DE1643274C3 - New peptides with ACTH effects - Google Patents

Description

acid or aspartic acid.

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

The main patent 14 93 559 relates to new ACTH- al? the previously known peptides as in-vivorkame Peptides with increased or extended tests, e.g. B. by determining the plasma corticosteroid, which are characterized in that they show steron values of hypophysectomized rats.

For example, the; D-Seri-Gly * -Lys ".» /?> - * <- _{groupj or} ^ special of the tert-butyloxycarbonyl-corticotropin has an un comparison to the natural residue. To protect the amino group in the guanido-corticotropin at least ten times higher and min- group of arginine is the nitro Grupps suitable; least 3 times prolonged effect on _{d; e.} but referred to the amino group of arginine must be in

The new peptides will not be protected after the 5 necessary for the reaction. The formation of peptides with a long chain length and the imino group of histidine can be produced by the benzylic 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 groups in a free group as well as the previous formation of smaller peptide units in the course of a functionally modified carboxyl linkage or, if appropriate, in a number of steps are split off according to methods known per se. _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 i ₅ A preferred method consists in that one ACTH-effective peptides with at least = for the first 3 Araiuosäuren HD-Ser-Tyr-Glv-OH N-terminus hm complete sequence of Naturally-containing tripeptide (the abbreviated notation borrowed adrenocorticotropic hormones thereby under- refers to L-amino acids, if the D- is not differentiated by the fact that it is designated as the first amino acid D-serine, as special) or the, in addition, 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 condenses up to at least the -amino group and the side chain to form amino acid 10 and then the decapepamino groups and the terminal carboxyl group tid with the rest of the peptide sequence, so z. B. with and optionally present side chain carboxyl production of the octadecapeptide amide are protected with the octo groups, the protective groups cleave off 25 peptide amide of amino acids 11-18, in the case of Nona and, if desired, the compounds obtained in decapeptide with the nonapeptide of the amino acids, their acid addition salts, derivatives or complexes 11-19, in the case of the eicosapeptide with the decapeptide of the transferred. The amino and / or amino acids 11-20, in the case of the dokosapeptide, are linked to the peptide units in such a way that a dodecapeptide of the amino acids 11-22, in the case of the tri-amino acid or a peptide with a protected α-amino-kosapeptide amide, is created with the Tridecapeptide amide of the group and activated terminal carboxyl group with amino acids 11-23, in the case of tetrakosapeptide amide with one amino acid or a peptide with free the 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, or an amino acid or peptide 35 to the octadecapeptide of amino acids 11-28, in the case of an activated a-amino group and protected termi- nonatriakontapeptide condensed with the nonakosa peptide of the naler carboxyl group with an amino acid or amino acids 11-39. In the case of this Konder.-a peptide with a free terminal carboxyl group, the coupling method used is preferably the protected α-amino group. The carbocarbodiimide method or the activated oxyl group method can be used, for example, by converting an ester, especially by means of the p-nitrophenyl ester, into an acid halide, azide, anhydride or imidazolide. In the latter case, the p-nitrophenyl isoxazolide or an activated ester such as cyano ester of the decapeptide does not need anything as such isolated to methyl ester, carboxymethyl ester, p-nitrophenyl ester, but can also be in the condensation or by reaction by means of a carbodiimide stage itself can be formed from the decapeptide with free carboxyl (optionally with the addition of N-hydroxysuccin group, p-nitrophenol and dicyclohexylearbodiimide imide) or Ν, Ν'-carbonyl-diimidazole.

den, the amino group, for example by reaction. As an N-terminal peptide fragment, however, one can

be activated with a phosphate amide. As £ e- any other peptide instead of the decapeptide, The most common methods are the Carbo- z. B. that containing the N-tennina! En amino acids diimide method, the azide method, the method of 50 tetrapeptide, pentapcptid, 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-terminal 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, one chooses for the condenser is by adding the amino acids in sequence 55 preferably the carbodiimide-hydroxysuccinimide 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, ins- The «-amino group of the N-terminal fragments!

special means by hydrolysis or reduction and all chain amino groups are before easily split off residues, the carboxyl group preferably by the tert-butyloxycarbonyl group, dii preferably by esterification, e.g. B. with methanol, side chain carboxyl groups through the tert-butyl tert-butanol, benzyl alcohol, p-nitrobenzyl alcohol, ester group protected. Also the terminal carboxyl or amide formation, the amino group is, for example, group of the C-terminal fragment, if si by introducing the tosyl, trityl, formyl, tri-amidated, preferably as tert-butyl esters fluoroacetyl, o-nitrophenylsulfenyl, phthalyl or 65 group. At the end of the synthesis: tert Carbobenzoxy group or colored protective groups, butyloxycarbonyl groups and tert-butyl ester groups like the p-phenylazo-benzyloxycarbonyl group and at the same time by acid hydrolysis, for example m the D - (r / - methoxyphenylazo) - beazyloxycarbonyl trifluoroacetic acid, are split off.

Depending on the procedure, the new compounds of an organic base, such as triethylamine, procaine, or compounds in the form of bases or their salts are obtained. Made from dibenzylamine, or other tertiary nitrogen bases. The polymers are known or can be obtained after being based on the salts in a manner known per se. From the latter, in turn, known processes can be made available, for example, by reaction with acids, 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. glulamic acid-a-carboxyanhydridic acids, such as hydrohalic acids, at- 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, sulfuric 10 molar ratio, e.g. B. 100: 1 (depending on the desired free · »or phosphoric acids, or organic acids, degree of polymerization), allow to react and split off the protective groups, acid, lactic acid, pyruvic acid, oxalic acid, z. B. the benzyloxy group with hydrogen bromide in ice-malonic acid, succinic acid, maleic acid, fumaric vinegar, the tert-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, defined chain length, 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-mandeic acid, salicylic acid, 4-amino-saJicylic acid, method, etc.).

2 - phenoxybenzoic acid, 2 - acetoxybenzoic acid, Me- 20 The concentration of the polymer in the pharmaceutical

Thanesulfonic 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. The

Naphthalenesulfonic 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 25 The concentration of the adrenocorticotropic active

Find. These contain the peptides in a mixture with peptides, 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.

those substances are in question that are not associated with the polypeptides.

react, such as B. gelatin, milk sugar, glucose, which are designated as follows:
Table salt, starch, magnesium stearate, talc, vegetable oils, benzyl alcohol, rubber, polyalkylene

glycols, petrolatum, cholesterol or other known system 40:

Drug carrier. The pharmaceutical preparations 35 n-butanol-ethanol-water (40:40:20)

can e.g. B. as lyophilisate or in liquid form Sy. «Tern 43A:

as solvents, 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, K. _{se-butanol-3%} ammonia (70:30)

Stabilizing, wetting or emulsifying agents. You con- 40

There are also other therapeutically valuable substances system ^52:.

contain n-butanol-glacial acetic acid-water (100: 10: 30)

So you can use them especially with the in the System 52A:

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)

Furthermore, to extend the effective 50 · _vstem joq.

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, ins- '

special ones that have a predominant part a ystem IUl:

of acidic «amino acids, such as glutamic acid or n-butanol-pyndm-glacial acetic acid-water (30: 20: 6: 24)

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

have, transfer. The polymers mentioned n-butanol-pyridine-acetic acid-water (42: 24: 2: 30)

and copolymers have free system 104 in the side chains:

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

oxyl group free or functionally modified, system 112 A:

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 c, 11 "> F ·

can. The molecular weight of the polymers can be “.butanol-pyridine-amcicic acid-water

between 10,000 and 100,000, preferably ,,, _{94 9} J _n -.

it is 200,000-80,000. One uses for the 6 ₅ ^^ aL ^ TZun ^ are used:

For the preparation of the preparations, it is advisable to use a waterless

Liches, physiologically acceptable salt, z. B. the BOC = tert-butyloxycarbonyl

Sodium or ammonium salt, or a salt with tBu = tert.-Butvl.

example 1

Rf (101) = 0.42 Rf (52A) = 0.25 Rf (112A) = 0.48

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

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

Lys-Val-Tyr-Pro-OH (D-Ser ^! -Gly ³ -Lys ^{17 18} - /? '- ² ' -

Corlicotropin)

270 mg BOC-D-Ser-Tyr-GIy-Met-GIu (OlBu) -His-Phe-Arg-Try-Gly-Lys (BOC) -Pro-Val-Gly-Lys- (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -Val-Lys (BOC) -Val-Tyr-Pro-Ossu are dissolved in 13 ml of 90% trifluoroacetic acid and left to stand for 30 minutes at 22 '. The solution is then concentrated to about 2 ml, diluted with 10 ml of water, 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 to convert it into the acetate and filtered through a column (0 = 15 mm; / = 15 cm) of weakly basic ion exchanger (Merck No. II) in the acetate form. The elual is concentrated to approx. 4 ml with the addition of a few ml of n-butanol and lyophilized. 230 mg of chromatographically and electrophoretically uniform acetate of D-Scr ¹ -Gly ³ -Lys ^I7 - ¹⁸ - /? ^I - ^M -Corticotropins as an amorphous, readily water-soluble powder. In the thin-layer chromatogram on aluminum oxide (DA) and on cellulose (DC) it shows the following Rf values:

THERE:

DC:

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 / 3'- ²⁴ -corticotropin, the compound has a greatly increased and prolonged corticotropic effect.

The tetracosa peptide derivative used as a starting material can be prepared 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 with stirring at -15 ° 12.5 ml of a 2-n. A solution of hydrogen chloride in ethyl acetate and then 1.13 g (11 mmol) of tert-butyl nitrite are added. 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 mmol) of H-Glu (OtBu) -His-Phe-Arg-Try-GIy-OH in 150 ml of dimethylformamide and 7.5 ml Water, the reaction mixture is stirred for 15 hours at 0 ° and then 50 ml of water are added. The separated amorphous product is filtered off with suction and washed with dimethylformamide / water (1: 5) and acetonitrile. After purification by reprecipitation from aqueous acetonitrile and equilibration with atmospheric humidity, 8.62 g (73%) of pure BOC decapeptide tetrahydrate with a temperature of 230-240 ° are obtained. [*]? = -15 ° -j -2 ° (c = 0.5 in pyridine-water 1 : 1). The substance according to thin-layer chromatogram uniform on silica gel; 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-GIy-Lys-
(BOC) -Lys (BOC) -Ly s (BOC) -Lys (BOC) -PrO-Val-Lys (BOC) -Val-Tyr-Pro-OtBu

ίο 326 mg BOC-D-Ser-Tyr-Gly-Met-GluiOtBiO-His-Phe-Arg-Try-GIy-OH and 350 mg H-Lys (BOC) -Pro-Val-Gry-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. One gives 112 mg of dicyclohexylcarbodiimide, it is stirred partially insoluble mixture during 3 '/ ₂ hours at 50 \ then again an equal amount of dicyclohexylcarbodiimide is added and 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 thus obtained is for final purification in the solvent system methanol-buffer-chloroform-carbon tetrachloride (30: 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 _mBn = 85; K = 0.42) are converted to 295 mg of protected tetrakosapeptide acetate in chromatographically uniform form as a white, amorphous powder with an indistinct decomposition point by concentrating to dryness and subliming away the ammonium acetate at 45 ° in a high vacuum gained approx. 205 °. It has the following Ri values on thin-layer silica gel plates:

Rf (100) = 0.61

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

Example 2

HD-Ser-Tyr-GIy-Met-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys-Lys · NH ₂

Lys ¹⁸ amide)

270 mg BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His- Phe-Arg-Try-Gly-Lys (BOQ-Pro-Va! -Gly-Lys (BOC) -LyS (BOC) - LyS (BOC) -LyS (BOC) -NH ₂ are dissolved in 13 ml of 90% trifluoroacetic acid, left to stand for 30 minutes at 22 °, and then concentrated to about 2 ml. It is diluted with 10 ml of water and concentrated again a volume of 3-4 ml lyophilized. The trifluoroacetate of the free octadecapeptide 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 exchangers (Merck No. II) filtered in the acetate form The eluate is concentrated to about 4 ml with the addition of a few ml of n-butanol and lyophilized. This gives 241 mg of chromatographically and electrophoretically uniform acetate of D-Ser'-Gly ^ Lys ^ - ^ -Corticotropin-Lys ^ -amids as an amorphous, water-soluble powder.

709 611/40

RF (52A) = r 0.15 Rf (IO!) = 0.33 RF (104) = 0.17 RF (112A) = 0.26

light chromatogram on aluminum oxide (DA) to stand room temperature for 30 minutes and on cellulose the following Rf values: let. It is then concentrated in a rotary evaporator

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

1 .1 ",, add 15 ml of water, concentrate again and lyophilize

5 sated. The trifluoroacetate of the free Tctrakosapeptide is thereby to be converted to the

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

(0 - 12mm; / 22cm) 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 F.luat will condense to approx. 5 ml 11.7 cm against the cathode. centered, lyophilized, at 40 ° in a high vacuum

Compared to /? '- ^{= 1} -Cor-, the compound has dried and then equilibrated with atmospheric moisture. ticotropin greatly increased and lengthened corticotropic. 279 mg acetate of the D-Ser -'-Gly'-Lys ¹⁷ · ¹⁸ effect are obtained. /? - '" ^2l -Corticotropin-Pro'- ¹ -amides as white, amor-

The octadecapic, readily water-soluble powder of an indistinct cerpeptide derivative used as the starting material can be produced as follows: settlement point at approx. 180 ".

chromatosramm on aluminum oxide (DA) and on

BOC-D-Ser-Tyr-Gly-Met-Glu (OtBu) -His-Phe- Cellulose (DC) has the following Rf values:

Arg-Try-Gly-Lys (BOC) -Pro-Val-GIy-Lys (BOC) -

Lys (BOC) -Lys (BOC) -Lys (BOC) · NH ₂ ² °>

Rf (52 A) - 0.27 Rf (IOl) = 0.66 Rf (IOl E) - = 0.36 RF (112E) = 0.15

2.9g 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 (BOC) -

PrO-VaI-GIy-LyS (BOC) -LyS (BOC) -LyS (BOC) -LyS- 25 The tetracosa used as starting material

(BOC) -NH ₂ (see No. C 40 616 IVb / 12qu, Case 5806 / peptide derivative can be prepared as follows:
5503/1; 2), are in 27 ml of pyridine, 0.7 ml of water

and 2.38 ml 1-n. Hydrochloric acid suspended and after addition _{1 7} . "_
there would be 1.07 g of dicyclohexylcarbodiimide at 50 ° ge ' ^z - ^Val -'y ^r -' ro-NH,
stirs. The mixture soon solidifies and after 30 18.8 g of Z-Val-Tyr-Pro-OtBu are dissolved in 95 ml of 90% -3h standing at 50 ° with a solution of iger trifluoroacetic acid and during one of a further 1.07 g of dicyclohexylcarbodiimide in Left to stand for 1ml hour at room temperature. Pyridine is added, the mixture is homogenized and then again concentrated to dryness and the oily oil is pulverized and left to stand at 50 ° for 4 hours. Finally residue by adding 100 ml of water and adding 300 ml of water, homogenized and filtered 35 rub at 0 °. After filtering it off, it becomes gelatinous and flaky precipitate. It is dried with constant weight over potassium hydroxide and dried and then reprecipitated in 100 ml of dimethylformamide at 70 ° from ethyl acetate-petroleum ether. The amorphous solved, with the greatest te :! of the dicyclo powder formed (17.01 g) remains insoluble together with 5.15 ml of trihexylurea and is filtered off. ethylamine in 170 ml of absolute tetrahydrofuran. The crude condensation product Juki 4 ° is dissolved from the filtrate, cooled to -15 °, and by adding 600 ml of benzo! and 400 ml of petroleum ether precipitated with a solution of 4.44 ml of isobutyl chlorocarbonate, filtered off and dried. (4.65 g) in 2.5 ml of absolute tetrahydrofuran were added. After 1.0 g of this crude product, for purification, stirring for 15 minutes at -10 °, is slowly added to each 75 ml of upper and lower phase of the solvent-107 ml of 0.64-n. Ammonia in absolute ethyl acetate to systems methanol - 2-n. Acetic acid-chloroform-Te- 45 and left for 1 hour at 0 ° uno. while carbon trachloride (10: 3: 7: 5) stir with gentle stirring for 6 hours at 20 °. The mixture is dissolved at high temperature and, according to Craig, concentrated over 135 steps, the sticky residue is distributed in 500 ml of ethyl acetate phase volume of 25 ml each. From the dissolved and successively with water, 2-N-soda solution dividing elements No. 88-110 (r _max = 100, K = 2.8) and again washed with water and to dryness one obtains with concentration to dryness and way 50 constricted. After the ammonium acetate has been reprecipitated twice, sublime is obtained .; at 45 ° in the residue of ethyl acetate-petroleum ether and a dry vacuum, a total of 490 mg of pure protected octane in a high vacuum, 14.4 g of an amorphous powder, decapeptide amide acetate as a white, amorphous powder. which behaves uniformly in thin-layer chromatography on silica gel. It has the following Rf values on silica gel plates: 55 R _f values:

Rf (52) = 0.55 _{Rf in the system} chloroform-methanol (9: 1) = 0.35

= ° ^'68 Rf (43 A) = 0.63

~ '60 2. H-Val-Tyr-Pro-NH ₂

ο τ- ^, Vi ^SP / l, ^e TT- ™ _ λ ¹⁴ ' ^{4g Z} - ^Val - ^T yr-Pro-NH ₂ are in 150 ml Me-

H-D-Ser-Tyr-GIy-Met-Glu-His-Phe-Arg-Trethanol and dissolved with 1.5 g of 10% palladium carbon Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys-Lys-Pro-Val- at normal pressure and under COj absorption up to Lys-Val-Tyr-Pro-NH ₂ (D Ser ^ Gly ^ Lys ¹⁷ · ¹⁸ - saturation (duration approx. 2 hours) hydrogenated.

^ - «- Corticotropin-Pro ^ -amid) ^{5 ffltrie} J ^en , ^the catalyst and concentration of the filtrate

, ^τατ dryness is the residue in 15 ml of methanol

2:93 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 and precipitated in petroleum ether, filtered off and in the high-

12th

vacuum dried at 40 °. 9.52 g of a matographically pure protected pentapeptide are obtained as amorphous powder, which is obtained as thin-layer chromato- white, amorphous powder. In the thin graph on cellulose plates, it shows the following Rf values: layer chromatogram on silica gel

Rf (40) = 0.73 ₅ ^on:

R _f (45) - o, 72 Rf in the system chloroform-methanol (9: 1) = 0.42

R, (54) = 0.70 Rf (43 A) = 0.68

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

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-NH ₂ are in

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

amide dissolved and with a solution of 5.76 g of Dicyclo- lO'Viger palladium carbon at room temperature under

hexylcarbodiimide was added in 100 ml of acetonitrile. One hydrogenates CO, absorption. The hydrogen uptake

stir for 12 hours at room temperature, it takes about 5 hours and it is filtered Then the precipitated dicyclohexylur- 15 trates off the catalyst and the solution evaporates

cloth 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 has the following R _t values on thin silica gel layers:

plate has the following Rf values: 2 ° R _f (52) - 0.40

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

Rr (43A) -0.68 7. Z-LyS (BOC) -LyS (BOC) -PrO-VaI-LyS (BOC) -

VaI-Ty r-Pro-N H ₂

4. H-Lys (BOC) -Val-Tyr-Pro-NH _{2 25 335 g Z. Lys} ( _B OC) -Lys (BOC) -PrO-O H (15.5 g of the protected tetrapeptide amide are prepared as indicated in Example 1) are dissolved in 20 ml of absolute 100 ml of methanol and, after the addition of 1.5 g Dimethylformamide is dissolved and, after the addition of 10% palladium carbon at normal pressure, it is cooled to 0.65 ml of triethylamine at -15 °. Saturation is left to hydrogenate (duration approx. 4 hours). 58 ml of pivaloyl chloride are added dropwise, the catalyst is filtered off and the filtrate 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 in 10 ml of methanol and 120 ml of ethyl acetate, add absolute dimethylformamide and stir the dissolved and mix by adding 120 ml of petroleum ether for 20 minutes at -10 °, for 5 hours and dried in a high vacuum at 40 °. It is kept at 0 ° and for 2 hours at 20 ° to obtain 11.9 g of an amorphous powder which is thinly colored In addition, 70 ml of ethyl acetate and 50 ml of water are added with a layer chromatogram on silica gel and the following Rr values are extracted, and the organic phase is extracted one after the other: with 0.1M citric acid, 2% ammonia solution

and water, dried 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

rr ,, τ ^^ , -τ- τ, -κ, TT gramm on Silicauel only slight impurities

5. Z-Val-Lys (BOC) -Val-Tyr-Pro-NH _{2 and is used without purification for} further reaction

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 °. ,,
cools and mixed with a solution of 4.57 ml of isobutyl 45 ^R ' ^{lm system} chloroform-methanol (9: 1) = 0.23 chlorocarbonate in 5 ml of ethyl acetate. One stirs _{TT TT} , "" .- ". ",,. _{T rn} , ^ \
for 15 minutes at -10 ° and then gives ⁸ · HL _y s (BOC) -Lys (BOC) -Pro-Val-Lys (BOC) -at about -5 ° a solution of 11.87 g of H-Lys (BOC) - vai-lyr-Pro-NM ₂
Val-Tyr-Pro-NH., In 10 ml of dimethylformamide. The 5.7 g of the above-described octapeptide derivative mixture is dissolved in 100 ml of methanol for 30 minutes at 0 ° and while being added, and then stirred for 5 hours at room temperature and then 550 mg of 10% palladium-carbon at normal pressure at 100% ml of water and 200 ml of n-butanol are added. hydrogenated to saturation. After the aqueous phase has been separated off, the organic catalyst and rinsing with methanol, the Irish phase is successively evaporated to dryness with 10% tartaric acid filtrate. The white, foamy solution (at 0 °), saturated sodium bicarbonate solution 55 residue is dissolved in 100 ml of chloroform, washed with and saturated sodium sulfate solution, over 20 ml of 2-n. Acetic acid extracted, the chloroform phase dried sodium sulfate and concentrated to dryness. then mL concentrated to 20 and from the peptide The foamy residue is switched to the pre-precipitated by adding 60 ml of petroleum ether and once from methanol-Essigester-petroleum ether and once at 50 ° in a high vacuum dried after Äquilibräeaus methanol-acetonitrile reprecipitated to yield 12, 15 g ^6o ren with the air humidity one obtains 5.21 g of an amorphous powder, which in the solvent system is amorphous, thin-layer-chromatographically uniform-methanol-0.1M-ammonium acetate-solution-chloro-borrowed powder, which according to titration and elementary form- Carbon tetrachloride (9: 3: 4: 4) with phase analyzes contains 0.63 mol of vinegar volumes of 25 ml each over 250 levels of multiplicative acid and 13 mol of H ₂ O per mol of octapeptide amide. It is distributed on silica gel . The following Rf values are obtained from the distribution elements No. 81-115 65 : 0max = 91; K = 0.57) become

Dryness and sublimation of the ammonium ace-Rf in the chloroform-methanol system (9: 1) = 0.08

tates in a high vacuum at 45 ° a total of 7.94 g chromium R _f (100) = 0.46

923

Tyr-Pro-NH ₂

3.91 g of Z-LyS (BOC) -PrO-VaI-GIy-LyS (BOC) -LyS- (BOC) -NHNH ₂ (cf. German patent 12 14 242) are dissolved in 40 ml of absolute dimethylformamide with gentle heating, cooled to -20 ° and at this temperature with stirring slowly successively with 6.58 ml of 2.15-n. Hydrochloric acid and with 0.74 ml of 5-n. Sodium nitrite solution added. The mixture is finally stirred for a further 15 minutes at -10 'and then a precooled solution of 4.07 g of H-Lys (BOC) -LyS (BOC) -PrO-VaI-LyS (BOC) -VaI-T) T-PrO is added -NIl ₂ (containing 2.5% CH ₃ COOH + 15.3 ";, H ₂ O) in 11 ml of dimethylformamide and 1.98 ml of triethylamine. The mixture is stirred for 1 hour at 0", whereupon the pH adjusted to 7.5 by adding triethylamine and left to stand overnight at 0 °. The reaction mixture is then concentrated to a volume of approx. 20 ml in a high vacuum, and 100 ml of water are added. The crude product precipitates in ready-to-use form. It is filtered off, washed with water and then reprecipitated three times from methanol-water and twice from methanol-benzene-petroleum ether. The material pre-cleaned in this way (4.72 g) is in the solvent system methanol-buffer-chloroform-carbon tetrachloride (12: 4: 9: 3) [buffer = - 28.5 ml glacial acetic acid + 19.25 g ammonium acetate in 960 ml water] subjected to a Craig distribution over 160 stages with phase volumes of 20 ml each. From the distribution _{elements No. 45-74} (/ - milx = 57; K = 0.55) a total of 3.3 g of chromatographically pure, protected tetradecapeptide as a white, amorphous powder from the decomposition point is obtained when the ammonium acetate is concentrated to dryness and sublimated away at 45 ° in a high vacuum gained approx. 210 °. It shows the following Ri values in the thin layer chromatogram on silica gel:

II. BOC-D-Ser-Tyr-Gly-Met-GluiOtBui-His-

Phe-Arg-Tyr-Gly-LysCBOQ-Pro-Val-Gly-LysiBOC) -LyS (BOC) -LyS (BOC) -LyS (BOC) -PrO-VaI-LyS-

(BOC) -Val-Tyr-Pro-NH ₂

355 mg BOC-D-Ser-Tyr-Gly-Mel-Glu (OlBu) -His-Phc-Arg-Tyr-Gly-OH and 400 mg H-Lys (BOC) -Pro-Val- (Jly-Lys (BOC ) -Lys (BOC) -Lys (BOC) -Lys (B0C) -Pro-Val-Lys (I? OC) -Val-Tyr-Pro-NH ₂ are in a mixture of 3.7 ml of pyridine and 0.273 ml of 1 -n hydrochloric acid is homogenized with heating to 50 ° The pasty, gelatinous mass is then mixed with a solution of 131 mg of dicyclohexylcarbodiimide in 0.2 ml of pyridine, flushed with nitrogen, homogenized again well and then for 3 hours at 50 ° After this time, a second portion of 131 mg of dicyclohexyl-carbodiimide in 0.2 ml of pyridine is added, again flushed with nitrogen, homogenized and left to stand for a further 3 hours at 50 °. Finally, 50 ml of water are added, homogenized , filter off the fine-grain gelatinous precipitate and dry it in a high vacuum at 45 ° To remove dicyclohexylurea and other lipophilic impurities, it is suspended twice in succession in hot methanol by adding Be nzol and petroleum ether precipitated, filtered off and dried. The so pre-cleaned product is multiplicatively distributed over 185 levels in the solvent system methanol - buffer - chloroform-carbon tetrachloride (10: 3: 6: 4) [buffer = 28.5 ml glacial acetic acid> 19.25 g ammonium acetate in 960 ml water], with phase volumes of 10 ml each. From the distribution elements No. 67-86 (r _{m! lx} = 77; K = 0.71) one isolates the ammonium acetate in the | High vacuum at 45 ° the pure, protected tetrakosapeptide amide as a white, amorphous powder with an indistinct decomposition point at approx. 220 °. It has the following Rf values in a thin layer chromatogram on silica gel:

Rf in the chloroform-methanol (9: 1) system = 0.17 Rf (43 A) = 0.62

Rr (100) = 0.87

10. H · LyS (BOC) -PrO-VaI-GIy-LyS (BOC) -

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

Val-Tyr-Pro-NH,

4.97 g of the protected Tetradekapeptidamids are dissolved in 100 ml of methanol and heated after addition of Ig 10% palladium carbon at room temperature and normal pressure with absorption of the carbon dioxide formed is hydrogenated to saturation. The catalyst is filtered off, the filtrate concentrated to about 15 ml and from it the hydrogenated tetradecapeptide by adding 50 ml of benzene and 150 ml of petroleum ether precipitated, filtered off and in a high vacuum dried at 45 °. 4.25 g of an amorphous powder are obtained, which is reflected in the thin-layer chromatogram behaves uniformly on Süicagel and has the following Rf values:

Rf (chloroform-methanol = 7: 3) = 0.42
j ^ f ($ -> \ = 0 41

_{Rf (1} öo) IO ₃₈

ΒΟΓ Tv! riv nrv, ⁱ - ^{ü (J (} -lyr-Gly-OCH ₃

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

Sun 10.12 g BOC-tyrosine. _"o

dissolved ester and after cooling to 20 ° with a solution of 3.84 g of glycine methyl ester in 10 ml of ethyl acetate offset. 10.4 g of dicyclohexylcarbodiimide in solid form are added and the mixture is stirred for 18 hours

the one at 25 °. It is then cooled to 0 ° after 30 minutes filtered off from the precipitated dicyclohexylurea and from the filtrate by concentration and Precipitation with petroleum ether isolates the crude dipeptide derivative as an amorphous, viscous mass. She is through still-

Mayy falling over from ethyl acetate-petroleum ether further purified and can then from the same solvent mixture be crystallized; 124-125 ° F.

The following Rf values are obtained in the thin-layer chromatogram on silica gel:

Rf (43 A) = 0.65

Rf (CHClg-methanol = 8: 2) = 0.68
Rf = 0.77

2. H-Tyr-Gly-OCHa hydrochloride

2 g of BOC-Tyr-Gly-OCH ~ are heated in 20 ml of absolute ethyl acetate dissolved and at 20 ° with 20 ml of 4-n. Hydrochloric acid is added to absolute ethyl acetate. After a few minutes, a precipitate begins to separate out. After 30 minutes, the reaction mixture is concentrated in vacuo to about 10 ml and completed the precipitation of the dipeptide ester hydrochloride by adding 50 ml of petroleum ether. One homogenizes, the amorphous product is filtered off, washed with petroleum ether and dried in a high vacuum at 30 °. The Dipeptide ester hydrochloride is obtained as a hygroscopic powder from F. approx. 110-115 (decomposition). It shows the following Rr values in the thin-layer chromatogram on silica gel:

Rt (43 A) 8: 2) = 0.33 Rr (CHCI ₃ -MethanoI - - 0.36 Rt (101) = 0.60

3. BOC-D-Ser-Tyr-Gly-OCHj

1.69 g of H-Tyr-Gly-OCHa hydrochloride are dissolved in 10 ml of absolute dimethylformamide with gentle heating and then cooled to 0 °. 0.84 ml of triethylamine, 1.23 g of BOC-D-Ser-OH and 1.65 g of dicyclohexylcarbodiimide are added in succession and the mixture is stirred for 20 hours at 25 ° and finally for a further hour at 0 °. The precipitated mixture of triethylammonium chloride and dicyclohexyl urine is filtered off and the filtrate is concentrated to an oily residue. By adding 30 ml of ethyl acetate and 100 ml of petroleum ether, the crude product is precipitated as a greasy mass. For pre-cleaning, it is dissolved again in a little ethyl acetate while warming and it is precipitated by adding a large amount of petroleum ether (1.8 g). The product, which is still impure according to thin-layer chromatography, is used for purification in the solvent system. Ethyl acetate-benzene - 0.05 - m. Aqueous ammonium acetate solution (2: 1: 2) with phase volumes of 10 ml each, distributed multiplicatively according to Craig. After 250 hours, the tripeptide derivative is isolated from distribution elements no. 45-74 (r _max = 60; K = 0.32) by evaporating the solvent and subliming away the ammonium acetate at up to 45 ° in a high vacuum as a chromatographically pure compound. Crystals from methanol-water, mp 180-182 °. The following Rf values are obtained in the thin-layer chromatogram on silica gel:

Rt (52) 8: 2) = 0.76 Rt (101) = 0.78 Rf (CHCL-methanol = = 0.58.

uniform by layer chromatography. It has the following Rf values on silica gel:

Rf (52) = 0.60

Rf (101) = 0.61.

5. BOC-D-Ser-Tyr-Gly-Nle-OCHj

3.09 g of BOC-D-Ser-Tyr-Gly-OH are in one Mixture of 5 ml of absolute dimethylformamide and

ίο 15 ml of acetonitrile dissolved with gentle warming. After cooling to 10 °, a solution of 1.23 g of freshly prepared norleucine methyl ester in 15 ml Acetonitrile added. Then 2.04 g of solid dicyclohexylcarbodiimide are added. The solution will be stirred for one hour at 10 ° and for 16 hours at room temperature. Excess dicyclohexylcarbodiimide is destroyed by adding 1.4 ml of glacial acetic acid and stirring for one hour at room temperature. The precipitated dicyclohexylurea is filtered off and evaporated to dryness at a bath temperature of 50 "in vacuo. The residue, a yellowish oil, is dissolved in 200 ml of ethyl acetate, the Solution with 70 ml 1-n. Citric acid solution, two times 70 ml 2-n. Natriui.ihydrogencarbonatlösung and twice 70 ml of saturated sodium chloride solution extracted, dried over solid sodium sulfate and evaporated. The white, foamy crude product is crystallized from ethyl acetate-petroleum ether and stored at 50 ° dried in a high vacuum. 3.50 g, m.p. 138-142 °; after repeated crystallization, F. 143-246 °.

It has the following Rf values in a thin-layer chromatogram on silica gel:

Rf (benzene-acetone = 1: 1) = 0.18.
BOC-D-Ser-Tyr-Gly-Nle-NH-NHj

4. BOC-D-Ser-Tyr-Gly-OH

685 mg of BOC-D-Ser-Tyr-Gly-OCH ,, are dissolved in 5 ml 1-n. Sodium hydroxide solution, left to stand for 10 minutes at 20 ° and then neutralized the solution Addition of 5 ml 1-n. Hydrochloric acid. The protected tripeptide is mixed with 50 ml of water-saturated n-butanol extracted, the butanol layer washed four times with 4 ml of water each time and concentrated to about 3 ml. Through Addition of 30 ml of petroleum ether gives a greasy precipitate which is crystallized from ethyl acetate can (M.p. 168 ° [decomposition]). The substance is thin - 3.30 g of BOC-D-Ser-Tyr-Gly-Nle-OMe are in 18 ml of absolute ethyl alcohol dissolved. 1.8 ml of hydrazine hydrate are added to the clear, yellow solution and the slightly cloudy solution was left to stand at room temperature for 24 hours. That crystallized out The product is filtered off and washed with cold absolute ethanol. After drying at Room temperature over concentrated sulfuric acid is dried in a high vacuum: 1.4 g, F. = 152- 161 ° (decomp.).

A further 0.5 g of F. 134.5-139 ° (Decomposition) can be obtained.

After recrystallization from methanol-ethanol = 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:

Rf (chloroform-methanol = 1: 1) = 0.64
Rf (chloroform-methanol = 8: 2) = 0.28

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

1.14 g (2 mmol) BOC-D-Ser-Tyr-Gly-Nle-NHNHj, are suspended in 10 ml of dimethylformamide and with stirring at -15 ° with 4 ml of a 2-n. solution of hydrogen chloride in ethyl acetate and 0.23 g (2.2 mmol) of tert-butyl nitrite were added. The reaction solution is stirred for a further 10 minutes at -15 °, and then by adding 1 ml of triethylamine weakly basic. The thus obtained

Solution of the BOC-i etrapeptide-a;: ids is added drop by drop to a solution, cooled to -10 °, of 1.42 g (1.6 mmol) of H-Glu (OtBu) -His-Phe-Arg-Try-Gly-OH given in 30 ml of dimethylformamide and 1.5 ml of water. The reaction mixture is 22 hours at 0 " stirred, then mixed with 100 ml of water, and the precipitated amorphous product is suction filtered, washed with dimethylformamide-water (1: 5), water and acetonitrile and dried. After cleaning by reprecipitation from aqueous acetonitrile and equilibration 1.44 g (S9%) of pure BOC decapeptide tetrahydrate with a temperature of 235-240 ° are obtained with atmospheric humidity receive; [a] f = -22 ° (c = 1 in pyridine-water 1: 1). The substance shows the following Rf values in a thin-layer chromatogram on silica gel:

Rf (52 A)
RF (101)

== 0.3-0.4
= 0.6-0.7

7. BOC-D-Ser-Tyr-Gly-Nle-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

300 mg BOC-D-Ser-Tyr-Gly-NIe-GIut (OtBu) -His-Phe-Arg-Try-GIy-OH and 310 mg H-Lys (BOC) -Pro-VaI-GIy-LyS (BOC) -LyS (BOC) -LyS (BOC) -LyS (BOC) -Pro-Val-Lys (BOC) -Val-Tyr-Pro -OtBu are with 6ml pyridine, 0.5 ml water and 0.15 ml 1-n. Hydrochloric acid added. A solution of 100 mg of dicyclohexylcarbodiimide is then added added in 0.75 ml of pyridine and stirred for 5 hours at 45-50 °. Then there will be more 75 mg of dicyclohexylcarbodiimide added and over Stirred at 45 ° overnight. The mixture of condensation product and dicyclohexyl urea is total precipitated by adding 75 ml of water, filtered off and dried. To separate the dicyclohexyl urea it is suspended or partially dissolved in ethanol twice in succession, and precipitated again by adding benzene and petroleum ether. The material pre-cleaned in this way is as in example 1 described purified by countercurrent distribution. After 120 distribution steps in the solvent system Methanol - buffer - chloroform - carbon tetrachloride (10: 3: 5: 4) [buffer: 28.5 ml of glacial acetic acid + 19.25 g ammonium acetate in 960 ml water] the pure, protected tetrakosapeptide is obtained from fractions 46-58 (distribution number K == 0.82). In thin-layer chromatography on silica gel plates, the protected peptide derivative shows the following Rf values:

Rf (100) = 0.61

Rf (chloroform-methanol 75:25) = 0.41

8. H-D-Ser-Tyr-Gly-NIe-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-Gly-Lysi-Lys-Lys-Lys-Pro-

Val-Lys-Val-Tyr-Pro-OH (acetic acid salt)

The cleavage of the protecting groups from the protected Peptide and the conversion of the trifluoroacetic acid into the acetic acid Sah: happens as in the example 1 described. The free tetrakosapeptide shows on paper electrophoresis (pyridine acetate buffer, pH = 6.35; Running time 1 hour; 2000 V) a running track 8.5 cm against the cathode. In thin-layer chromatography on aluminum oxide, the Connection in system 101 has a: n Rf value of 0.41.

Example 5

Dissolve 11.12 mg of glacial acetic acid, 1.21 mg of sodium acetate, 2.5 mg of sodium chloride and 2.0 mg of D - Ser ¹ - GIy ^ - £ 'i7,? B «ι-:". Corticotropin - hexaacetate in 0, 7 ml of distilled water and make up to 1 ml with distilled water. The solution is autoclaved at 120 ° for 20 minutes. The pH after sterilization is 3.7.

Example 6

Dissolve 0.5 mg of D-Ser ^l -Gly ³ -Lys "- ¹⁸ - ^ _: ²¹ -corticotropin hexaacetate in 0.7 ml of physiological sodium chloride solution and acidify the solution with 0.1 N hydrochloric acid to pH 3.2 It is made up to 1 ml with distilled water and heat-sterilized as in Example 4.

Example 7

In 0.8 ml of distilled water, 7.05 ml

Glycine 'and 6.08 mg of sodium chloride dissolved and

0.055 ml 0.1-n. HCl added. Then will

hexaacetate in the solution and made up to 1.0 ml with distilled Wisser.

The solution is nitrated in the usual way, filled into ampoules and autoclaved at 120 ° for 20 minutes. The pH is 3.6.

Example 8

In 0.8 ml of distilled water, a solution of 10.16 mg of citric acid (with 1 mol of water of crystallization), 0.097 ml 1-n. Sodium hydroxide solution, 3.54 mg sodium chloride and 0.51 ml 0.1-n. Hydrochloric acid produced. In this lo-

Solution, 4.0 mg of D-Ser ^{1 -Gly 3} -Lys ¹⁷ ' ^I8 - ^ ¹ ~ ²¹ -Corticotropin-hexaacetate are dissolved and made up to 1.0 ml with distilled water.

The solution is filtered in the usual way, filled into ampoules and autoclaved at 120 ° for 20 minutes.

piano. The pH is 3.6.

Example 9

A suspension is made from the following components from:

y ^

tropine hexaacetate 1.0 mg

ZnCl ₂ 5.25 mg

NaHPO ₄ : 2 H ₂ O 1.05 mg

NaCl 2.0 mg

Benzyl alcohol 10.0 mg

NaOH ad pH 8.3

Distilled water to 1.0 ml

Example 10

Make a suspension of the following components from:

D-Ser ^l -Gly ³ -Lys ^{17 '18} - /?' - ²⁴ -Cortico-

tropine hexaacetate 1.0 mg

ZnCl ₂ 6.30 mg

Na ₂ NPO ₄ · 2 H ₂ O 1.26 mg

NaCl 1.5 mg

Benzyl alcohol 10.0 mg

NaOH pH 8.3

Distilled water ad 1-00 ml

Example 11

Make 0.5 ml of a hydrochloric acid solution of 0.5 mg D-Ser ^{1 -Gly 3} -Lys ^{17> 18} - ^ ^l ~ ²⁴ -corticotropin hexaacetate, 5.25 mg ZnCl * 1.05 mg Na ₂ HPO ₄ · 2 H ₂ O and 2.0 mg of NaCl with a pH of 3.0 and add the solution to 0.5 ml of an aqueous solution of 10 mg of benzyl alcohol which contains enough sodium hydroxide solution that a suspension with a pH of 8.3 is obtained.

Example 12

A dry vial is made from the following components: I ₅ ,

40

Example 14

2.0 g of poly-L-glutamic acid with an average molecular weight of approx. 39,600 are in approx. 5.7 ml 10% sodium hydroxide solution dissolved, so that the pH of the solution 7.4. In this solution, s, C mg

Water made up to 10 ml. The solution is filtered sterile. It contains per ml:

tropine hexaacetate 2.0 mg

ZnSO ₄ • 7 H ₂ O 5.0 mg

Na ₃ PO ₄ • 12 H ₂ O 5.5 mg

Mannitol 40.0 mg

Before use, the contents of the dry vial are mixed with 1 ml of distilled water. You get one Suspension of pH 7.6.

Example 13

5 mg of poly-L-glutamic acid with an average molecular weight of 39,600 are in 5 ml of 0.1-n. Sodium hydroxide solution dissolved. The solution is filtered, a solution of 2.5 mg D-Ser ¹ -Gly ³ -Lys ^I7 - ¹⁸ - /? ^l - ^2l -Corticotropinhexaacetat added, then acidified with acetic acid or hydrochloric acid to pH 4 and fills ml with water to 10th A poly-L-glutamic acid-D-Ser ¹ -Gly ³ -Lys ^{17 '18} - /? ¹ - ^{!! 4} -corticotropin complex finely divided. The suspension contains 0.5 mg of poly-L-glutamic acid and 0.25 mg of ^ "" - corticotropin as a complex compound per ml.

yy ^

tropine hexaacetate 4.0 mg

Poly-L-glutamic acid 200.0 mg

Sodium hydroxide solution up to pH 7.4

Merthiolate 0.02 mg

Aqua dest. ad 1.0 ml

Example 15

A solution ampoule is made from the following components:

yy ^

tropine hexaacetate 2.0 mg

Gelatin 280.0 mg

Phenol 5.0 mg

least. Water to 1.0 ml

Example 16

A sterile filtered aqueous solution of D-Ser ¹ -GIy ³ - Lys ¹⁷ * ¹⁸ -ß ¹ - ² * - corticotropin - hexaacetate is mixed under aseptic conditions with sodium polyphloretin phosphate and sodium chloride and filled into vials and lyophilized, so that a dry vial containing the following components

and 0.2 mg of merthiolate and dissolved with distilled yy ^

tropine hexaacetate 0.2 mg

Sodium polyphloretin phosphate

(86.5%) 23.20 mg

NaCl 12.28 mg

Before use, the contents of the dry vial are mixed with 2 ml of distilled water, contained in a solution ampoule, mixed.

Example 17

In a manner analogous to that in Examples 5-16, preparations are made which contain, as an adrenocorticotropically active peptide, D-Se ^ -Gly ^ Lys ^ - ^ - ^ - ^ - Corticotropin-Pro ²⁴ -amid, D-Ser ¹ -Gly ³ - Lys "- ¹ » - / J ¹ - ^ie -Corticotropin-Lys ¹⁸ - amide, D - Ser ¹ - GIy ³ - Never ⁴ - Lys ¹⁷ · ¹⁸ - / 3 ¹ - ²⁴ - Corticotropin, D - Ser ¹ - GIy ³ - Never ⁴ - Lys ¹⁷ · ¹⁸ -Z? ¹ - ²¹ -Corticotropin -Pro ²⁴ -amid or D-Ser ¹ -GIy ³ -NIe ⁴ - Lj "> ¹⁷ · ¹⁸ - ß ^l ~ ^ia - Corticotropin - Lys ¹⁸ - amide included.

Claims (8)

as the first amino acid an amino acid with D-configuration claims: have figuration. The present invention relates to 1. Adrenocorticotropically active peptides according to adrenocorticotropically active peptides according to the main patent 14 93 5J9 with a chain length 5 main patent with a chain length of 18-39 of 18-39 amino acids and the N-terminus amino acids and the N-terminus complete to complete sequence of the natural Corti sequence of the natural corticotropins, but in the cotropine, but in which one or more of the one or more of the amino acids in the positions amino acids in the positions 1-5, 17, 18 1-5, 17, 18 and 25 in a known manner against others and 25 _{can be exchanged for other 10} La-amino acids in a known manner, the da-La-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, characterized by 17- and 18-position amino acids having L-lysine, characterized in that they are the 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 production of the compounds listed as 17- and 18-position 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 of 18-25 amino acids. borrowed adrenocorticotropic hormones, e.g. B. from ß-Cor- 3. D-Seryl-L-tyrosyl-glycyl-L-methionyl-L-glut-20 ticotropin (pig), corticotropin A (pig), coramyl-L-histidyl-L-phenylalanyl-L-arginyl-L-trypticotropin (Cattle), Λ-corticotropin (sheep). In particular, 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 aut. L-Iysyl-L-valyl-tyrosyl-L-proline, its acid addi- In addition to those defined, there may be other known ones ionic salts and complexes. 35 deviations from the sequence of natural 4. D-Seryl-L-tyrosyl-glycyl-L-methionyl-L-glut- adrenocorticotropic hormones be present. So amyl-L-histidyl-L-phenylaianyi-L-arginyl-L-trypto- can in particular be the fourth N-terminal amino acid phyl-glycyl-L-lysyl-L-prolyl-L-valyl-glycy 1-L- by L-norvaline or L-norleucine, the fifth aminolysyl-L-lysyl-L-lysyl-L-lysyl-L-prolyl- L-valylic acid be replaced by L-glutamine, the 25th amino acid by L-lysyl-L-valyl-L-tyrosyl-L-prolinamide, its acid- 30 L-valine. Particularly noteworthy in addition salts and complexes. are related to their adrenocorticotropic effects 5. D-Seryl-L-tyrosyl-glycyl-L-methionyl-L-glut- D-Ser'-Gly ^ Lys '^^ - ^' - ^ - Corticotropin-Lys ^ -amid, amyl-L-histidyl- L-phenylalanyl-L-arginyl-L-tryp-D-Ser'-Gly'-Lys ¹⁷ · ¹⁸ - /? ¹ - ^ - Corticotropin and D-Ser ¹ -tophyl - glycyl - L - Iysyl - L - prolyl - L - valyl - glycyl - Gly'-Lys ¹⁷ · ^ΙΒ -β ^τ " ²⁴ -Corticotropin-Pro ^Z4 -amide. L -Iysyl-L-lysyl-L-lysyl-L-lysine-amide, its acid- 35 As acid addition salts are particularly salts of addition salts and complexes. Therapeutically applicable acids, such as sakic acid, 6. D-Seryl-L-tyrosyl-glycyl-L-norleucyl-L-glut-acetic acid, but especially sparingly soluble salts, such as amyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryp sulfates, phosphates or sulfonates, tophyl - glycyl - L - Iysyl - 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 which the C-terminal carboxyl group is amidated, L - iysyl - L - valyl - tyrosyl - L - proline, its acid - to understand. 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 phate and / or zinc hydroxide. 45 or organic substances to adrenocorticotropically effective 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 corticotronines, 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 in a known manner against other niche substances that prolong the effect of her-L-a-amino acids can be exchanged, and call for, are for example non-antigenic gelatine, the first amino acid D-serine, the third amino 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, polyphenoen {speaking protected peptides produced len and polyalcohols, especially polyphloretinphosund, if desired, the compounds in their phat and phyiic 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