and lyophilized. 241 mg of chromatographically and electrophoretically pure acetate des is obtained D-Ser'-Gly'-Lys ^ '^ - Corticotropin-Lys ^ -amids as amorphous, water-soluble powder. It shows in the thin

609530/488

9 ίο

light chromatogram on aluminum oxide (DA) at room temperature for 30 minutes

jnd the following Rf values on cellulose: let. It is then concentrated in a rotary evaporator

_f 30 ° bath temperature to a volume of approx. 3 ml,

DA: Rf (52A) = 0.1j _{adds 15 m} jv / water, constricts again and lyophilizes

Rf (101) = 0.33 5 _S at. The trifluoroacetate of the free tetra-

Rf (104) - 0.17 kosapeptide used for conversion to the

TLC 'Rf (112A) = 0.26 acetate dissolved in about 4 ml of water and passed through a column

(0 = 12 mm; / = 22 cm) of weakly basic

In paper electrophoresis (pyridine acetate buffer, ion exchanger (e.g. Merck No. II) in the acetate pH 6.1.16 volts / cm) the compound migrates in 2 hours ίο is filtered. The eluate is condensed to approx. 5 ml 11.7 cm against the cathode. centered, lyophilized, at 40 ° in a high vacuum after-The compound has ^{dried compared to ^ '~ 24} -Cor- and then equilibrated with humidity. ticotropin greatly increased and lengthened corticotropic. 279 mg acetate of the D-Ser- ¹ -Gly ³ -Lys ¹⁷ · ¹⁸ effect are obtained. / P- ²⁴ -Corticotropin-Pro "-amides as a white, amor-The octadecaphe used as starting material, readily water-soluble powder of unsharp Zerpeptidderivat can be produced as follows: settlement point at approx. 180 °. It has.

chromatogram on aluminum oxide (DA) and on

BOC-D-Ser-Tyr-GIy-Met-GluODtBuVHis-Phe- Cellulose (DC) has the following Rf values:
Arg-Try-Gly-Lys (BOC) -Pro-Val-Gly-Lys (BOC) -

Lys (BOC) -Lys (BOC) -Lys (BOC) · NH ₂ ^a °

Rf (52 A) = 0.27 RF (101) = 0.66 Rf (101 E) ^ 0.36 RF (112 E) = 0.15

2.9 g finely powdered BOC D-Ser-Tyr-Gly-Met- DC:
Glu (OtBu) -His-Phe-Arg-Try-GlyOH (shown as
described in Example 1) and 2.1 g H Lys (BC) C) -

Pro-VaI-GIy-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys- 25 The tetrakasa-

(BOC) -NH ₂ (see No. C40616IVb / 12qu, Case 5806 / peptide derivative can be prepared as follows:
5503/1 + 2), in 27 ml of pyridine, 0.7 ml of water

and 2.38 ml 1-n. Hydrochloric acid suspended and after addition.

1.07 g of dicydohexylcarbodiimide at 50 ^{Cl would give} '^ - ^ »- ^ -" οινη.,

stirs. The mixture will soon solidify and after 30 18.8 g of Z-Val-Tyr-Pro-OtBu will be in 95 ml 90% -

3 hours standing at 50 ° with a solution of iger trifluoroacetic acid and dissolved during a

left to stand by a further 1.07 g of dicydohexylcarbodiimide in 1 ml at room temperature. Man

Pyridine is added, homogenized and then again concentrated to dryness and pulverized the oily

left to stand at 50 ° for 4 hours. Finally residue by adding 100 ml of water and 5!

300 ml of water are added, the mixture is homogenized and filtered 35 rub at 0 °. After filtering it is

the gelatinous, flaky precipitate. He wire! constant weight dried over potassium hydroxide and

dries and then reprecipitated in 100 ml of dimethylformamide at 70 ° from ethyl acetate-petroleum ether. The amorphous

dissolved, with most of the dicyclo powder formed (17.01g) together with 5.1: 5 ml of tri-

hexylurea remains insoluble and is filtered off. ethylamine in 170 ml of absolute tetrahydrofuran

The crude condensation product is dissolved from the filtrate, cooled to -15 ° and for 5 minutes

by adding 600 ml of benzene and 400 ml of petroleum with a solution of 4.44 ml of isobutyl chlorocarbonate

ether precipitated, filtered off and dried. (4.65 g) in 2.5 ml of absolute tetrahydrofuran were added. To

For purification, 1.0 g of this crude product is slowly added while stirring at -10 ° for 15 minutes

75 ml each upper and lower phase of the solvent- 107 ml 0.64-n. Ammonia in absolute ethyl acetate too

systems methanol - 2-n. Acetic acid-chloroform-Te-45 and leaves for 1 hour at 0 ° and during

Stir carbon trachloride (10: 3: 7: 5) with gentle stirring for 6 hours at 20 °. The mixture becomes strong

warm dissolved and concentrated according to Craig over 135 steps, the sticky residue in 500 ml of ethyl acetate

Phase volumes of 25 ml each distributed. From the dissolved and one after the other with water, 2N soda solution

dividing elements No. 88-110 (r _m «= 100, K == 2.8) and again washed with water and dried to dryness

is obtained by concentration to dryness and path s *> constricted. This is obtained after folding over twice

sublime the ammonium acetate at 45 "in the high- of the residue from ethyl acetate-petroleum ether and dry

vacuum a total of 490 mg of pure protected octane in a high vacuum 14.4 g of an amorphous powder,

decapeptjdamide acetate as a white, amorphous powder. which is uniform by thin-layer chromatography

Behaves in thin layer chromatography on silica gel. The following result on Sflicager plates it has the following Rf values: 55 Rr values:

Rf (52) = 0.55 _{Rl in the} chloroform-methanol (9: 1) system = 0.3i

Rf (96) -0.68 R ₁ (43 A) = 0.63

Rf (100) = 0.45 _fc 2. H-VaI-TyT-PTO-NH ₈

^{Example 3} throw 14.4g Z-VaI-TyT-PrO-NH ₈ in 150ml Me

H-D-Ser-Tyr-Gry-Met-Glu-His-Phe-Arg-Trethanol dissolved and 1.5 g of 10% Palladium alcohol

Gly-Lys-Pro-Val-jGly-Lys-Lys-Lys-Lys-Pro-Val- at normal pressure and under COj absorption up to zni

Lys-Val-Tyr-Pro-NHj (D Ser ¹ -Gry ⁸ -) Lys ¹ '· ¹⁸ - saturation (duration approx. 2 hours) hydrogenated. After Ab

fli- "€ rnrii", hm ^. Ov _n M „π, ΑΛ ⁵ filter the catalyst and concentrate the filtrate

F- "-Corbcotropin-Pro '^ - anud) _{m Trodme} ^ ^ _{Röckst £ md} ^ ₁₅ ^ _Methan0

293 mg of protected tetrakosapeptide amide and 40 ml of ethyl acetate are dissolved by adding 120 ml

dissolved in 14 ml of 90% trifluoroacetic acid, precipitated in petroleum ether, filtered off and found high

Rr (40) = 0.73 RF (45) = 0.72 RF (54) = 0.70

11 12

/ vacuum dried at 40 °. 9.52 g of a matographically pure protected pentapeptide are obtained as imorphous powder, which is obtained as thin-layer chromato- white, amorphous powder. It shows in thin graphics behaves uniformly on cellulose plates: layer chromatogram on silica gel, the following Rf values

on:

Rt in the chloroform-methanol (9: 1) system = 0.42 Rt (43 A) = 0.68

3. Z-Lys (BOC) -Val-Tyi-Pro-NH ₂ ⁶ · H-Val-Lys (BOC) -Val-Tyr-Pro-NH _s

9.74 g of Z-Lys (BOC) -OH and 9.52 g of H-Val-Tyr- io 5.72 g of Z-Val-Lys (BOC) -Val-Tyr-Pro-KH ₂ are in

PrO-NH ₂ are dissolved in 40 ml of absolute dimethylform- 75 ml of methanol and, after adding 600 mg

amide and dissolved with a solution of 5.76 g of dicyclo 10% palladium carbon at room temperature

hexylcarbodiimide was added in 100 ml of acetonitrile. One _{hydrogenates CO 2} absorption. The hydrogen uptake

stir for 12 hours at room temperature, fil- is finished after about 5 hours, and it is filtered

Then the precipitated dicyclohexylur- 15 trates off the catalyst and the solution evaporates

fabric and concentrate the solution to dryness. The dry one. 4.74 g of pentapeptide amide are obtained as

solid foamy residue is obtained twice from vinegar - amorphous, chromatographically uniform powder,

ester petroleum ether reprecipitated and yields 16.02 g of amorphous in the thin layer chromatogram on silica gel

phes tetrapeptide derivative, which shows the following Rf values on silica gel thin layer:

plates has the following Rf values: * ° R, (52) = 0.40

Rf in the chloroform-methanol (9: 1) system = - 0.29 ^{Rf (100) = 0 <35}

R, (43 A) = 0 68 7 · Z-LyS (BOC) -LyS (BOC) -PrO-VaI-LyS (BOC) -

Val-Tyr-Pr ₀ -NH ₂

4. H-Lys (BOC) -Val-Tyr-Pro-NH _{2 a5 335 g of Z} -Lys (BOC) -Lys (BOC) -Pro-OH (15.5 g of the protected tetrapeptide amide are prepared in as in Example 1 given) are in 20 ml absolute

Dissolve 100 ml of methanol and after adding 1.5 g of dimethylformamide, and after adding 10% palladium-carbon, cool it to 0.65 ml of triethylamine at -15 ° at normal pressure. Allow saturation to hydrogenate. (Duration approx. 4 hours). Then add dropwise 0.58 ml of pivaloyl chloride at -15 °. The catalyst is filtered off and the fi lt is concentrated. After 6 minutes, a pre-cooled solution is added, the crude product obtained is obtained from 3.34 g of H-Val-Lys (BOC) -Val-Tyr-Pro-NH ₂ in 10 ml then add absolute dimethylformamide in 10 ml of methanol and 120 ml of ethyl acetate and stir the solution and mix it with 120 ml of petroleum ether for 20 minutes at -10 ° for 5 hours and dry in a high vacuum at 40 °. The one at 0 °, and for 2 hours at 20 °. To obtain 11.9 g of an amorphous powder, which has been worked thinly, 70 ml of ethyl acetate and 50 ml of water are layer chromatographed on silica gel and the following Rr values are extracted, and the organic phase is extracted one after the other: with Ο, Ι-M. citric acid , 2% ammonia solution

and water, dries over sodium sulfate and

Rt in the system chloroform-methanol (9: 1) = 0.08 the solvent evaporates. The powdery amorphous one Rf (43A) = 0.27 40 residue (5.74 g) shows in thin layer chromatography

< τ \ i 1 τ roc \ r ^ νιτ r> χτυ grams on silica gel only shows minor impurities

5. Z-Val-Lys (BOQ-Val-Tyr-Pro-NH ₂ * _{nd is without purification} ^ _x further reaction a

5.28 g of Z-VaI-OH and 3.07 ml of triethylamine are added.

dissolved in 50 ml of absolute ethyl acetate to -10 °. _ _x _ ,,. _w .,. _{/ n,. ft} "

cools and ^stem with a solution of 4.57 ml Tsobutyl- 45 ^{R <S} y ⁱⁿ chloroform-methanol (9: 1) = 0.23 chlorocarbonate in 5 ml Essigester. One stirs "", ",, *, -, -. τ, ^ λ, - ^ ™ ir ι τ rn ^ ™

still for 15 minutes at -10 ° and then gives ⁸ "H-LyS (BOCVLyS (BOC) -PrO-VaI-LyS (BOC) -

at approx. -5 ° a solution of 11.87 g of H-Lys (BOC) - vai-iyr-rro-iNrtj

Val-Tyr-Pro-NHj in 10 ml of dimethylformamide. The 5.7 g of the octapeptide derivative described above

Mixture is for 30 minutes at 0 ° and while 5 »are dissolved in 100 ml of methanol and after addition, stirred for 5 hours at room temperature and then of 550 mg of 10% palladium carbon at normal pressure! mixed with 100 ml of water and 200 ml of n-butanol. Hydrogenated to saturation After filtering off de! After separation of the aqueous phase, the orga-catalyst and rinsing with methanol is there! Niche phase successively evaporated to dryness with 10% tartaric acid Ffltrat . Acetic acid extracted, the chloroform phase sodium sulfate dried and concentrated to dryness then concentrated to 20 ml and the peptic from it dried After equilibrium, reprecipitated to methanol acetomer and yields 12.15 g of ⁶ O ren with the air humidity, 5.21 g of an amorphous putver is obtained, which is amorphous in the solvent system, methanol-0.1 M. borrowed powder, which according to titration and elemental form carbon tetrachloride (9: 3: 4: 4) with phase analyzes per mole of octapeptide amide 0.63 moles of vinegar volumes of 25 ml each over 250 levels of multiplicative acid and 13 moles of H. _x contains O. Obtained on silica gel is distributed. The following Rr values are obtained from distribution elements No. 81-115 6s: (»max = 91; K = 0.57) when narrowing down to

Dryness and sublimation of the ammonium ace- Rr in the chloroform-methanol system (9: 1) = 0.0

tates in a high vacuum at 45 ° a total of 7.94 g chromium R ₁ (100) = 0.4

13 14

11. BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His-

Phe-Arg-Tyr-Gly-Lys (BOC) -Pro-Val-Gly-Lys (BOC;

Lys (BOC) -Lys (BOC) -Lys (BOC) -Pro-Val-Lys-

(BOC) -Val-Tyr-Pro-NH »

3.91 g Z-Lys (BOC) -Pro-VaI-GIy-Lys (BOC) -Lys- 355 mg BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His

(BOC) -NHNH ₈ (cf. German Patent 12 14 242) are Phe-Arg-Tyr-Gly-OH and 400 mg of H-Lys (BOC) -Pro den with gentle warming in 40 ml of absolute Di-Val-Gly -Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) methylformamide dissolved, cooled to -20 ° and at Pro-Val-Lys (BOC) -Val-Tyr-Pro-NH ₂ are in a this temperature with stirring successively long-io mixture of 3.7 ml of pyridine and 0.273 ml of in. Salt sam with 6.58 ml of 2.15-n. Hydrochloric acid and homogenized with 0.74 ml of acid while warming to 50 °. Tue <5-n. Sodium nitrite solution added. Finally, the pasty-gelatinous mass is stirred with a solution for 15 minutes at -10 ° and then a pre-cooled solution of 4.07 g of H-Lys (BOC) of 131 mg of dicyclohexylcarbodiimide in 0.2 ml of Pyri is added. din was added, flushed with nitrogen, again gui Lys (BOC) - Pro - VaI - Lys (BOC) - VaI - Tyr - Pro - NH ₂ 15 and then homogenized for 3 hours at 50 '(containing 2.5% CH ₃ COOH + 15.3% H ₂ O) in 11 ml. After this time, a second dimethylformamide and 1.98 ml of triethylamine are added. Portion of 131 mg of dicyclohexyl-carbodiimide ir The mixture is stirred for 1 hour at 0 °, 0.2 ml of pyridine is added, flushed again with nitrogen, whereupon the pH is sipped by adding triethylamine and left for a further 3 hours at 50 ^c adjusts to 7.5 and lets stand at 0 ° overnight. The 21 stand. Finally, 50 ml of water are added, the homogeni reaction mixture is then concentrated in a high vacuum, the fine-grain gelatinous precipitate is filtered to a volume of approx. 20 ml and concentrated with 100 ml and dried at 45 ° in a high vacuum. Added to the water. The crude product precipitates in the gallbladder removal of dicyclohexylurea and other forms. It is filtered off, washed twice with water for gel-lipophilic impurities and then suspended three times in hot methanol from methanol, precipitated from benzene and petroleum ether by adding water and twice from methanol-benzene-petroleum ether, filtered off and reprecipitated. The material pre-cleaned in this way (4.72 g) is dried. The pre-purified product is in the methanol-buffer-chloroform-solvent system methanol- buffer -chloroform-carbon tetrachloride (12: 4: 9: 3) [buffer = 28.5 ml carbon tetrachloride (10: 3: 6: 4) [buffer = 28.5 ml of glacial acetic acid + 19.25 g of ammonium acetate in 960 ml of water 30 glacial acetic acid + 19.25 g of ammonium acetate in 960 ml of water] ser] a Craig distribution over 160 levels with over 185 levels multiplicatively distributed, with phase-phase volumes of each Subjected to 20 ml. From the volumes of 10 ml each. From the distribution _{elements distribution elements No. 45-74 (/ - max} = 57; K No. 67-86 (r _ma , = 77; K = 0.71) are isolated at = 0.55 ) when concentrated to dryness and concentrated to dryness and sublimated away of the sublimating ammonium acetate at 45 ° in the high monium acetate in the | High vacuum at 45 ° the pure, vacuum a total of 3.3 g of chromatographically pure protected tetrakosapeptide amide obtained as a white, amorphous protected tetradecapeptide as a white, amorphous powder with an unsharp decomposition point at approx. Powder with a decomposition point approx. 210 °. It 220 °. In the thin-layer chromatogram, it shows the following Rf values in the thin-layer chromatogram on silica gel: the following Rf values: 40

Rf (chloroform-methanol = 7: 3) = 0.42

Rf in the system chloroform-methanol (9: 1) = 0.17 jy / 52) = 0 41

Rf (43 A) = 0.62 Rf ( ₁₀₀ ) _{= 0 '38}

Rf (100) = 0.87 ₄₅

Example 4

10. H Lys (BOQ-Pro-Val-Gly-Lys (BOQ-Lys (BOC) -Lys (BOC) -Lys (BOQ-Pro-Val-Lys-

Val-Tvr-Pro-NHi _{50 IQU g BO} C-Tyrosine get hot in 45 ml vinegar

ester dissolved and after cooling to 20 ° with a

4.97 g of the protected tetradecapeptide amide are dissolved in a solution of 3.84 g of glycine methyl ester in 10 ml of acetic acid with heating in 100 ml of methanol and ester is added solid form and stirred for 18 hours at room temperature and normal pressure with absorption 55 den at 25 °. Then it is cooled to 0 °, after 30 mides of the carbon dioxide that has formed, the precipitated cyclohexylurea is hydrogenated to saturation. The catalyst is filtered off, the filtrate is filtered off and concentrated from the filtrate by concentration and to about 15 ml, and the hydrogenated precipitation with petroleum ether is the crude Dipeptide derivative as tetradecapeptide isolated by adding 50 ml of benzene and an amorphous, viscous mass. It is filtered off through another 150 ml of petroleum ether which has been precipitated out and further dried 60 times under vacuum from ethyl acetate petroleum ether at 45 °. This gives 4.25 g of a purified one and can then be made from the same amorphous powder that crystallizes in the mixture of thin-layer chromatographs; 124-125 ° F. gram behaves uniformly on silica gel and the following

Has Rf values: Receiving the following Rr values:

«5

Rf (43 A) = 0.18 RF (52) = 0.41 Rf (IOO) = 0.33

Rr (43 A) = 8: 2) = 0.65 Ri (CHCls-methanol = 0.68 R ₁ . Λ DD

2. Include H-Tyr-Gly-OCHs-hydrochloride by layer chromatography. She shows

Silica gel has the following Rf values:

2 g BOC-Tyr-Gly-qCHj, under mentioning in _{Rf f52} > = 0 60

Dissolve 20 ml of absolute ethyl acetate and dry it at 20 ° with tj * «« ™ n <i

20 ml 4-n. Hydrochloric acid is added to absolute ethyl acetate. 5 * "^ ¹⁰¹ ' ⁼ °' ⁶¹ ·

After a few minutes a precipitate begins, _ _ _ _ _ ",,. ,, _Λ ^ τ _Τ

to fall. After 30 minutes, the reaction is concentrated ⁵ - BOC-D-Ser-Tyr-Gly-Nle-OCH,

Mix in a vacuum to approx. 10 ml and complete 3.09 g of BOC-D-Ser-Tyr-Gry-OH in one

constant precipitation of the dipeptide ester hydrochloride mixture of 5 ml and absolute dimethylformamide rids by adding 50 ml of petroleum ether. Man homo- ίο 15 ml of acetonitrile dissolved with gentle warming, The amorphous product is filtered off, washed with. After cooling to 10 °, a solution of 1.23 g is obtained Petroleum ether and dries in a high vacuum at 30 °. The freshly made norleucine methyl ester in 15 ml Dipeptide ester hydrochloride is added as hygroscopic acetonitrile. Then 2.04 g of solid Powder obtained from F. approx. 110-115 (decomposition). It added dicyclohexylcarbodiimide. The solution will be shows in the thin-layer chromatogram on silica gel 15 for one hour at 10 ° and for 16 hours the following Rf values: stirred at room temperature. Excess di-

R Wi Al - n «cyclohexylcarbodiimide is obtained by adding 1.4 ml

'* · ' ~ ^{U> JJ} Glacial acetic acid and stir for one hour at room temperature

Rf (CHClj-methanol = 8: 2) = 0.36 destroyed. The precipitated dicyclo- Rf (101) = 0.60. ao hexylurea and evaporates at 50 ° bath temperature

temperature to dryness in a vacuum. The residue, a

3 ROC n <! T π new yellowish oil, is dissolved in 200 ml of ethyl acetate, the

- er-1 yr-uiy-LK-Hs solution with 70 ml 1-n. Citric acid solution, twice

1.69 g of H-Tyr-Gly-OCHa hydrochloride are added to 70 ml of 2-n. Dissolve sodium hydrogen carbonate solution and warm it twice in 10 ml of absolute dimethyl aj times 70 ml of saturated sodium chloride solution extrafo: mamid and then cool it to 0 °. The mixture is added, dried over solid sodium sulphate and 0.84 ml of triethylamine and 1.23 g of BOC-D- evaporated one after the other. The white, foamy raw product will Ser-OH and 1.65 g of dicyclohexylcarbodiimide to and from ethyl acetate-petroleum ether and crystallized at 50 ° the mixture is stirred for 20 hours at 25 ° and dried in a high vacuum. 3.50 g, m.p. 138-142 °; finally for an hour at 0 °. The resulting temperature, after repeated crystallization, has a temperature of 143-246 °. Divorced mixture of triethylammonium chloride It shows in the thin-layer chromatogram on silica gel

and dicyclohexylurea is filtered off and the following Rf values are obtained: the filtrate is concentrated to an oily residue. ^ j fl) _enz0 ] acetone = 1 x 1) = 018

By adding 30 ml of ethyl acetate and 100 ml of petrol-BOC-D-Ser-Tyr-Gly-Nle-NH-NH, "

Ather, the crude product turns out to be a greasy mass

pleases. For pre-cleaning, it is dissolved again under 3.30 g of BOC-D-Ser-Tyr-Gly-Nle-OMe in

Warm up in a little ethyl acetate and it is dissolved by adding 18 ml of absolute ethyl alcohol. To clear it, add a lot of petroleum ether (1.8 g). The thin yellow solution is added to 1.8 ml of hydrazine hydrate, which is still impure product by layer chromatography, and the slightly cloudy solution is left to stand for 24 hours at room temperature for cleaning in the solvent system of ethyl acetate. The crystallized benzene - 0.05 - m. Aqueous ammonium acetate solution ized product is filtered off and washed with cold abso- (2: 1: 2) with phase volumes of 10 ml each according to Craig lutem ethanol. After drying at multiplicative distributed. After 250 hours, isolated room temperature concentrated sulfuric acid from the distribution elements Nr.45-74 (r _m ax = 60; is dried under high vacuum: 1.4 g, mp - 152- K = 0.32), the tripeptide derivative by evaporation 4P 16l ° (dec.).

of the solvent and subliming away the Am- from the mother liquor can by concentration, FiI-

moniumacetates up to 45 ° in a high vacuum as chromate and dry an additional 0.5 g F. 134.5-139 ° graphically pure connection. Crystals from methanol (decomposition) can be obtained. Water, m.p. 180-182 °. In thin-layer chromato- After recrystallization from methanol-ethanol =

grams of silica gel give the following Rf values: 50 1: 1, mp 172-177 ° (decomposition).

The substance crystallizes from aqueous solvents with 1 mol of water of crystallization. Chromatographically uniform substance, Rf values in the thin-layer chromatogram on silica gel:

⁵⁵ Rf (chloroform-methanol = 1: 1) = 0.64 4. BOC-D-Ser-Tyr-Gly-OH Rf (chloroform-methanol = 8: 2) = 0.28

685 mg of BOC-D-Ser-Tyr-Gly-OCH _{3 are dissolved} in 6. BOC-D-Ser-Tyr-Gly-Nle-Glu (OtBu) -Hi _S -5 ml of 1-n. Sodium hydroxide solution, left for 10 minutes at 60 rne-Arg- 1 ry-Uiy-Utt 20 ° and then neutralized the solution by 1.14 g (2 mmol) of BOC-D-Ser-Tyr-Gly-Nle-NHNH, addition of 5 ml 1-n. Hydrochloric acid. The protected tri are suspended in 10 ml of dimethylformamide and the peptide is mixed with 50 ml of water-saturated n-butanol while stirring at -15 ° with 4 ml of a 2-n. Solution extracted, the butanol layer washed four times with 4 ml of hydrogen chloride in ethyl acetate and with water and concentrated to about 3 ml. 65 0.23 g (2.2 mmol) of tert-butyl nitrite were added. The reac-addition of 30 ml of petroleum ether gives a lubricant solution is stirred for a further 10 minutes at -15.degree. The substance is made slightly basic in thin ethylamine. The thus obtained

609530/488

RF (52) = 8: 2) = 0.76 RF (101) = 0.78 Rf (CHCl.-methanol = 0.58.

17 ¹⁸

Solution of the BOC tetaapeptide azide is added dropwise to Example 5

to a solution, cooled to -10 °, of 1.42 g of _Mhi

(1.6mmol) H-GMolu ^ His-Phe-Arg-Try-Gly-OH _ ^ _% 1ΜΪ «« 5 ^ ί ^ ίί! ΪΤ »

(1.6 mmol) H-GluiQtBu ^ His-Phe-Arg-Try-Gly-OH Dissolve 11, g g

m 30ml DimethyL-mamid and 1.5ml water - 2.5 mg sodium chloride and 2.0 mg D - Ser * - y

give. The reaction mixture is 22 hours at 0 ° 5 Lys "-" - ^ - "- Corucotropm-hexaac" tat in 0 ^ 7 ml

stirred, then treated with 100 ml of water, and the distilled water and filled with desUlhertem water

Excreted amorphous product is suctioned off, up to 1 ml. The solution is at 120 for 20 minutes

with dimethylformaniide water (1: 5), water and autoclaved. The pH after sterilization is 3.7.

Acetonitrile washed and dried After cleaning Example 6

by reprecipitation from aqueous acetonitrile and equili- io ..... ^ ₁ , ...

1.44 g (89%) are dissolved in 0.7 wi I physio.ogischer Nataurnchlond-

pure BOC decapeptide tetralydrate from F. 235-240 ° solution 0.5 mg D-Ser ^ Gly ^ Lys ^^ - ^ - ^ - Cortico-

obtain; [«]? = -22 ° (c = I in pyridine-water 1: 1). tropine hexaacetate and acidify the solution with 0.1 n salt

The substance shows acid at pH 3.2 in the thin-layer chromatogram. With distilled water is on

the following Rf values on silica gel: 15 1 ml made up and heat-stenlized as in Example 4.

Rf (52A) = 0.3-0.4 'Example 7

Rf (IOl) = 0.6-0.7 _{in ogml distilled water becomes 705ml}

and 6.08 mg of sodium chloride dissolved and

Sä- ^{d mifdestiIlie} " ^em

300 mg BOC-D-Ser-Tyr-Gly-Nle-GlutiOtBui-His-25 The solution is filtered in the usual way, in

Phe-Arg-Try-Gly-OH and 310 mg H-Lys (BOQ-Pro ampoules filled and auto-

VaI-GIy-LyS (BOQ-LyS (BOCO-LyS (BOC) -LyS (BOC) - piano. The pH is 3.6. Pro-VaI-Lys (BOC) -Val-Tyr-Pro-OtBu are mixed with 6 ml

Pyridine, 0.5 ml water and 0.15 ml 1-n. Hydrochloric acid. Example 8 puts. Then a solution of 100 mg Dicydo- 3 °

hexylcarbodiimide in 0.75 ml of pyridine is added and a solution of 0.8 ml of distilled water is added

Stirred for 5 hours at 45-50 °. Then another 10.16 mg of citric acid (with 1 mol of crystal water),

75 mg of dicyclohexylcarbodiimide were added and over 0.097 ml of 1-n. Sodium hydroxide solution, 3.54 mg sodium chloride

Stirred at 45 ° overnight. The mixture of condensate and 0.51 ml of 0.1-n. Hydrochloric acid produced. In this lo-

sation product and dicyclohexyl urea is dissolved, 4.0 mg of D-Ser'-Gly'-Lys ¹⁷ · ¹⁸ - /? ¹ - ²⁴ ^ «! -

completely precipitated by adding 75 ml of water, dissolved cotropin-hexaacetate and with distilled

filtered off and dried. To separate the di-water, make up to 1.0 ml.

Cyclohexyl urea is used twice in succession. The solution is filtered in the usual way, suspended in hot water in ethanol, or partially dissolved, and bottled and paved again at 120 ° for 20 minutes by adding benzene and petroleum ether. The pH is 3.6. falls. The material pre-cleaned in this way is cleaned as described in Example 1 by countercurrent distribution. After 120 distribution steps in the solution example y means system methanol - buffer - chloroform - tetra - A suspension is made from the following compochloro carbon (10: 3: 5: 4) [buffer: 28.5 ml of iron peas: vinegar + 19.25 g ammonium acetate in 960 ml water]
the pure, protected tetrakosapeptide from the D-Ser ¹ -Gly ³ -Lys ^{17> 18} - / 5 ¹ - ²⁴ -Cortico-

Fractions 46-58 obtained (distribution number K = 0.82). tropine hexaacetate 1.0 mg

With thin layer chromatography on silica gel plates ZnCl ₂ 5.25 mg

the protected peptide derivative shows the following Rf values: 50 NaHPO ₄ : 2 H ₂ O 1.05 mg

NaCl 2.0 mg

Rf (100) = 0.61 benzyl alcohol 10.0 mg

Rf (chloroform-methanol 75:25) = 0.41 NaOH ad pH 8.3

Distilled water to 1.0 ml

8. HD-Ser-Tyr-Gly-Nle-Glu-His-Phe-Arg- ⁵⁵

Try-Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys-Lys-Pro Example 10

Val-Lys-Val-Tyr-Pro-OH (acetic acid salt)., "". _o . . ,, "

A suspension is made from the following components

The splitting off of the Schultz groups from the genent: protected peptide and the transfer of trifluoro- 6 °

acetic acids in the acetic acid salt happens as in Bei- D-Ser ¹ -Gly ³ -Lys ^17iI8 - / S ¹ ~ ^a4 -Cortico-

game 1 described. The free tetrakosapeptide shows tropine hexaacetate 1.0 mg

in paper electrophoresis (pyridine acetate buffer, ZnCl ₂ 6.30 mg

pH = 6.35; Running time 1 hour; 2000 V) one run- Na ₂ NPO ₄ · 2 H ₂ O 1.26 mg

stretch of 8.5 cm against the! cathode. At the thin 65 NaCl 1.5 mg

Layer chromatography on aluminum oxide indicates the benzyl alcohol 10.0 mg

Compound in system 101 has an Rf value of 0.41 NaOH pH 8.3

on. Distilled water to 1.00 ml

19 20

Water made up to 10 ml. The solution becomes sterile Example 11 filtered. It contains per ml:

0.5 ml of a hydrochloric acid solution of D-Ser ¹ -Gly ³ -Lys ^{l7 '18} - / S ¹ - * ⁴ -Cortico-

0.5 mg D - Ser ¹ - GIy ³ - Lys ¹⁷ * ¹⁸ -ß ^l ~ ** - Corticotropin- 5 tropine-hexaacetate 4.0 mg

hexaacetate, 5.25 mg ZnQ ₈ , 1.05 mg Na ₄ HPO ₄ · 2 H ₁ O poly-L-glutamic acid 200.0 mg

and 2.0 mg of NaCl from pH 3.0 and the solution is sodium hydroxide solution to pH 7.4

to 0.5 ml of an aqueous solution of 10 mg of benzyl merthiolate 0.02 mg

alcohol that contains so much caustic soda * that a sus- Aqua dest. ad .., 1.0 ml

pension of pH 8.3 is obtained. io _. . ,,.

Example 15

_D. . , One makes a solution ampoule from the following

B e ι s ρ ι e 1 12 components:

A dry vial is made up of the following components her: x5 D-Ser'-Gly ^ -Lys ^ - ^ - ^ - ^ - Cortico-

tropine hexaacetate 2.0 mg

D-Ser ^ Glya-Lys ^^ - ^ - Cortico- Gelatine 280.0 mg

tropine hexaacetate 2.0 mg phenol 5.0 mg

ZnSO ₄ · 7 HjO 5.0 mg dist. Water to 1.0 ml

Na ₃ PO ₄ -UH ₈ O 5.5 mg ω _. . ,.,

Mannitol 40, OmI example 16

A sterile filtered aqueous solution of D-Ser ¹ -

Before use, the contents of the dry vial are mixed with GIy ³ - Lys ¹⁷ · ¹⁸ - ß ¹ - ** - corticotropin - hexaacetate, 1 ml of distilled water is obtained under aseptic conditions with sodium poly suspension of pH 7.6. as phloretrnphophat and sodium chloride mixed and

filled into vials and lyophilized so that a dry

_,. . ,, "Kenvials is obtained, the following components

Example _{II contains}

5 mg poly-L-glutamic acid from the middle molecule- _{D Se]} .i Gly ³ -Lys ^{17 '18} -i? - ¹ - ²⁴ -Cortico-

lar weight 39 600 are in 5 ml 0.1-n. Caustic soda 30 t ^. ^^ .. ^P 0.2 mg

solved. The solution is filtered and a solution of sodium polyphloretin phosphate is added

2.5 mg D - Ser ¹ - Gly »- Lys ¹⁷ · ¹⁸ - jS * -» «- Corticotropin- (86 5 ° / ig) .... 23.20 mg

hexaacetate added, then acidified with acetic acid or NaCl 12 28 mg

Hydrochloric acid to pH 4 and make up to 10 ml with water '

on. A poly-L-glutamic acid-D-Ser ¹ - 35 is dropped

Gly ³ -Lys ^{17 '18} - / S ¹ - ⁱ⁴ -Corticotropin complex finely mixed 2 ml of distilled water, contained in a solution

hands out. The suspension contains 0.5 mg poly-ampoule per ml, mixed.

L-glutamic acid and 0.25 mg / ^ - "- corticotropin as _Ώ.. ,., -

Complex compound. B e 1 s ρ 1 e 1 17

40 In a manner analogous to that in Examples 5-16

. . the preparations manufactured as adrenocorticotropic

B e ι s ρ 1 e I 14 effective peptide D-Ser ^ Gly ^ Lys ^ - ^ - ^ - ^ - Cortico-

2.0 g poly-L-glutamic acid with a medium tropine-Pro ^M -amide, D-Ser ¹ -Gly ⁱ -Lys "- ^U - / S ¹ - ¹⁸ -Cor-

Molecular weight of approx. 39,600 are contained in approx. 5.7 ml of ticotropin-Lys ¹⁸ -amide, D-Ser ¹ -Gly ^s -Nle ⁴ -Lys ^{17 18} -

10% sodium hydroxide solution, so that the pH of the solute 45 ß ^l ~ ^u ■ corticotropin, D - Ser ¹ - Gly ^s - Never «- Lys ¹⁷ · ¹⁸ -

solution is 7.4. In this solution 4.0 mg ß ^l ~ ^u - Corticotropin - Pro * ⁴ - amid or D - Ser ¹ - GIy ³ -

D-Se ^ -Gly ^ Lys ^ - ^ - ^ - ^ - Corticotropin-hexaacetat Never ⁴ - Lys ¹⁷ · ^1β -ß ^l ~ ¹⁶ - Corticotropin - Lys "- amid ent-

and 0.2 mg of merthiolate dissolved and kept with distilled.

Claims (8)

1 2 that they have as the first amino acid an amino acid with claims: D-configuration. The present invention relates to adreno- 1. Adrenocorticotropically active peptides according to corticotropically active peptides according to the main main application DT-AS 14 93 5S> 9.2 with a 5 patent application with a chain length of 18-39 Chain length of 18-39 amino acids and more complete towards the amino acids and the N-terminus N-terminus towards the complete sequence of the natural sequence of the natural corticotropins, but in the borrowed corticotropins, in which, however, one or more of the amino acids in the positions several of the amino acids in the positions 1-5, 1-5, 17, 18 and 25 against others in a known manner 17, 18 and 25 in a familiar way? can be exchanged for other io L-a-amino acids, the da-L-a-amino acids can be exchanged, their are characterized by the fact that they are the first amino-C-terminal Amides and acid addition salts acid D-serine, as the third amino acid glycine and as and complexes of these compounds, thus having 17- and 18-position amino acids L-lysine, characterized in that they ate first amino and their C-terminal amides, acid addition salts acid D-serine, as the third amino acid glycine and 15 and complexes and processes for the preparation of the compounds listed as 17 and 18 amino acids L-lysine. point. The new links are chain length 2. Peptides according to claim 1 with a chain of 18-39 amino acids from the N-terminus of the natural length from 18-25 amino acids. borrowed adrenocorticotropic hormones, e.g. B. from /? - Cor- 3. D-Seryl-L-tyrosyl-glycyl-L-methionyl-L-glut- so ticotropin (pig), corticotropin A (pig), coramyl-L-histidyl-L-phenylalanyl-laiginyl-L-trypticotropin (Cattle), «-corticotropin (sheep). In particular phyl-glycyl-L-lysyl-L-prolyl-L-valyl-glycyldere they have a chain length of 18-29 or 39, L-lysyl-L-lysyl-L-lysyl-L-lysyl-L-prolyl-L-valyl- preferably 18-25, especially 24 amino acids. L-lysyl-L-valyl-tyrosyl-L-proline, its acid addi- In addition to those defined, other known ones can also be used ionic salts and complexes. 35 deviations from the sequence of natural 4. D-Seryi-L-tyrosyl-glycyl-L-methionyl-L-glut- adrenocorticotropic hormones be present. So amyl-L-histidyl-L-phenylalanyl-L-arginyl-L-trypto- can in particular be the fourth N-terminal amino acid phyl-glycyl-L-lysyl-L-prolyl -L- \ alyl- glycyl- L- by L-norvaline or L-norleucine, the fifth aminolysyl-L-lysyl-L-lysyl-L-lysy '.- L- prolyl-L-valylic acid by L-glutamine, the 25th amino acid L-lysyl-L-valyl-L-tyrosyl-L-prolinamide, its acid-30 L-valine replaced. Particularly noteworthy in addition salts and complexes. in terms of their adrenocorticotropic effects 5. D-Seryl-L-tyrosyl-glycyl-L-methionyl-L-glut-D-Ser ¹ -GIy ³ -Lys ^l '- ¹⁸ - / J ¹ - ^{| 8} -Corticotropin-Lys " ⁸ -amide, amyl -L-histidyl-L-phenylalanyl-L-arginyl-L-tryp- D-Se ^ -Gly'-Lys ¹⁷ · "- / ^ -" - Corticotropin and D-Ser ¹ -tophyl - glycyl - L - lysyl - L - prolyl - L - valyl - glycyl - Gly'-Lys ¹ '· »" - / ^ - "- Corticotropin-Pro ^ -amid.
L-lysyl-L-lysyl-L-lysyl-L-lysin-amide, its acidic 35 Acid addition salts are especially salts of addition salts and complexes. therapeutically applicable acids, such as hydrochloric acid, 6. D-Seryl-L-tyrosyl-glycyl-L-norleucyl-L-glut-acetic acid, but especially sparingly soluble salts such as amyl-L-histidyl-L-pheiiylalanyl-L-ariiinyl-L-tryp-sulfates, phosphates or sulfonates, to mention
tophyl-glycyl-L-lysyl-L-prolyl-L-valyl-glycyl- Among amides are in particular peptide amides, in L-lysyl-L-lysyl-L-lysyl L-lysyl-L-prolyl-L-valyl-40 those the C-terminal carboxyl group is amidated, L - lysyl - L - valyl - tyrosyl - L - proline, its acid - to be understood. addition salts and complexes. Among complexes are the complex-like ones, in theirs 7. Complexes of the peptides according to one of the compounds that have not yet been clarified Proverbs 1-6, with zinc phosphate, zinc pyrophos- understand that with the addition of certain inorganic ones phate and / or zinc hydroxide. 45 or organic substances too adrenocorticotropic 8. Processes for the production of new peptides with seed peptides arise and especially those, ACTH effect, characterized in that they give them a prolonged effect. Such one in a known manner peptides with an inorganic substance are compounds that are different from Chain length of 18-39 amino acids and to metals such as calcium, magnesium, aluminum, N-terminus to derive the complete sequence of the natural cobalt and in particular of zinc borrowed corticotropins, in which, however, one or all of the sparingly soluble salts, such as phosphates and pyromultiple of the amino acids in positions 1-5, phosphates and hydroxides of these metals. Orga-17, 18 and 25 apply in a known manner to other niche substances which prolong the effect of L-α-amino acids exchanged rope can, and call for, for example, non-antigenic gelatine, the first amino acid D-serine, the third Ammo 55 ζ. B. oxypolygelatin, polyvinylpyrrolidone and carbic acid Glycine and L-lysine in 17 and 18 positions, oxymethylcellulose, also sulfonic acid or phos, or their C-terminal amides from the phosphoric acid esters of alginic acid, dextran, polypheno-corresponding protected peptides produced len and polyalcohols, especially polyphloretinphosund, if desired, the compounds in their phat and phytic acid, as well as polymers and co-acid addition salts or complexes transferred. 60 polymers of amino acids, e.g. B. Protamine and in particular of amino acids that contain a predominant proportion of acidic α-amino acids, such as glutamine