DE2027346A1 - Process for the production of ACTH-effective peptides - Google Patents

Description

CIBA AKTIENGESELLSCHAFT, BASEL (SWITZERLAND)

Case 679I / E '·' ■

Germany

Process for the production of ACTH -ν * irksamen
Peptides.-

The present invention relates to the production of ACTH-effective peptides which can be used orally.

It has been found that oral ACTH-effective peptides are obtained if ACTH-effective peptides which have an amino acid with a D configuration as the first amino acid or their amides N ^a -acylated, in particular with an alkanoyl radical with up to 30 carbon atoms. The subject of the invention are therefore ACTH-effective peptides, which are the first
Amino acid have an amino acid with D-configuration
and whose α-amino group is aylated, in particular

009851/2239

by an alkanoyl radical with at most } 0 carbon atoms, for example propionyl, butyryl, valeryl, caproyl, pelargonyl, lauroyl, myristoyl, palrr.itoyl, behenyl ,. and their amides, and acid addition salts of these compounds, and processes for their preparation.

As ACTH-effective peptides with a D-amino acid The first amino acids to be mentioned are e.g. peptides with the sequence of the natural corticotropins, but D-serine am N-terminus, e.g. D-Ser -β-corticotropin, also synthetic Peptides with a shorter amino acid sequence and / or with a modified amino acid sequence and an N-terminal D-amino acid. The last-mentioned synthetic peptides mainly include those with 18-28 amino acids from the amino end of the natural corticotropins counted, if necessary with Deviations from the natural sequence with regard to one or more of the amino acids 1-5, 11, 17 * J-8, 19 and 25 · The first amino acid can e.g. also be D-alanine or

2
D ~ be threonine; Thyrosine can ζ οB. by phenylalanine,

■ 5 ·. 4th

Serine- 'e.g. by glycine, methionine by a-lower alkyl-a-

aminoacetyl, e.g. through norvaline or norleucine, leucine, valine or a ~ aminobutyric acid, glutamino acid ⁵ e.g. through glutamine, lysine ¹¹ through ornithine, lysine ¹¹ * ¹⁵ 'through ornithine, arginine''through ornithine or lysine, proline ¹ ^ through valine, amino acid ^ to be replaced by valine »As examples of the new peptides are mentioned N ^a - * acyl-, eg

009851/2239

U - / '. LkaMoylbiuva'-G from D-Ser -Corticotropin-Arg -amid, from D-Ser ¹ -Oni ^' ^] -ß ¹ -Cortieotropin-Orn -amid, from D-Ser ¹ -Oly ^ - Lys ¹⁷ - » ¹ ^ - (J ¹ ~ ^1Ö -Corticotropin-T.ys ¹ -amid, from D-Ser ¹ -NIo 'Iy: v ¹ ^ ^fl8 -ß ¹ " ^lß Corticotropin-I ₁ ys ¹ -amid, from D-Sei ^ -Lys ^1? '-Corti-

oo'.tvi! ji.-I.yiJ -ια.Λό, from D-llcv -p ~ -'- COr-Ueolropi.n, from D-Si-r "-ß" ~ - ^> -rro ^^ - arnit], from I) - £ ei ' ¹

ccirupin, D-Üor ¹ -Lyis ^{1 'lc} -VaI ¹ Z' ¹ - ^ ¹ ^ ³ ~ -Coi'f loot ropin- ■

10 1 ^ 17 18 IO -1-10 λ

VaI - -amid, from Π-Ser- -NIe -I.ys' '-VaI' -β ^ -Corticotro- i

jo Il -pn 20

]! in-Val --amid, from D -. 'icr'-,' ³ . '-Corticotropin-Val "-amid, from D.-Sor ¹ -Lys" ^l ''' ^lo - _l 3 ¹ " ^{? 0} -Corlieo-1.ropin-Val ^{i? 0} - £ r; ad _J. Von D -Ser ¹ -ö-Aiainol.-utyi'yl'-GIu (NIi.,) .'- ^> -ß ¹ " ^{i? 0} -CorticoLropin-Val ' ^{? 0} -amid _J

from D-Ser ~ Lya -ß '--Corticotropin-Pro r-arnid, from D-Ser -a-Arninohutyryl ^l -ß ¹ " ²⁰ -Corticotropin-Yal ^{t? 0} -ar.iid,

of D-Ser -0 -Corticotropin, of D-Ser -Lys '-'- ß Corticotropin-Lys ²¹ -amide, of D-Ser ¹ -Lys ¹⁷ ' ¹ ^ ¹ " ²² -

Cortieotropin-Val "-amid, from D-Ser -ß" - ^ - Corticotropin,

from D-Ser -β ^ -Corticotropin-Tyr ^ -arnide, from D-Ser -AIa ■ - ™

β ~ ² ^ -Corticotropin-Tyr ² ^ -amid, from D-Ser ¹ -Gly ^ -ß ¹ " ² ^ - Corticotropin-Tyr ^ -amid, from D-Ser ¹ -Lys ¹ ^ * ¹ -ß ¹ " ² 5 Cortico

tropine-Tyr ^ -amid, from D-Ser -ß -Corticotropin, from D-Ser ₂ " ² ^ corticotropin, from D-Ser ¹ -Orn ^{17 '18} ^ ^3- " ² ^ -Cor.ti-

•. ι "^ i -24 ι

cotropin, from D-Ser -Gly - ^ - ß -Corticotropin, from D-Ser Orn ^{11 '15} " ¹⁸ ^ ¹ " ^ -Corticotropin, from D-Ser ¹ -Nle ^ -Q ¹ " ² ** -

11-24 24

Corticotropin, from D-Ser -ß -Corticotropin-Pro -amide,

D-AIa ¹ ^ ¹ -Corticotropin, from D-Ser ¹ -Gly ^ -Lys ¹⁷ ' ^l8 -ß

l-? 4 1 4 17 18 1-24

-Corticotropin ,, from D-Ser -Nie -Lys '' -ß -Cortico-

009351/2239

ph ι 17 l8 1 ~? ^ι \? h

tropin-Pro -amide, D-Ser -Orn '' -β -Corticotropin-Pro -

1 H 17 18 i-? 4 24

amide, from D-Ser -Nie -Om ^f '-ß -Corticotropih-Pro -amide,

1 "^ 17 iP 1 -pil p / i

from D-Sor -Gly ^ -Lys- ¹ '' · ¹⁰ ^ "-Corticotropin-Pro -amide, from D-Ser ¹ -Lys ^{1 /} '' ¹ -ß ¹ " ^2j -Corticotropin, from D-Ser ¹ - Lys ¹⁷ ' ² ^ -ß

1 -PÜ P ^ 1 Ji ■ P ^ 1 _ρς

⁴ -Corticotropin-Pro-amide, from D-Ser -HIe -VaI ³ -β ^ - corticotropin-VaI ²⁵ -amide, from D-Ser ¹ ^ Va -Val ²⁵ -ß ¹ ~ ²⁵ -Corticotropin-Val ²⁵ -amide , from D-Ser ¹ -Nva * '- Lys ¹⁷ ' ^l8 Corticotropin-Val -'- amide, from D-Ser -ß -Corticotropin-Val amide, from D-Ser ¹ -NIe ^ -Lys ¹ ^ ¹ -Val ²⁵ -ß ¹ " ²⁵ -Corticotropin-Val ²⁵ -

ι ι -p6 ti -Pfl

amide, from D-ßer -ß -Corticotropin, from D-Ser -ß -Corticotropin, from D-Ser -ß "^ -Corticotropin, from D-Ser -ß"

1 1 - ^ Q

Corticotropin, from D-Ser -ß ^ - -Corticotropin, N ^a -alkanoyl-, especially acetylderiyate of the D-type of the N-terminal amide of the peptide with the first 18 amino acids of the β-corticotropin with lysine residues in place of the Arginine residues in 17 and 18 positions (D-Ser ¹ , Lys ¹⁷ ' ¹ -ß

-Corticotropin-Lys -amide) and the corresponding peptides with norleucine in place of methionine, furthermore corresponding N ^a derivatives of D-Ser, Lys '' -ß ~ -corticotropin and its amide.

Salts in particular are used as acid addition salts of therapeutically usable acids such as hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, long-chain fatty acids, to call. ■ '

009 551/2239

BATH ORIGINAL

■■■:. '.;. ■ ■;., - 5 - ■■■.'^; ■. _: ■ "

The manufacturing process according to the invention of the new peptides, their amides and acid addition salts ■ characterized by the fact that '

1) from ACTH-effective peptides as the first amino acid an amino acid with D-configuration and in which the acylated a-amino group and at least the side chain amino ■ groups and the terminal and side chain carboxyl groups are protected are, splits off the protecting groups or '

2) ACTH-effective peptides, the first amino acid to be an amino acid with D-configuration, N-acylated and, if desired, the compounds obtained are converted into their acid addition salts.

The starting materials are used for the production of long-chain peptides, especially the ACTH-effective Peptides, methods described.

Used as a protective group for variant 1) of the process and for the intermediates of variants 1) and 2) the groups known for peptide synthesis, especially those known for the synthesis of ACTH sequences and any newly proposed groups, e.g. those in French Patent No. 15 54 051 described groups. Examples of amino protective groups that may be mentioned are optionally substituted aralkyl groups such as Diphenylmethyl or triphenylmethyl groups, or acyl groups such as formyl, trifluoroacetyl, phthaloyl, p-toluenesulfonyl, ben-

'0098 51/2239

BATH ORIGINAL

zylsulfonyl, benzenesulfenyl, o-nitrophenyiaulfenyl, or before especially those derived from carbonic acid or thiocarbonic acid Groups such as, where appropriate, in the aromatic radical by halogen atoms, Nitro groups, nicderalkyl or lower alkoxy or lower carbalkoxy groups, substituted carbobenzoxy groups, e.g. carbobenzoxy, p-bromo- or p-chlorocarbobenzoxy., p-nitrocarbobenzoxy, p-methoxycarbobenzoxy, colored benzyloxycarbonyl groups such as p-phenylazo-bs.nzyloxycarbonyl and p- (p'-methoxy-phenylazoj-benzyloxycarbonyl, Tolyloxycarbonyl, 2-phenyl-isopropyloxycarbonylj 2-tolyl-isopropyloxycarbonyl and above all 2-p-diphenyl-isopropyloxycarbonyl (cf. French Fatent No. 15 5 ^ 051 (Case βΐθό), also aliphatic oxycarbonyl groups such as allyloxycarbonyl ,, cyclopentyloxyearbonyl, tert, Ainyloxycarbonyl, adamantyloxyearbonyl and primarily tert. Butyloxycarbonyl, further e.g. carbamoyl, thiocarbarnoyl, N-phenylcarbamoyl and -thiocarbamoyl «,

The carboxyl groups are protected, for example, by amide or hydrazide formation or by esterification. Suitable for esterification are, for example, lower, optionally substituted alkanols such as methanol, ethanol, cy & nmethyl alcohol or in particular tert-butanol, also aralkanols such as aryl-lower alkanols, e.g. optionally substituted benzylal

BATH ORIGINAL

009851/2239

koholc v; ie p-nitrobenzyl alcohol or p-methoxybenzyl alcohol, Phenols and thiophenols such as p-nitrothiophenol, 2,4i5-trichlorophenol, p-cyanophenol, or p-foethanesulfonylphenol, further e.g. N-hydroxysuccinimide and H-hydroxyphthalimide.

The Ilydroxyeruppcn the side chains, e.g. the serine and / or tyronine rusts can e.g. with benzyl alcohol or preferably with tert * - Butanol can be protected, but they are not necessary

to be protected. To protect the amino group in the guanidino group of the arginine are mainly the nitro group and the tosyl group to use, but needs the guanidine group not protected against grounding. Likewise, the imino group needs of the histidine does not necessarily need to be protected, but it can be advantageous to protect them, e.g. by benzyl, Trityl, adamantyloxycarbonyl or those in Ber. 100 (I967), 3838-38 ^ 9 described 2,2,2-trifluoro-l-tert-butyloxycarbonylaminoethyl or -l-benzyloxycarbonylaminoethyl groups. The protective groups are split off in a known manner by hydrogenolysis or hydrolysis, especially acid hydrolysis, in a single stage or, if appropriate, in several stages.

- 0093 5 1 2 239 - _b ad

Preferably used in variant l) des Process a starting peptide in which the amino groups of the side chains by the tert.Butyloxycarbonylgruppe and the Carboxyl groups of the side chain and the C-terminal acid, if they are not amidated, by the tert-butyl ester group are protected. These protective groups are expediently split off using trifluoroacetic acid.

In variant l) of the process, the N-lower alkanoyl group itself can be used as the α-amino protective group during the synthesis of the starting material. Either you use them from the beginning to protect the amino group of the first amino acid or, preferably, you first build an N-terminal peptide fragment, for example the sequence 1-4 or _; 1-1.0, with an α-amino protective group customary in the synthesis of ACTH peptides, e.g. the tert-butyloxycarbonyigruppe,

then splits off this group and leads to the lower alkanoyl group into the peptide fragment, for example by means of an activated ester of lower alkanoic acid.

009851/2239

BATH ORIGINAL

For the purpose of producing the starting peptides, the amino acids are linked individually in the desired sequence or after prior formation of smaller peptide units. The amino acid and / or peptide units are linked in such a way that an amino acid or a peptide with a protected cc-amino group and an activated terminal carboxyl group with an amino acid or a peptide with a free ct-amino group and free or protected, e.g. esterified or amidated terminal carboxyl group, or that one reacts an amino acid or a peptide with activated a-amino group and protected terminal carboxyl group with an amino acid or a peptide with free terminal carboxyl group and protected a-amino group »The carboxyl group can be converted, for example, into an acid azide , -anhydride., -imidazolide, -isoxazolide or an activated ester, such as cyanomethyl ester, carboxymethyl ester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, 8-hydroxyquinoline ester, N-hydroxypiperine ester or by reaction using a carbodiimide (given if necessary with the addition of N-hydroxysuccinimide) or N, N ¹ -carbonyldiimidazole, the amino group can be activated, for example, by reaction with a phosphite amide, as the most common methods

009851/2239.

to be mentioned are the carbodi'imide method ^ the azide method, the activated ester method and the anhydride method, also the Merrifield method.

Free, functional, not involved in the reaction Groups are appropriately protected, in particular by means of residues that can be easily split off by hydrolysis or reduction, as mentioned above »

In variant 2) of the method, the starting peptide is attached to the α-amino group by known methods selectively acyllert, preferably by means of an activated ester of lower alkanoic acid, e.g. by means of a phenolic or Thiophenol esters such as p-nitrophenyl .- * 2,4j, 5-trichlorophenyl ~ or p-nitrothiophenyl ester or by means of the N-hydroxysuccinimide or N-hydroxyphthalimide ester.

Depending on the method of operation, the new compounds are obtained in the form of bases or their salts. The bases can be obtained from the salts in a manner known per se will. The latter, in turn, can be reacted with acids that lead to the formation of therapeutically usable saline

se suitable gain projection are salts such "B ₀ such acid with inorganic acids such as hydrogen halides, such as hydrochloric acid or hydrogen bromide, perchloric acid, - '''''nitric acid or Thioeyansaure ^ sulfur or Phopfooi''' - ^z ' '■ "acid, or organic acids, such as formic acid ^ ₁ acetic-'". · Acid, propionic acid, glycolic acid ^. Lactic acid ^ pyruvetteq- '

• ■

acid, oxalic acid, malonic acid, succinic acid ^ maleic acid,

009851/2239. .

Fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, Hydroxymaleic acid ,. Dihydroxymaleic acid, benzoic acid, Phenylacetic acid, winiinobenzoic acid, 4-hydroxybenzoic acid, Anthranilic acid, cinnamic acid, mandelic acid, salicylic acid, ^ -amino salicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, methanesulfonic acid, Ethanesulfonic acid, hydroxyethanesulfonic acid, dynzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid or sulfanilic acid, · ■ ·

The peptides obtained according to the method can. ä find use in the form of pharmaceutical preparations. These contain the peptides mixed with a pharmaceutical, organic or inorganic carrier material suitable for oral application. For the same, substances that do not react with the polypeptides come into consideration, such as gelatin, milk sugar, glucose, starch, cellulose, for example "Avicel" (microcrystalline cellulose) and cellulose derivatives such as carboxymethyl cellulose, methyl or ethyl cellulose, talc, magnesium stearate, gum , Polyalkylene glycols, water, mono- or polyhydric alcohols such as ethanol, isopropanol, glycerine, hexites, vegetable oils and other fatty acid esters such as arachid oil, cottonseed oil, almond oil, olive oil, castor oil, ethyl oleate, isopropyl myristate, isopropyl palmitate, "Cetiol V" ( Oleic acid esters of liquid fatty alcohols), "Miglyol" of "Labrafac" (triglyceride mixture of fatty acids with 8-12 carbon atoms), "Labrafil M 2735" or "Labrafac WL I219" (mixtures of glycerine and polyoxyethylene fatty acid esters),

00985 1/2239

BATH ORIGINAL

"(Sorbitan fatty acid ester)," Tween "(polyoxyethylene sorbitan monooleate) or other known excipients. The pharmaceutical preparations can be, for example, tablets. Dragees or capsules or in liquid form as solutions, suspensions or emulsions. If applicable, are they sterilized and / or contain auxiliaries such as Preservatives, stabilizers, wetting agents or emulsifiers. They can also contain other therapeutically valuable substances.

The pharmaceutical preparations contain for example °. »5 to" 50-iT.g aes peptide for oral use e.g. once or several times a day.

The invention is illustrated in the example below described. The temperatures are given in degrees Celsius.

The following systems are used in thin layer chromatography:

System ¹ IQ
System 43A
System 43C
System 4jE
System 52
System 52A

n-butanol-ethanol-water (40: ^J 10: 20) tert. Amyl alcohol isopropanol V / water (100 ί4θ ■: 10) tert. Amyl alcohol isopropanol V / water (51; 21j28) tert. -Amyl alcohol-isopropanol-V / water (32 % 32:36) n-butanol-glacial acetic acid-water (75s7j, 5; 2l) n-butanol-glacial acetic acid-water (67:10:23) '

BATH

System 87: isopropanol-glacial acetic acid-water (77: 4: 19) System 89: ethyl acetate-acetone-water (72: 24: 4).

-i sec. butanol-glacial acetic acid-water (67:10:23) : Ethyl acetate-pyridine-glacial acetic acid-water (62: 21: 6: 11) ί n-butanol-pyridine-glacial acetic acid-water (38: 24: 8: 30) : n-butanol-fyridine-glacial acetic acid-water (42: 24: 4: 30)

System 101B: n-butanol-pyridine-glacial acetic acid-water (40: 24: 6: 30)

System 102A

System 96 System 100 System 101 System 101A

System 102E

Ethyl acetate-methyl ethyl ketone-formic acid-water (50; 30: 10: 10) 'V.

Ethyl acetate, methyl ethyl acetate, glacial acetic acid, water (50:30: 10:10)

System 104: chloroform-methanol ~ 17% aqueous ammonia (41:41:18)

n-butanol-pyridine-ammonia (26 ^ ig) -Viasser (42: 24: 4: 30) n-butanol-pyridine-ammonia (26 # ig) -V / water (40: 24: 6: 3ο) n-butanol-pyridine-ammonia (2-6 ^ ig) -V / water (38: 24: 8: 30) Esslgester-Pyridine-Formic Acid-Water (63: 21: 10: 6) Isopropanol-ammonia (26 ^ ig) -water (70:10:20) Isopropanol-ammonia (26 ^ ig) -water (85: 5ί10)

System UlA System HlB ■ System HlC System II5 system 121 System 121A

BOC = tert. Butyloxycarbonyl
tBu = tert. Butyl
Ac = acetyl

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Example Is,

Acetyl-D-Ser-Tyr-Ser-Met-Glu-His-Pfo © -Arg-Trp-Gly-Lys-Fro-Val-GIy-Lys-Lys-Lys-Lys-Pro-VaI-Lys-VaX-Tyr -Pro-OH

mg Acetyl -D-Sei'-Tyr-Ser-Mefc-Glu (ObBu) -His-Phe-Arg-Trp-Gly-Lys (BOC) -P-ro-Val-Gly-Lys (BOC) -Lys (BOC ) Lys (BOC) -. Lys (BOC) -Pro-Val-Lys- (BOC) 461r.Tyr-JPro-OtBu are dissolved in 7 ml of 90% trifluoroacetic acid and 1.5 Hours-left at room temperature. Then, with good cooling, 30 ml of peroxide-free ether are added; the precipitated product is filtered off and vialed with ether. The white residue is dissolved in 10 ml of water and transferred to the acetic acid salt through a sauce (1.6 cm, 15 cm ) of an ion exchanger (Dowex 2 ~ X 8, acetate form). The eluate followed by UV control is lyophilized and yields 114 mg of a white powder * ^;

The preparation is uniform in thin-layer chromatography and shows the following Rf values?
On aluminum oxide (from Camag with 8 % gypsum additive), ^Rf KpA ~ - ^ ** ^

In the UV spectrum in 0.1 ~ n _f sodium hydroxide solution, the product has the following maxima!

^ max ^{= 2} 83 · ηιμ ^: (£ «2650), 290 m μ (3700) In paper electrophoresis (Schleieher and Schill 1. 20 ^ 3 * 1 hour, 2000 V) at pH 1.9, the substance migrates 12.5 ^c ™

009351/2239

■ - ■■: - 15 -

towards the cathode. ■ ■

The starting material can be prepared as follows: 1) AeOH-HD-Ser-Tyr-Ser-Met-HH-NHg

4.0 g of DOC-D-Ser-Tyr-Ser-Met-NH-NHg become ice-cold in 32 ml 90 J & lger trifluoroacetic acid dissolved and 30 minutes at room temperature ditched. By repeated dilution with water with udder cooling and concentration on a rotary evaporator the trifluoroacetic acid is removed. ·

The residue obtained is dissolved in 20 ml of water and the solution is left to exchange the trifluoroacetate against acetate ions by a column (2.6 cm. 40 cm) one strongly basic ion exchangers in the acetate form (Dowex .2.-X 8,) flow and elute with water.

The eluate is followed on the flow UV analyzer and the fractions containing the product are combined and lyophilized. 3.4 g of a white amorphous lyophilizate are obtained which shows the following Rf values in thin-layer chromatograms:
on silica gel: Rf ₁ ^ = 0.29, Rf ₁₀₁ = 0.64, Rf ₅₂ = 0.19 x

2) Acetyl-D-Ser-Tyr-Ser-Met-NH-NHg

3.3 g of AcOH-H-D-Ser-Tyr-Ser-Met-NH-NHg are in 190 ml of absolute Pyridine dissolved and 1.6 g of 4-nitrophenyl acetate in 45 ml of pyridine are added. 1.26 ml are added to this solution Triethylamine and let stand at 24 ^ for 25 hours.

Then the pyridine is on the Rotations-Ver-009 351/22 39

steamer sucked off under vacuum, the residue 3 times with each 100 ml of ether shaken and this by filtration

removed.

The product can be represented by purely

Recrystallization from ethanol-water (8: 1) or acetonitrile

Water (9'1)

It shows the following Rf values in thin-layer chromatograms:

on silica gel: Rf ^ ₀ = 0.38, R ^ ₁₀₁ = 0.69, Rf ₅₂ = 0.38.

3) Acetyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-OH

2.80 g of acetyl-D-Ser-Tyr-Ser-Met-NH-NH (5.1 mmol) are in 23 ml of absolute dimethylformamide cooled to -15 °. To do this, drips 4.50 ml of hydrogen chloride in ethyl acetate are added with thorough stirring (2, .6-n. .11.7 mmol) and then 0.65 ml t-butyl nitrite (5.6 mmol) in 10 ml of ethyl acetate and allowed to react for 10 minutes at a temperature of -I3 to -16. Then 1.6 ml of triethylamine (11.6 mmol) are added dropwise and the entire solution is filtered through a G-3 glass suction filter in a meanwhile prepared, cold solution (-16 °) of 3.66 g of H-GIu (OtBu) -His-Phe-Arg-Trp-Gly-OH (3.7 mmol) and 0.65 ml of triethylamine in II5 ml of dimethylformamide. The mixture is then stirred for one hour while cooling with ice and sodium chloride then put in the refrigerator (2-5 °) overnight Tomorrow the precipitate is filtered off, with small portions washed cold dimethylformamide-water mixture and dried.

The crude product can be obtained by recrystallization from acetonitrile -

009851/2239

Water. (1: 1) purified; be "The pure product, shows the following Rf values in thin-layer chromatograms. " on silica gel.? Rf _52n = 0.16, Rf ₁₀₁ » 0.55, Rf ₁₀₀ = 0.05 *.

4) Acetyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-Lys • (BOC) -PrO-VaI-GIy-LyS- (BOC) -LyS (BOc) -LyS- (BOC) -LyS (BOc) -

Pro-Val-Lys (Boc) - ^Val - ^T y ^r - ^pr ° - ^otBu / "» "* '· f

mg H-Lys- (Boc) -Pro-VaI-GIy-Lys (Boc) -Lys (Boc) -Lys (BOG) -Lys (Boc) -Pro-Val-Lys (Boc) -VaI-Tyr-Pro- OtBu are added together with 264 mg of acetyl-D-Ser-Tyr-Ser-Met-GlIi (OtBu) -His-Phe-Arg-Try-Gly-OH with the addition of 0.18 ml of 1.-n. Hydrochloric acid suspended in water in a mixture of 4 ml of absolute pyridine and 0.2 ml of water. 93 ^m S of dicyclohexylearbodiimide are added and the mixture is stirred at a bath temperature of 50. After 4 hours, another 93 mg of dicyclohexylcarbodiimide are added. The mixture is stirred at 50 ° overnight, then placed in the refrigerator and the precipitated dicyclohexylurea is filtered off.

The product is obtained from the filtrate by precipitation with a lot of benzene as if it were a white amorphous powder. The product can be purified by column chromatography on silica gel: 258 mg of crude product ^ dissolved in chloroform-methanol (9 ^ 1) are drawn up on a column (1.4 cm, 33 cm) of silica gel and.

00985.1 / 2239

with a mixture of chloroform-methanol with increasing Methanol content eluted. At a ratio of the mixed solvent from approx. IiI the substance leaves the column. the pure, fractions are combined and evaporated. In thin

Layer chromatograms show the substance the following Rf value on silica gel
on aluminum oxideϊ

009851/2230

Example 2 ^: _:

ι- · _: ■ ■ ■ · '. ■■' · ._ ', ■■

Acetyl-D-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro - VaI-Gly-Lys-Lys-Lys-Lys-NIIg

φ ■ I - I Il ■ II, m i I ■■ I Ι Il! ■ !! ■! ■■ - '. *

8'IO mg of acetyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-Lys (BOC)! Pro-VaI-GIy-Lys (BOC) -LyS are dissolved (BOC) -LyS (BOC) -LyS (BOC) -NH ₂ with stirring in 20 ml of 90% trifluoroacetic acid and the solution is stirred under a nitrogen atmosphere for a further 5 minutes at room temperature. Then 120 ml of peroxide-free ether are added with cooling and stirring within 10 minutes and the precipitated trifluoroacetate of the octadecapeptide amide is filtered off, washed with ether and dried in vacuo. The colorless product is then dissolved in 15 ml of water, slowly poured into a column filled with 20 ml of moist araberlite (acetate form) to convert it into the acetate and poured with water

eluted. No: h Lyophilizing the eluate will be 680 mg chromatographically pure acetyl-D-Ser -Lys '' - β-corticotropin-Lys -amid obtained in the form of the pentaacetate.

The product v / eist an optical rotation of Ca] _n = -59 ° + (c S = 1 in aqueous acetic acid) and UV maxima at 283 and 290 μm (in 0.1-η .HCl). In thin-sheet chromatography on aluminum oxide, the following Rf values are obtained;

Rf (52 A) 0 ^ 1

Rf "(101). OA

Rf (101 B) 0.25

00985 r / 22.39

The starting material can be prepared as follows; 1 g of N-AcGtyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp- GIy-OH is added to a mixture of 50 ml of dimethylformamide and 0.12 ml of 6, 5 * · "· Hydrochloric acid and dissolved with a solution of 1.2 g of H-Lys (BOC) -Pro-VaI-GIy-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -NH ₂ (cf. the French brevet special de medicament 58 ^ 9 M) in 5 nil dimethylformamide After adding 21.5 mg of dicyclohexylcarbodiimide and 160 mg of N-hydroxysuccinimide, the reaction mixture is stirred for 20 hours at 45 ° and then with 250 ml of ether was added. the precipitated reaction product is filtered, washed with ether and dried in vacuum. For purification of the crude product 1.8 g of a countercurrent distribution in the solvent system methanol-chloroform -Puff he ^ f Tetrachlorkohlenstof I0i'j>i7'A (buffer = 57 ml of glacial acetic acid -l x 7.7 g of ammonium acetate in 95O ml of water) -with phase volumes of 20 ml each subject. After a total of 370 steps v / ill from the elements I78-I95 (Max = l86j K = I ₁ 02) by Concentration to dryness and sublimation of the arr.monium acetate at HO in a high vacuum, the thin-layer light chromatographically uniform octadecapeptide derivative: ⁴ ; available as a morphous powder. It has the following e;

cagfeli Rf (5 '^) 0.3 ■

Bf CiOO)

BATH ORIGINAL

Example 3:

Myristoyl-D-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val -GIy ^ Lys-Lys-Lys-Lys.-NHp (acetate).

268 mg myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly_, Lys (BOG) -Pro-Val-Gly-Lys (BOC) -Lys (BOC) - Lys (BOG) -Lys (BOG) -NH ₂ are dissolved in 20 ml of JO ^ iger trifluoroacetic acid at 0 ° and left to stand closed for 16 hours at room temperature. The batch is stirred into 200 ml of freshly purified, peroxide-free ether cooled to f -16, the white precipitate is filtered off and washed with ether. This octadecaptidamide trifluoroacetate is dissolved in aqueous acetic acid and amberlite on a column (22 cm; 10 mm). CG-45 (weakly basic ion exchanger in acetate form) converted into the acetate salt. The eluate, which is checked for substance by means of a UV flow analyzer, is then lyophilized. There are 238 mg of white, voluminous lyophilizate. Thin layer chromatography on aluminum oxide (Camag):

Rf ₅₂ = 0.40: Rf _101A = 0.40: Rf _101B = 0.50: Rf _111B - 0.37: Rf ₁₁₁₀ = 0.34.

UV (0.1-N NaOH) I _{m v} = 283 nm (Q = 65ΟΟ), 29Ο nm (έ = 2300). In the electrophoresis on Selecta-Cellulose prefabricated plates (Schleicher and Schüll No. 1440) the connection at 200 V moves the following distances against the cathode for 1 hour:

009851/2239

at pH 1.9 (acetic acid / formic acid buffer) 5> 6 cm,

at pH 6.4 (pyridine acetate buffer) with tail formation 5.9 cm, at pH 9 * 1 (ammonium carbonate buffer) with tailing 3.4 cm.

The Ausgangsmafcerial can be made as follows ι
1) Myristoyl-D-Ser-OCH, ..

10.0 g of D-serine methyl ester hydrochloride (64.5 mmol) ^ in 30 ml of dimethylformamide while cooling with 20.7 S (84 mmol)

Myristic acid chloride and 18.8 ml (169 mmol) of N-methylmorpholine, each dissolved in 15 ml of dimethylformamide, resulting in a thick suspension which, diluted with a further 50 nil of dimethylformamide, is stirred overnight at room temperature. It is then taken up in a lot of chloroform and extracted three times with dilute hydrochloric acid, three times with dilute soda solution and five times with saturated sodium chloride solution, dried over sodium sulfate and evaporated. Substance P is dissolved in 30 ml of ethanol, 600 ml of ether / petroleum ether (1: 3) are added, and the mixture is left to stand overnight in the refrigerator. The precipitated product is filtered off and dried. It melts at 75-76 °. The rotation Ca] ^ ⁰ = + 8 ° + 0.5 ° (c = 2 in ethanol). The thin-layer chromatogram on silica gel is 0.68; Rf ₈₉ = 0.65; Rf ₅₂ = 0.72; Rf ^ _02E = 0.86

009851/2239

2) Myristoyl-D-Ser-hydra / .id

III ■ - ■ ■■■. Jl ■ I .1 ■ II-. ι III II ■ I I ■ «I— .-I— I— ■ l I I -. ,

25.0 β (76 mmol) of myristoyl-D-Ser-OCm are mixed in 250 ml of methanol at 0 ° with 10 ml of hydrazine hydrate and left to stand for 17 hours at room temperature. The product is filtered off, washed with ether, briefly boiled in 00 ml of methanol and, after cooling to room temperature, filtered and dried. Nudelchen vom P. I69-I7I ⁰ . In the thin-layer chromatogram on SiIicagel, Rf ₅₂ = 0.63; Rf _{from 100 to} 0.75; Rf ₉₆ = 0.66; Rf in methanol-chloroform (1: 1) = 0.58. ·. f

3) myristoyl-D-Ser-Tyr-Ser-Met-OCH,

7.67 g (23.3 mmol) of myristoyl-D-Ser-hydrazide are finely powdered in 4'0 ml of dimethylformamide aufgeschlä.T, mt and under nitrogen

- 39 ° with 19.4 ml of hydrogen chloride in dioxane 3.0-N (58.2 mmol) stirred. The thick suspension is diluted with 20 ml of dimethylformamide and 1.99 g of sodium nitrite dissolved in 4 ml of water are added at -20 °. The mixture is stirred for 30 minutes at -20 to -10 °, diluted gradually with 50 ml of chloroform and then 5.0 ml of triethylamine and 8.67 g (20.9 mmol) of H-Tyr-Ser-Ket-OCH in solid Shape too. It is cooled in such a way that the temperature never exceeds

ο ■

- 15 rises. Then add 3, Q ml of triethylamine

added and stirred for one hour at low temperature, for two hours at O ^'J vinl 1? Hours be: ¹ 24. The product is obtained by adding the reaction mixture _; filtered, the filtrate a-rt in a vacuum and "in a lot of Essi, -ster several times kit verd, SaI ^ -

badoriginal

Shake out acid solution, dilute soda solution and half-saturated saline solution. The ethyl acetate solution is evaporated and the crude product is treated with activated charcoal in ethanol. The tetrapeptide derivative separates out of the ethanolic solution in pure form when it is left to stand for a long time in the refrigerator. P. = 165-168 ° (dec.); α ^ ° = -12 ° + 0.5 ° (c = 2 in dimethylformamide) In the thin-layer chromatogram on silica gel, Hf ^ ,, _c - 0.75 » Rf _100. = 0.86; Rf _121A -0.73.

4) Myristoyl-D-Ser-Tyr-Ser-Met-hydrazide. ■

4.88 g of myristoyl-D-Ser-Tyr-Ser-Met-OCH, (6.86 mmol) are dissolved in 100 ml of methanol and left to stand in the refrigerator at 0 ° with 10 ml of hydrazine hydrate for 48 hours. The hydrazide precipitates out voluminously. It is filtered off, washed with methanol and then recrystallized from 450 ml of methanol. In the thin-layer chromatogram on silica gel, Kf ii? «- 0.49; ^Rf 102E ⁼ ° ^{'51; Rf} 96 = 0.70.

5) Myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-OH

899 mg (1.26 mmol) of finely powdered myristoyl-D-Ser-Tyr-Ser-Met-hydrazide are suspended in 8 ml of abs »dimethylformamide, cooled and treated with 1 ^ 16 ml of hydrogen chloride solution in dioxane (3, Q-Nj 3.48 mmol) were added. To which now clear

BATH ORIGINAL

The resulting solution is added at -22 ° 0.24 ml of t-butyl nitrite (2.0 mmol) and stirred for 15 minutes, the temperature reaching -16. After cooling again, 0.25 ml of triethylamine is added. filtered and the filtrate runs into the pre-cooled solution of 1.13 S (1.14 mmol) H-Glu (OtBu) -His-Phe-Arg-Trp-Gly-OH and 0.16 ml of triethylamine in 26 ml of dimethylformamide. The reaction mixture is stirred for half an hour under nitrogen at about -10 °, with 0.30 ml of triethylamine being added in three portions so that the pH is about 8. The mixture is then stirred for a further 24 hours at room temperature, filtered from the precipitated product after cooling in the refrigerator, washed with acetonitrile, acetonitrile / water (1: 1) and with water and dried over phosphorus pentoxide in a high vacuum. In the thin-layer chromatogram on silica gel, Rf ^ _ = 0.51 * Rf ₈₇ = 0.69; Rf ₉₆ = 0.48; Rf ₁₀₁ = 0.63; Rf ₁₂₁ = 0.70.

6) Myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-LyS- (BOC) -PrO-VaI-GIy-LyS (BOC) -LyS (BOC) -LyS (BOC) -LyS (BOC) -NH ₂

1 * 23 g (0.78 mmol) of myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His- · Phe-Arg-Trp-Gly-OH are finely powdered in 9 ml of dimethylformamide under nitrogen with 0 , 26 ml of hydrogen chloride in dioxane (3.0-N, 0.78 nmol) was stirred. For this purpose 1.21 g (0.85 mmol) H-Lys (BOC) -Pro-Val-GIy-Lys (BOC) -Lys (BOC) -Lys (BOC) -liys- (BOC) -VIl ₂ and IIS mg of N-hydroxysuecinimide given in solid form. At room temperature, 220 mg of dicyclohexylcarbodiimide added (l, 06 mmol) is added and stirred · after another with 9 ml of dimethylformamide comparable

00985 1/2239

thinned, 21 hours at 35 bath temperature. The suspension gradually goes into solution, some dicyclohexylurea begins to deposit in crystalline form. The whole reaction mixture is then stirred into 600 ml of purified ether / toluene mixture (1: 1), filtered and the residue is dried in a high vacuum. The crude product thus obtained, 2.35 g *, is methanol / ammonium acetate buffer / chloroform / carbon tetrachloride in the system (10: 3: 7: ^j O multiplicatively distributed over 250 levels. The myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBi) -His-Phe-Arg-Trp - Gly-Lys (BOC) -Fro -Val-Gly-Lys (BQC) -LyS (BOC) -LyS (BOC) -LyS (BOC) -NH ₂ is then in vessels 38 to 60 (r, = 46, K = 0.22 phase volume 20 ml) and is isolated in good purity. In the thin-layer chromatogram on Silcagel it is: Rf ₅ ^ = 0.57, ^Rf ₁₀₀ ^β 0 »5 ^, ^ C ° ^ = 0.50.

example

Myristoyl-D-Ser-Tyr-Ser-Met-Glu-His-Fhe-Arg-Trp-Gly-Lys-Prο-VaI-Gly-Lys-Lys-Lys-Lys-Pro-VaI-Lys-VaI-Tyr- Pro-0H

505 mg myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp Gly-Lys (BOC) -Pro-Val-Gly-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (BOC) -Prο-Va1-Lys (BOG) -VaI-Tyr-Prο-OtBu are poured over with 20 ml of ice-cold, r trifluoroacetic acid, dissolved and 2 hours at

009851/2239

BATH

Left to stand at room temperature. The batch is then poured into 100 ml of peroxide-free, absolute ether which is cooled with an ice / common salt mixture. The precipitate is filtered off, washed with ether and dissolved in a little dilute acetic acid. This solution of the myristoyl tetracosapeptide trifluoroacetate is converted into a solution of the corresponding acetate on a column of Amberlite CG- ^} \ ^f j (Acetaform, column: 1 ^; 4 mm; 23 em) and the eluate is lyophilized. 113 mg of a voluminous, white lyophilizate are obtained. In the thin layer chromogram on Alox (Camag) is ΠΓ. «.« = 0.59 * ^Rf ₅ 2A ^{= O} ' ⁵⁸ ' ^Rf lllB = ° '38 ·

The starting material can be prepared as follows

will;

Myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-Lys (BOC) -Prο-VaI-GIy-Lys (B0C) -Lys (BOC) -Lys ( BOC) -Lys (B0C) -Pro-Val-Lys (B0C) -Val-Tyr-Pro-0tBu

1 ^ 39 g (0.89 mmol) of myristoyl-D-Ser-Tyr-Ser-Met-GIu (OtBu) -His-Phe-Arg-Trp-Gly-OH are in 10 ml abs. Dimethylformamide under nitrogen with 0.30 ml of hydrogen chloride in dioxane (3.0-N, 0.9 mmol) touched. 2.0.0 g (0.89 mmol) of H-Lys are added to the suspension

00985 1/2 23 9 _ßAD

(DOC) -PrO-VaI-GIy-LyS (DOC) -LyS (BOC) -LyS (BOC) -LyS (BOC) -PrO-VaI-Lys (BOC) -VaI-Tyr-Pro-OtBu dissolved in 10 ml Dimethylformamide and 175 mg of N-hydroxypuccinimide, and 2 ¹ mg of dicyclohexylcarbodiimide in 7 ml of dimethylformamide. The mixture is stirred for 22 hours at 35 ° -40 °, during which dicyclohexylurea is deposited from the solution. This is filtered off at room temperature and the filtrate

■ s

stirred into 400 ml of peroxide-free ether. A fine precipitate is formed, which is isolated and dried. This crude product is subjected to countercurrent distribution in the system methanol / buffer / chloroform / carbon tetrachloride (10: 3: 7: ^] O. Buffer: 19 g of ammonium acetate and 29 ml of glacial acetic acid are made up to one liter with water. After 1200 distribution steps, the myristoyl-D is found -Ser-Tyr-Ser-Met-GIu (OtBu) -His-Fhe-Arg-Trp-GIy-LyS (BOC) -PrO-VaI-GIy-LyS (BOC) -LyS (BOC) -LyS (BOC) - LyS (BOC) -Prο-VaI-Lys (BOC) -VaI-Tyr-Pro-OtBu in the vessels 72 - 95 (K ⁵ ^ = 0.08) and is by evaporating the solution "and driving off the ammonium acetate in a high vacuum Isolated in a thin-layer chromatogram on silica gel in chloroform / methanol (75:25) Rf = 0.61; Rf ^ = 0.63; Rf ₁₀₀ = 0.63.

009851/2239

BATH ORIGINAL

. - 29 -

Example 5 ϊ

Valeryl-D-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Lys-Lys-NHp (Acetate).

420 mg Valeryl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-Lys (BOC) -Pro-Val-Gly-Lys (BOC) -Lys (BOC) - Lys (BOC) -Lys (BOC) -NH ₂ are dissolved in 25 ml of ice-cold, 70% trifluoroacetic acid and left to stand closed for 15 hours at room temperature. g The clear solution is then placed in 300 ml freshly cleaned, peroxide and stirred at -L6 °-cooled ether, and the white precipitate filtered off and washed with ether. This octadecapeptide amide trifluoroacetate is dissolved in a little aqueous acetic acid and converted into the acetate salt on a column (23 cm} lh mm) Amberlite CG-45 (weakly basic ion exchanger in acetate form). The eluate is lyophilized. 338 mg of white, voluminous lyophilizate are obtained.

Thin layer chromatography on aluminum oxide (Camag): \

Rf ₅₂ = 0.22; Rf _101A . = 0.25; Rf _101B = 0.45; Rf ₁₀₄ = O, ₇ 4; ^Rf iiiA = ° '50.

UV (0.1 N NaOH) ^ = 282 nm (£ = 6750), 289 nra (g = 6600).

In electrophoresis on Selecta cellulose plywood sheets (Schleicher and Schüll No. 1440) the connection migrates to 200 V for 1 hour against the cathode:

009851/2239

at pH 1.9 (acetic acid / formic acid buffer) 6.2 cm, at pH 6.4 (pyridine acetate buffer) 5.2 cm,

at pH 9 * 1 (ammonium carbonate buffer) 3 * 5 cm.

The compound migrates onto cellulose acetate film (Cellogel) 280 V in 30 min. At pH 1.9 (acetic acid / formic acid buffer) 6.9 cm to the cathode.

The starting material can be produced as follows:
1) Valeryl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-OH

This decapeptide derivative is made in the same way as that Myristoyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-Gly-OH described in Example 3 » The intermediate products show the following characteristics:

Valeryl-D-Ser-OCH-. : In the thin-layer chromatogram on silica gel, Rf _43C = 0.70; Rf ₈₉ = 0.66; Rf _102E = 0.73; Rf ₅₂ = 0.62;

in chloroform-methanol (1: 1), Rf «is 0.79.

Valeryl-D-Ser-NH-NH ₂ : P. 184-185 ° Ca] ^ ⁰ = + 22 ° + 1 ° (c = 1 in methanol). In the thin-layer chromatogram on silica gel, = 0.48; Rf ₈₉ = 0.27; Rf _102E = 0.56; Rf ₅₂ = 0.48; in chloroform-methanol (1: 1), Rf = 0.44. Valeryl-D-Ser-Tyr-Ser-Met-OCH ™: In the thin-layer chromatogram on silica gel in Rf ₁₀₀ = 0.70.

Valeryl-D-Ser-Tyr-Ser-Met-HH-IML ·: in the thin-layer chromatogram on silica gel, Rf _n / - = 0.55; Rf

96 ⁼ "'^' ^nx 52A ^{= U} ' ^3X * ⁿⁱ 100

0.52; Rf _101A = 0.66.

0 Q 9 8 5 1/2 2 3 9

BATH ORIGINAL

Valeryl-D-Scr-Tyr-Ser-Met-GliiiOtBuJ-IIiü-Phe-Arg-Trp-Gly-OH: In the thin-layer chromatogram on silica agcl, Rf ₉₆ -. O, H '( ; Rf _{1 # vl} β 0.61; Rf ₁₀₁ = 0.70.

2) Valei ^ yl-D-Ser-Tyr-Ser-Met-GluiOtBuJ-His-Phe-Arg-Trp-Gly-Ly s (BOC) -Pro -VaI -G Iy -Ly s (BOC) -Lys (BOC ) -Lys (BOC) -Lys (BOC) -NH ₃

1.36 β (0.95 mmol) Valeryl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Ar {s-Trp-Gly-OH v.-earth in 10 ml of dimethylformamide slurried and stirred under nitrogen at room temperature with 0.33 hydrogen chloride in dioxane (3.0 N; 0.99 mmol). Most of the insoluble substance goes into solution. One then gives 1. ^ 0 g (0.99 nsMol;) H-Lys (BOC) -Pro-Val-Gly-Lys (BOC) -Lys (EOC) -Lys (BOC) -Lys (BOC) -NH ₂ to, stir for 10 min. and add 202 mg of N-hydroxysuccinimide and 28 ^ 4 mg of dicyclohexylcarbodiinide in solid form. After dilution with 10 ml of dimethylformamide, the mixture is stirred for 23 hours at 35 ° C. bath temperature. During this time, some dicyclohexylurea separates out in crystalline form. It is filtered off at room temperature and washed three times with 2 ml of Λ dimethylformamide. The filtrate is stirred into 200 ml of toluene, and the precipitate is filtered and washed with toluene and ether. This crude product, 2.8 g, is purified by countercurrent distribution in the system methanol / ammonium acetate buffer / chloroform / carbon tetrachloride (10: 3: 7: 4). After 200 steps there is the Valeryl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe- ·

0 0 S 8 5 1/2 2 3 9

Arg-Trp-Gly-Lys (BOC) -Pro-VaI-GIy-Lys (BOC) -Lys (BOC) -Lys (BOC) -Lys (130C) -NH ₀ in vessels 50 to 70 (r - = 59 , K = 0.42, phase volume 20 ml) and is isolated in good purity. In the thin-layer chromatogram on silica gel: yst RfV ^ Rf ₁₀₀ = 0.44, Rf _102A = 0.39-.

Example 6:

Pelargonyl-D-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Fro-VaI-Gly-Lys-Lys-Lys-Lys-NH ₂ (acetate).

379 mg Pelargonyl-D-Ser ~ Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-GIy-LyS (BOC) -PrO-VaI-GIy-LyS (BOC) -LyS (BOC) - LyS (BOC) -LyS (BOC) -NH ₂ are dissolved in 30 ml of ice-cold, 70% trifluoroacetic acid and stored closed for 18 hours at room temperature. The clear solution is then in 300 ml of peroxide-free, abs. Aether of -15 ° is stirred in, the white precipitate is filtered off, washed with ether and, dissolved in a little dilute acetic acid, run through a column (25 cm; 16 mm) Amberlite CG-45 (weakly basic ion exchanger in acetate form). The eluate is collected under UV control and lyophilized. 329 mg of white, voluminous lyophilizate are obtained. Thin layer chromatography on aluminum oxide (Camag):

⁼ ° ' ^{1 | 2; Rf} 96 ⁼ ° ^{'68; Rf} 101 ^{= 71; Rf} 101B ^Ä ° ^'53;

009851/2239

> o, 4 ₃ .
UV (Q, 1 -N NaOH) = 282 nm. (£ = 64-00) V 290 nm (£ = 6200).

ITIcI-X .__

In electrophoresis on Selecta-Cellulose prefabricated plates (Schleicher and Schüll No. 1440) the connection moves at 200 V the following distances against the cathode for 1 hour: at pH 1.9 (acetic acid / formic acid buffer) 5 * 9 cm * at pH 6.4 (pyridine acetate buffer) 5.1 cm, at pH 9 * 1 (ammonium carbonate buffer) with tail formation 3 * 2 cm. The starting material can be produced as follows will: · /

1) Pelargonyl-D-Ser-Tyr-Ser-Met-GIu (OtBu) -His-Phe-Arg-Trp-GIy-OH

This deeapeptide derivative is prepared quite analogously to the myristoyl-D-Ser-Tyr-Ser-Met-GIu (OtBu) -His-Phe-Arg-Trp-GIy-OH described in Example 3. The intermediate products show the following characteristics ^: · . '' ■

Pelargonyl-D-Ser-OCH: P. 48-4-9 °, · Ea] ^ ⁰ = + 11 ° + 0.5 °

(c = 2 in ethanol). In the thin film chromatogram on Silcägel

is Rf ₁₀₂ E ~ 0 * 83; ^Rf 52 ^{= 0>} ^ 9 * ^{in Cnloroi> 0} rm-methanol (l: l) is Rf = 0.83, '■ --.- ■ ,.

Pelargonyl-D-Ser-NH-NHg: mp 170-172 °; in thin-layer chromatography

grams of silica gel is Rf ₁₀₂ E = 0.69; Rf ₅₂ = 0.51; Rf ₁₀₀

0.63; in chloroform-methanol (1: 1). Rf = 0.50.

0098 5 1/22 3 9

Pelargonyl-D-Ser-Tyr-Ser-Met-OCH,: m.p. = 154-159 ° (dec.) ; [A. = -13 ° 0.5 ° (c - 2 in dimethylformamide); in the thin-layer chromatogram on silica gel, RiY ™ = 0.70; R ^ ₁₀₀ - O>87;

RfcoA = 0.80.
52A

Pelargonyl-D-Ser-Tyr-Ser-Met-NH-NHp: in the thin-layer chromatogram on silica gel, Rf ₅₂ A ⁼ ° * ^ ^{Oi Rf} 4 "5E ⁼ ° ^'62 > ^ ioo"°> 56.

Pelargonyl-D-Ser-Tyr-Ser-Met-GIu (OtBu) -His-Phe-Arg-Trp-Gly-OH: In the thin-layer chromatogram on Silcagel, Rf ₂ , -, "= 0, ^ 5 ^ Rf _52A = O , J, h; Rf ₁₀₀ = 0.15.

2) Pelargonyl-D-Ser-Tyr-Ser-Met-GIu (OtBu) -His-Phe-Arg-Trp-GIy-Ly s (BOC) -Pro-VaI-GIy-Ly s (BOC) -Ly s (BOC) -Ly s (BOC) -Lys (BOC) -

g (1.0 mmol) of pelargonyl-D-Ser-Tyr-Ser-Met-Glu (OtBu) -His-Phe-Arg-Trp-GIy-OH are suspended in 11 ml of dimethylformamide and 0.3 * 1 under nitrogen ml of hydrogen chloride in dioxari (3.0-N, 1.0 mmol) was stirred. One adds successively in solid form: 1.5 ¹ I g (1.1 mmol) H-LyS (BOC) -PrO-VaI-GIy-LyS (BOC) -Lys (BOC) -Lys (BOC) -Lys ( BOC) -NH ₂ , 150 mg N ^ -hydroxysuccinimide and 278 mg dicyclohexylcarbodiimide. After dilution with 12 ml of dimethylformamide, the mixture is stirred for 27 hours at 35 ° C. bath temperature, the suspension is stirred into 750 ml of ether / toluene (1: 1) and the precipitate is filtered off and dried in a high vacuum. This crude product, 2.9 Z * , is purified by countercurrent distribution in the system methanol / ammonium acetate buffer / chloroform / carbon tetrachloride (1Ο: 3: 7 ·: 4). After 25Ο steps there is that

009851/2239

■ - 35 -

Pelarconyl -D-Scr.-Tyi'-Scr-Kct-Glu (OtDu) -Ilia -Phe -Arg-Trp-Gly-Ly5 (IOC) -Pro-Val-GIy-Lys (BOC) -Lys (EOC) -Lys (BOC) -Lys (BOC) -NHp in vessels 51 to 76 (K = Q, 33 r. "= 62). The protected

Max

Octadecapoptidamide shows the following Rf values in thin layer chromatography on Siiicagel: Rf ^ ₂ = 0, ^ 8, Rf ₁₀₀ = 0.46.

009851/2239 '

BATH

Claims (1)

Patent claims: Process for the preparation of adrenocorticotropically active peptides, their amides and acid addition salts, thereby marked that one 1) from ACTH-effective peptides, which are the first amino acid have an amino acid with D-configuration and in which the α-amino group is aylated and at least the side chain amino groups and the terminal and side chain carboxyl groups are protected, deprotected or deprotected 2) ACTH-effective peptides which have an amino acid with D configuration as the first amino acid, N ^a -acylated and, if desired, the compounds obtained are converted into their acid addition salts. 2. The method according to claim 1, characterized in that the amino protective group is the tert. Is butyloxycarbonyl group. 3. Method according to claim 1 or 2, characterized in that that the carboxyl protecting group is the tert. Butyl ester group is.t. 0098 51/2239 BAD ORSQiNAL 4. 'The method according to any one of claims 1-3, characterized characterized 'that one the protecting groups by means of acids splits off. 5. The method according to any one of claims 1-4, characterized characterized in that the N -acyl group is an alkanoyl group with up to 30 carbon atoms. 6. The method according to any one of claims 1-4, characterized in that the N ^a -acyl group is the acetyl group. 7 · The method according to any one of claims 1-6, characterized characterized. that one of a protected N -acylated Peptide or peptide amide starts from 18-28 amino acids of the N-terminal sequence of natural corticotropins, in which optionally contains one or more of the amino acids 1 - 5, 11 * 15 * 16, 17, 18, 19 and 25 against other natural amino acids or their homologues are exchanged and in which the first amino acid is the D-form of a natural ei-amino acid. 8. The method according to any one of claims 1-6, characterized in that one of a protected N -acylated Starting out peptide or peptide amide which has 18-28 amino acids of the -N "-terminal sequence of natural corticotropins, in which, however, replaces the first amino acid with D-serine, D-alanine, D-proline or D-threonine and optionally the second amino 009851/2239 . BAD OFdGINAL acid by L-phenylalanine and / or the third by glycine or L-ilanine and / or the fourth by L-valine, L-leucine, L-norvaline, L-norleucine or La-aminobutyric acid and / or the fifth by L-glutamine and / or the 11th or 11th and. and 15. to l6. or 11. and I5. to 18. by L-ornithine and / or the 17. and 18. is replaced by L-ornithine or L-lysine and / or the 19th and / or 25th by L-valine. _; .-> 9. The method according to any one of claims 1-6, characterized in that one ^{starts from a protected N a} -alkanoyl-substituted peptide or peptide amide, the first N-terminal amino acid of which is D-serine. 1 10. The method according to any one of claims 1-9 *, characterized in that one starts from a protected N -alkanoyl-substituted peptide or peptide amide which has 18-25 amino acids and whose first amino acid is D-serine. 9iifs3QOXmA sale siu'BzoaimA 11. The method according to any one of claims 1-6, characterized in that one ^{proceeds from a protected N a} -alkanoyl-substituted peptide ^ deg.Pejptidamid ^. acids of the N- ^ rmin ₈ le, n sequence of course ^ _b Corticotro ^ n§ _s _ _8χ Ί Jl has, in ^ r ^ j ^ gggh Serine by B-Se ^ t ^ un ^ / o ^ gf ^ J ^ ji ^ durGh _f L-Norvaline _fci o ^ e ^ _x L-Norleucine and / or by L-Glutaminju ^ gder arginine '\ or L-Ly.siA _r and ^^ r _c amino acid ²⁵ .by ^ " 009851/2 2 3 p ^BAü 12. Process for the preparation of new adrenocorticotropically active peptides as described in the examples. 13. Adrenocorticotropic feptides which are used as first amino acid have an amino acid with D configuration and whose α-amino group is acylated, their amides and acid addition salts. 14. Adrenocorticotropically active peptides which have an amino acid with a D configuration as the first amino acid and whose α-amino group is substituted by an alkanoyl radical with up to J> 0 carbon atoms, their amides and acid addition salts. 15. Adrenocorticotropic peptides which have an amino acid with a D configuration as the first amino acid and the α-amino group of which is substituted by an acetyl radical, their amides and acid addition salts. 16. N ^a acylated peptides or peptide amides with 18-28 amino acids of the N-terminal sequence more natural , Corticotropins, possibly one or more of amino acids 1 -5 * H * 15 * 16 # 17 * 18, 19 and 25 against others natural amino acids or their homologues are exchanged and in which the first amino acid is the D-form of a 009851/223 = 9 BAD - 40 is natural amino acid, and its acid addition salts. 17. N ^a -Acylated peptides or peptide amides with 18 28 amino acids of the N-terminal sequence of natural corticotropins, in which, however, the first amino acid is replaced by D-serine, D-alanine, D-proline, or D-threonine and optionally the second by L -Phenylalanine and / or the third by glycine or L-alanine and / or the fourth by L-valine, L-leucine, L-norvaline, L-norleucine or La-aminobutyric acid and / or the fifth by L-glutamine and / or the 11th or 11th and 15 to 16. or 11. and I5. to l8. by L-ornithine and / or the 17th and 18th by L-ornithine or L-lysine and / or the 19. and/ or 25. is replaced by L-valine, and its acid addition salts, ^{18.N a} -acylated peptides or peptide amides with l8 - Amino acids of the N-terminal sequence of natural corticotropins, 1 4 in which, however, serine through D-serine and / or methionine through L-norvaline or N-urleucine and / or glutamic acid L-Glutarr.in and / or Arginine '* by L-Ornithine or L-Lysine 25 ■ - and / or amino acid is replaced by L-valine, and their Acid addition salts. 19. N ^a -Acylated peptides or peptide amides with 18 amino acids of the N-terminal sequence of natural corticotropins, ■ in which, however, the first amino acid by D-serine and / or the 00 9 ^ ^1/2229 BAD υί-uötNAL fourth 'by L-norleucine and / or the 17th and 18. by L-lysine and / or the 25th is replaced by L-valine, and their acid addition salts. 20. Compounds according to claims 13-19 * wherein the acyl group is an alkanoyl group of up to 30 carbon atoms is. 21. i ^ -Acetyl-D-Ser ^ Lys ¹⁷ * -ß ¹ -Corticotropin and its acid addition salts. 22. · N ^a -Myristoyl-D-Ser ¹ -Lys ^{17> 18} -β ¹ -Corticotropin and its acid addition salts. 23. N ^a -acetyl-D-Ser ¹ -Lys ^{17r # l8} -ß ^{1 l8} -Corticotropin- -1 Q ' Lysamide and its acid addition salts. 24. N ^a -Myristoyl-D-Ser ¹ -Lys ¹⁷ * ^l8 -ß ¹ " ^{: L8} -Corticotropln- 18th
Lysamide and its acid addition salts. 25. N ^a -Pelargonyl-D-Ser ¹ -Lys ^{17 '18} -β ¹ " ¹⁸ -Corticotropin- 18th
Lys-amide and its acid addition salts. . 26. N ^a -Valeryl-D-Ser ³ ^ -LyS ¹⁷ ' ^l8 -ß ¹ " ^l8 -Corticotropin- 18th
Lysamide and its acid addition salts. 0 098 51/2239 27 · The compounds described in the examples. 28. Therapeutically applicable acid addition salts of the peptides or peptide amides mentioned in claims I3-27, 29. Pharmaceutical preparations which contain the compounds mentioned in claims I3-28. 009851 / 22.3