DE1210877B - Process for the production of a decapeptide - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

C07C

German KL: 12 q - 6/01

Number: 1210 877

File number: C 32151IV b / 12 q

Filing date: February 15, 1964

Opening day: February 17, 1966

In the patent application C 24191 IVb / 12q (German Auslegeschrift 1205 546) the production of a new decapeptide of the formula L-Seryl-L-tyrosyl-L-seryl-L-methionyl-L-glutaminyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine and the corresponding compound which has the remainder of glutamic acid instead of the glutamine residue. These compounds exhibit the secretion of the adrenocorticotropic hormone (ACTH) from the anterior pituitary stimulating effect (CRF effect). The compounds and especially their derivatives with Protected amino or carboxyl groups can also be used as intermediates for the production of medicinal products, which have a longer chain of amino acids, e.g. B. the ACTH or the peptide that the comprises the first 24 amino acids of ACTH and how this has an adrenocorticotropic effect, is used will.

It has now been found that the process for the preparation of the decapeptides mentioned, in which the Tetrapeptide of amino acids 1 to 4 condensed with the hexapeptide of amino acids 5 to 10, improved can be. The hexapeptide derivative y-tert-butyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine used in this process

OtBu

Z -

I.
GIu His

■ NH - NH ₂ (N ₃ ) Z -

Phe -OH

Process for the production of a decapeptide

Addition to registration: C 24191IV b / 12 q - Interpretation document 1205 546

Applicant:

CIBA Aktiengesellschaft, Basel (Switzerland)

Representative:

Dr.-Ing. Dr. jur. F. Redies, Dipl.-Chem. Dr. rer. nat. B. Redies and Dr. rer. nat. D. Türk, patent attorneys, Opladen, Rennbaumstr. 27

Named as inventor: Dr. Robert Schwyzer, Riehen; Dr. Heinikapier, Birsfelden; Dr. Beat Iselin, Riehen (Switzerland)

Claimed priority: Switzerland of February 21, 1963 (2207)

z - Arg -OH H - Try GIy z - Arg Try GIy H - Arg Try GIy

-OCH ₃

OtBu

Ζ - Phe Arg Try GIy Η - Phe Arg Try GIy

- OCH,

- OCH.

-OCH ₃

-OCH ₁

Z - GIu His

Phe Arg

Try GIy

- OCH ₃

OtBu

Z - GIu His

OtBu

Η -

GIu His

Phe

Phe Arg

Arg

Try GIy - OH

Try GIy

- OH

can be obtained with advantage if carbobenzoxy-L-arginine is condensed with L-tryptophyl-glycine methyl ester, the carbobenzoxy group is split off from the tripeptide derivative obtained, the free tripeptide ester is condensed with carbobenzoxy-L-phenylalanine, the carbobenzoxy group is split off from the tetrapeptide ester obtained, condensed the free tetrapeptide ester with carbobenzoxy- (y-tert-butyl) -L-glutamyl-L-histidine-azide, in the resulting carbobenzoxy- {γ - tert. - butyl) - l - glutamyl-L-histidylx-phenyl-alanyl-L-arginyl-L-tryptophyl-glycine methyl ester saponifies the methyl ester group and removes the carbobenzoxy group by hydrogenation (see table); Z means the carbobenzoxy group.

The hexapeptide derivative described can, as shown in the main patent application C 24191 IVb / 12q, with the tetrapeptide derivative tert-butyloxycarbonyl-L-seryl-L-tyrosyl-L-seryl-L-methionine azide to the decapeptide derivative tert. - Butyloxycarbonyl ^: L-seryl-L-tyrosyl-L-seryl-r-methionyl-y-tert.-butyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine. From this, the free decapeptide can be prepared as described in the main patent, or the derivative can be used as a starting material for the preparation of the above-mentioned tetrakosapeptide.

The invention is described in the following example. The temperatures are in degrees Celsius specified.

The following systems were used for paper chromatography:

35

40

System 52: n-butanol-acetic acid-water
100:10:30.

System 54: sec-butanol-isopropanol-monochloroacetic acid-water 70:10: 3 g: 40.

System 56: sec-butanol-isopropanol-5% veronal sodium water 100: 15: 10: 60.

System 87: isopropanol-formic acid-water
400: 20: 100.

System 100: ethyl acetate-pyridine-acetic acid-water
60: 20: 6: 1.

System 101: n-butanol-pyridine-acetic acid-water 30: 20: 6: 24.

example

Step 1. Carbobenzoxy-L-arginyl-L-tryptophyl-

glycine methyl ester hydrochloride

40 g (0.128 moles) of L-tryptophyl glycine methyl ester hydrochloride and 41.5 g (0.135 mol) of carbobenzoxy-L-arginine are in 650 ml of absolute pyridine at 40 ° stirred until a slightly cloudy solution is obtained. This is cooled to room temperature and 39.6 g of dicyclohexylcarbodiimide (0.192 mol) were added. After stirring for 1 hour, overnight at Stored at room temperature. The next day it is cooled to 0 ° and the dicyclohexylurea is sucked off. The filtrate is concentrated strongly in vacuo, the thick oil is dissolved in chloroform and the solution is added dropwise while stirring in a lot of ether. An amorphous precipitate is obtained. One decants the Ether off and wash the residue well with ether. After drying in vacuo at 50 °, 77.3 g are obtained of a pale reddish brown powder (100% of theory = 77.1 g). The product obtained will without further cleaning for. used the next level.

Step 2. L-arginyl-L-tryptophyl-glycine-methyl ester

77.2 g (0.128 moles) of carbobenzoxy-L-arginyl-r-tryptophyl - glycine - methyl ester - hydrochloride are dissolved in 800 ml of ln acetic acid and mixed with 5 g of activated charcoal touched. After about 10 minutes, the mixture solidifies in a gelatinous manner. By adding 40 ml of glacial acetic acid and Warming to 40 ° the substance is brought back into solution and the activated charcoal is sucked off through kieselguhr. After adding 5 g of 10% palladium-carbon, the filtrate is hydrogenated until the theoretical level is reached after about 6 hours Amount of hydrogen is absorbed (2.91). After filtering off the catalyst, the filtrate is Amberlite IRA-400 (trade name), acetate form, is added and the mixture is stirred until there are no more chloride ions can be proven. The ion exchanger is filtered off with suction, washed with water and the filtrate in the Vacuum evaporated to dryness. 74.5 g of L-arginyl-L-tryptophyl-glycine methyl ester diacetate remain behind in the form of a yellowish foam.

Conversion into the ditosylate

34.5 g (0.0625 mol) of L-arginyl-L-tryptophyl-glycine methyl ester diacetate are dissolved in 200 ml of methanol, and 23.8 g of p-toluenesulfonic acid.1H ₂ O (0.125 mol) are added. The solution is evaporated to dryness in vacuo, the residue is completely dehydrated with ethanol and dried in vacuo. The ditosylate is obtained as a beige foam (48 g).

Step 3. Carbobenzoxy-L-phenylalanyl-L-arginyl-L-tryptophylglycine methyl ester acetate

45 g (58 mmol) of L-Argmyl-L-tryptophyl-glycine methyl ester ditosylate, 26 g (87 mmol) of carbobenzoxy-L-phenylalanine and 8.1 g (58 mmol) of triethylamine are dissolved in 450 ml of acetonitryl at room temperature. 19.3 g (95 mmol) of dicyclohexylcarbodiimide are then added and the mixture is stirred for a further ₂ hours at room temperature. After standing in the refrigerator overnight, the dicyclohexylurea is suctioned off. The filtrate is strongly concentrated in vacuo, then the substance is precipitated as a viscous product with ether. After the solvent has been poured off, the residue is dried in vacuo at 50 °. The crude product obtained (65 g) is converted into the acetate for purification. The crude product is dissolved in 250 ml of tert-butanol at 50 °, and 200 ml of water are added. The slightly cloudy solution is filtered through an Amberlite IRA-400 (trade name) ion exchange column, acetate form, which has been prewashed with 50% tert-butanol. It is then rewashed with 50% tert-butanol until swab samples with Sakaguchi are negative. The filtrate is evaporated to dryness in vacuo at 50 ° and 49.2 g of carbobenzoxy-L-phenylalanyl-L-arginyl-l-tryptophyl-glycine-methyl ester acetate are obtained as a pale brown foam as residue. The Rf value in system 52 is 0.83, in thin-layer chromatogram system 101: 0.7. The product is used for the hydrogenation without further purification.

Step 4. L-phenylalanyl-L-arginyl-L-tryptophyl-glycine methyl ester

49 g (63.4 mmol) of carbobenzoxy-L-phenylalanyl-L - arginyl - L - tryptophyl - glycine - methyl ester - acetate

are dissolved in 600 ml of 90% methanol, mixed with 4.9 g of 10% palladium carbon and hydrogenated with absorption of the carbon dioxide with potassium hydroxide. After ₄ hours, 1130 ml of hydrogen have been taken up and the hydrogenation stops. The catalyst is filtered off and the filtrate is evaporated to dryness in vacuo. The residue, 39 g == 96% of theory, gives the following values in paper chromatography:

System: 52 54 56 87
Rf: 0.45 0.65 0.78 0.5

In the case of paper electrophoresis in in-acetic acid, 7 V / cm, 5 hours, the walking distance is 18 cm.

Stage 5. Carbobenzoxy- (y-tert-butyl) -L-glutamyl-

L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-

glycine methyl ester

17.12 g (34.9 mmol) carbobenzoxy (γ-tert-butyl) -L - glutamyl - l - histidine - hydrazide (patent application C 24691 IVb / 12q) in 106 ml of dimethylformamide cooled to -5 °, then treated with 16 ml of ice-cold 6N hydrochloric acid, and 7.7 ml are added dropwise at -5 to -10 ° ice-cold 5N sodium nitrite solution within 2 to 3 minutes to. The mixture is left to react for 5 minutes at -5 °. The ice-cold one is then added with vigorous stirring freshly prepared solution of 16.3 g of t-phenylalanyl-L - Arginyl - L - tryptophyl - glycine - methyl ester - acetate (25.5 mmol) in 140 ml of dimethylformamide, containing 12.03 ml of N-triethylamine, in addition. The mixture is stirred for a further 2 hours in the cold bath and then for 15 hours at 0 °. The salt which has crystallized out is filtered off and 11 ethyl acetate is added to the dimethylformamide solution. 1.9 g of axis contaminated with triethylamine hydrochloride separates. The filtered clear Solution is evaporated to a small volume in vacuo and then the crude still with starting material and by-products contaminated hexapeptide esters precipitated with a lot of ethyl acetate. The precipitation after drying gives 28 g of crude product. 27 g of this product are added to the mixture Methanol-chloroform 1: 9 (810 g) applied to an alumina column and with 201 of the same Solvent mixture eluted. 11 g (41.5%) of amorphous are isolated in a thin-layer chromatogram pure carbobenzoxy hexapeptide ester. Rf 0.3 in system 100; positive reaction to Pauly, Ehrlich and Reindel-Hoppe.

The carbobenzoxyhexapeptide ester shows in low-voltage electrophoresis pH 2.4 (in-acetic acid) at 300 volts after one hour a walking distance of 15.5 cm.

Acidified with 10% acetic acid. One leaves another 30 minutes stand at 0 °, the carbobenzoxy-hexapeptide is filtered off and washed thoroughly with water. After this Drying in a high vacuum Yield: 8 g (90% of Theory), m.p. 204 ° (decomposition). Another 780 mg of less pure carbobenzoxy-hexapeptide can be obtained from the mother liquor preserved, F. 180 °. The carbobenzoxyhexapeptide can be recrystallized from 90% methanol be, m. 206 °. In the thin-layer chromatogram on silica hydroxide, the Rf values are: 52 / 0.2 and in the system 101 / 0.75.

Step 7. y-tert-Butyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine

7.96 g (7.36 mmol) carbobenzoxy (γ-tert-butyl> L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophylglycine are in 300 ml of acetic acid (90%) in the presence of 1.5 g of 10% palladium-carbon catalyst hydrogenated for 5 hours at normal pressure and room temperature. The one freed from the catalyst The solution is evaporated to dryness in vacuo at 40 °. The residue is reprecipitated from methanol-ether. Yield: 6.9 g of hexapeptide-γ-tert-butyl ester. The product is identical to that described in the main patent identical.

The hexapeptide-y-tert-butyl ester can after Process described in the main patent with tert-butyloxycarbonyl - l - seryl -L- tyrosyl -L- seryl -L- methionine hydrazide (-azid) to tert-butyloxycarbonyl-L-seryl-L-tyrosyl-L-seryl-L-methionyl- (y-tert-butyl) -L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl -L-tryptophyl-glycine be condensed.

Step 6. Carbobenzoxy- (γ-tert-butyl) -L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine

9.6 g (8.7 mmol) of carbobenzoxy-hexapeptide methyl ester are saponified in 150 ml of 75% dioxane with 18 ml of 1N sodium hydroxide solution for 20 minutes at room temperature. Substance is deposited during the saponification. The saponification mixture is mixed with 150 ml of ice-cold water and with 30 ml

Claims (1)

Claim: Process for the preparation of a decapeptide of the formula L-seryl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-l-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine by condensation of tert. - Butyloxycarbonyl-L-seryl-l-tyrosyl-L-seryl-r-methionine azide with y-tert-butyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine according to the method of the main patent application C 24 191 IVb / 12g (German Auslegeschrift 1 205 546), characterized in that the hexapeptide derivative to be used as the starting compound γ - tert. - Butyl-L - glutamyl - L - histidyl - L - phenylalanyl - l - arginyl-L-tryptophyl-glycine by condensing carbobenzoxy-L-arginine with L-tryptophyl-glycine methyl ester, splitting off the carbobenzoxy group from the tripeptide derivative obtained, the free tripeptide ester is condensed with carbobenzoxy-L-phenylalanine, the carbobenzoxy group is split off from the tetrapeptide ester obtained, the free tetrapeptide ester is condensed with carbobenzoxy- (y-tertbutyl) -L-glutamyl-L-histidine azide, in the resulting carbobenzoxy - (7 - tert-butyl) -L-glutamyl-lJ-histidyl-l-phenylalanyl-l-arginyl L-tryptophyl-glycine methyl ester saponifies the methyl ester group and removes the carbobenzoxy group by hydrogenation. 609 508/243 2.66 © Bundesdruckerei Berlin