DE1214242B - Process for the preparation of a tetracosapeptide - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

C07c

German class: 12 q- 6/01

Number: 1214242

File number: C 30563IV b / 12 q

Filing date: July 27, 1963

Opening day: April 14, 1966

The subject of patent 1196 666 is the manufacture of a new tetrakosapeptide of the formula L-seryl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-L-histidyl -L- phenylalanyl - l - arginyl - l - tryptophyl - glycyl-L-lysyl-L-prolyl-L-valyl-glycyl-L-lysyl-L-lysyl-L-arginyl-L-arginyl-L-prolyl-L -valyl-L - lysyl -L- valyl - l - tyrosyl-L-proline as well as the corresponding compound which has the remainder of glutamine instead of the glutamyl residue, and their derivatives, acid addition salts and heavy metal complexes. Have these connections high adrenocorticotropic (ACTH) potency.

It has now been found that the process for the preparation of L-seryl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycyl-L -lysyl-L-prolyl-L-valyl-glycyl-L-lysyl-L-lysyl-L-arginyl - l - arginyl - l - prolyl - l - valyl - l - lysyl-L-valyl-L-tyrosyl -L-proline, in which the decapeptide of amino acids 1 to 10 is condensed with the tetradecapeptide of amino acids 11 to 24, can be improved. The tetradecapeptide derivative N ^s -tert-butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycyl-tert-butyloxycarbonyl-L-lysyl-tert-butyloxycarbonyl-L-lysyl-L-arginyl- L-arginyl-L-prolyl-L-valyl-tert.-butyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline tert.-butyl ester is advantageously obtained if L-proline tert.- butyl ester condensed with carbobenzoxy-L-valyl-L-tyrosine azide, the carbobenzoxy group is split off from the tripeptide derivative obtained, the free tripeptide ester is condensed with carbobenzoxy-N ^ tert.-butyloxycarbonyl-L-lysine, the carbobenzoxy group is split off from the tetrapeptide ester obtained free tetrapeptide ester is condensed with carbobenzoxy-L-valine, the carbobenzoxy group is split off from the pentapeptide ester obtained, the L-valyl-tert-butyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-prolintert-butyl ester with carbobenzoxy-L-arginyl -L-arginyl-L-proline condenses to the protected octapeptide ester, from which the carbobenzoxy group is removed ltet and the resulting free octapeptide ester with carbobenzoxy-N ^ -tert.-butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycyl-tert. -butyloxycarbonyl-L-lysyl-tert. -butyloxycarbonyl-L-lysine azide, which by condensation of carboron benzoxy-N ^E -tert.-butyloxycarbonyl-L-rysyl-L-prolyl-L-valyl-glycine with N ^e -tert.-butyloxycarbonyl-L-lysyltert. -butyloxycarbonyl-L-lysine methyl ester to carbobenzoxy -N ⁶ -tert. -butyloxycarbonyl-L-lysyl-L-prolyl-L - valyl - glycyl - tert. - butyloxycarbonyl - L - lysyl - tert-butyloxycarbonyl-L-lysine methyl ester and conversion of the ester into the hydrazide and azide is obtained, condensed to the tetradecapeptide ester and finally the carbobenzoxy group is removed from this.

Method of manufacture
a tetracosapeptide

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

Named as inventor:

Dr. Heinikapier, Birsfelden;

Dr. Robert Schwyzer, Riehen (Switzerland)

Claimed priority:
Switzerland of August 1, 1962 (9208),
dated February 21, 1963 (2209)

splits (see figure). BOC means the tert-butyloxycarbonyl group, Z is the carbobenzoxy group.

The tetradecapeptide derivative obtained can, as described in the main patent 1196 666, with the decapeptide derivative tert-Butyloxycarbonyl-L-seryl-L-trosyl-L-seryl-L-methionyl-y-tert-butyl-L-glutamyl-L-histidyl- • L-phenylalanyl-L-argmyl-L-tryptophyl-glycine or condensed with the corresponding compound tritylated on the histidine residue to give the tetrakosapeptide derivative, from which, as described in the main patent, all protective groups are split off in one step will.

609 558/399

BOC

BOC BOC BOC

-Lys Pro VaI GIy-

-OH H-

BOC

I -Lys Lys4-0CH ₃ Z-

-Arg-

-OH Z-

BOC
I. Z- I.
-Lys Pro VaI GIy BOC
I. BOC Z- I.
-Lys Pro Lys Lys- BOC
I. VaI GIy BOC BOC
I. Lys Lys-

-OCH ₈

-NH-NH ₂

Z-- Arg

Z-

-Arg

Z-

BOC BOC

-Arg — OH H-

Z-

-Arg

H-

-Arg

Arg

Arg

-Per-

-OCH, Ζ — VaI-

-OH Z-

-Lys-

-OH Z-

-VaI Tyr-

-OCH ₃ H- Pro-

-OtBu

Per-

-OCH ₃

VaI Tyr — NH — NH ₂ (N ₃ )

PrO-OCH ₃

Per-

Z-

-VaI Tyr

-OH,

H-

BOC

Per-

-OH

BOC

BOC

-VaI Tyr

Per-

Per-

Z- I.
-Lys H- I.
-Lys VaI Tyr Per- BOC
I. VaI Tyr Per-

-VaI Z- -VaI BOC
I. VaI Tyr Per- H- Lys BOC
I. VaI Tyr Per- Lys

-Arg Arg Per VaI Lys VaI Tyr Per- BOC
I. -Arg Arg Per VaI I.
Lys VaiTyr Per-

OtBu OtBu

-OtBu -OtBu -OtBu --OtBu -OtBu -OtBu -OtBu -OtBu

-Lys Pro VaI GIy

Lys Lys

Arg

Per

Lys

VaI Tyr

Per-

BOC

BOC BOC BOC

H-Lys Pro VaI GIy

Lys Lys

Arg

Per

Lys

VaI Tyr

Per-

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

The following systems were used for paper chromatography:

System 40: n-butanol-ethanol-water = 2: 2: 1. System 43: tert-amyl alcohol — isopropanol—

Water = 100: 40: 55. System 45: sec-butanol - 3% ammonia

= 100: 44.
System 49: sec-butanol — triethylamine — diethyl

barbituric acid — water — isopropanol

= 100: 0.8: 1.8 g: 60:10. System 53: n-butanol-formic acid-water

= 480: 6: 314.
System 54: sec-butanol — isopropanol — mono-

chloroacetic acid — water

= 70:10: 3 g: 40. System 56: sec-butanol - isopropanol 5 ° / _ö Ve- ^ao

ronal — sodium in water

= 100: 15: 10: 60. System 87: isopropanol-formic acid-water

= 400: 20: 60.
System 100: ethyl acetate — pyridine — acetic acid—

Water = 60: 20: 1 system 101: n-butanol — pyridine — acetic acid—

Water = 30: 20: 6: 24 System 102: ethyl acetate — methyl ethyl ketone—

Formic acid — water

= 50: 30:10; 10.

example

Stage 1. L-proline tert-butyl ester a) Hydrochloride

35

In a pressure bottle, 5 g of dry L-proline, 150 ml of dry, alcohol-free chloroform, 5 ml of pure, concentrated sulfuric acid and 150 ml of liquid isobutylene are shaken at room temperature until solution occurs (about 3 to 4 days). After cooling to -15 °, the pressure vessel is opened and the isobutylene is distilled off in vacuo with exclusion of moisture. The remaining chloroform solution is washed with 200 ml of 20% potassium carbonate solution while cooling with ice and dried with sodium sulfate. After the sodium sulfate has been filtered off with suction, the L-proline tert-butyl ester content of the chloroform solution is determined by titrating a sample with dilute hydrochloric acid. The remaining chloroform solution is then cooled to -15 ° and the precisely calculated amount of hydrochloric acid in alcohol is added. Evaporation and trituration of the residue with ether gives 6.88 g of crystalline L-proline tert-butyl ester hydrochloride (76 ° / ₀ of theory) with a melting point of 107 to 108 °. The product is extremely hygroscopic.

b) free ester

60

To convert it into the free ester, the hydrochloride is dissolved in as little water as possible, the solution Covered with a lot of ether and made strongly alkaline by adding concentrated potassium carbonate solution posed. The ester which is separated out is taken up in the ether; the ethereal solution gives way Drying and evaporation of oily L-proline tert-butyl ester, which can be further processed directly.

Step 2. Carbobenzoxy-L-valyl-L-tyrosine-azide

12.9 g (0.3 mol) of carbobenzoxy-L-valyl-L-tyrosine hydrazide (H. Schwarz, FM Bump us and LH. Page, J. Am. Chem. Soc, 79, p. 5697 [1957] are in dissolved in a mixture of 600 ml of 2N hydrochloric acid and 1200 ml of dimethylformamide and, after cooling to -15 ° with stirring, 70 ml of a 5 normal aqueous solution of sodium nitrate are added.The solution is ^{stirred for a further 5 minutes at -10 °} and then in plenty of ice water The precipitated azide is filtered off, dissolved in cold ethyl acetate, the solution is washed at 0 ° with saturated sodium bicarbonate solution and ice water, briefly dried over magnesium sulfate and used directly for the further conversion.

Step 3. Carbobenzoxy-L-valyl-L-tyrosyl'-L-proline tert-butyl ester

51.5 g of L-proline tert-butyl ester are dissolved in the solution of carbobenzoxy-L-valyl-L-tyrosine azide obtained after stage 2 in about 2.91 ethyl acetate at 0 ° and for 5 hours at 0 ° and Left in the dark for 15 hours at 22 °. The reaction mixture is then concentrated to half its volume in a 5 l flask at 40 °. The crystalline carbobenzoxy-L-valyl-L-tyrosyl-L-proline tert-butyl ester precipitates out, which is filtered off through a glass frit, with a little ethyl acetate, then with benzene (50 ml) and with benzene-petroleum ether 1: 1 ( 75 ml) and finally dried at 55 ° in vacuo. Yield: 79.5 g, mp 173 ° to 175 °. The filtrate (volume about 1.51) is cooled in a separating funnel and washed at 0 ° as follows: twice with 450 ml of citric acid 10 ⁰ I ₀ ; three times 300 ml of saturated sodium chloride solution; twice with 250 ml of saturated sodium bicarbonate solution; three times with 150 ml of water. After adding 200 ml of benzene, the ethyl acetate phase is evaporated to dryness, 100 ml of benzene are added again and the mixture is again evaporated to dryness. The residue is dissolved in 25 ml of acetonitrile by heating, 45 ml of benzene are added and, after inoculation, left to crystallize overnight at 0 °. The precipitate is filtered off, washed with a little benzene and finally with petroleum ether and dried at 55 ° in vacuo. Yield: 29 g, mp 163 ° to 167 °. A single recrystallization of this 29 g from 430 ml of hot acetonitrile gives 19.2 g of pure material with a melting point of 173 ° to 175 °, and working up the remaining mother liquor a further 3 g with a melting point of 172 ° to 173 °. Paper chromatography: after 1 hour at 40 ° with concentrated hydrochloric acid; 200 γ on Whatman I, staining with ninhydrin and Pauly: Rf (40) = 0.65; Rf (45) = 0.48.

Level 4. L-valyl-L-tyrosyl·L-proline * tert.-butyίester

28.35 g carbobenzoxy-L-valyl-L-tyrosyl-L-proline tert-butyl ester from mp 173 to 175 ° are suspended in 250 ml of methanol (dist.) and after adding 3 g Palladium carbon 10% with absorption of the forming Hydrogenated carbon dioxide in the shaker. The hydrogen uptake is finished in less than 2 hours, and after a total of 4 hours, the catalyst is filtered off through a G4 suction filter and 25 ml Washed out methanol. The clear, colorless solution is concentrated in vacuo and dried at 45 °. the amorphous, foamy mass is finely pulverized and in a high vacuum at 45 ° to constant weight

7 8

post-dried. 21.3 g (98.5 ° / ₀ ) of chromate are obtained, cooled to -10 °. This is done with stirring and

graphically pure L-valyl-L-tyrosyl-L-prohn-tert-butyl cooling at -10 ° for about 1 ^x / ₂ minutes 4.57 ml

ester of about 100 ° (fuzzy) [α] χ> = -47.4 ° isobutyl chlorocarbonate added dropwise. Rinse with 12 ml

(c = 2 in methanol). Ethyl acetate and stirred for a further 15 minutes

Paper chromatography: directly or after hydrolysis 5 at -10 °. 21.92 g of N ^s -tert-butyloxy are then added with concentrated hydrochloric acid for 1 hour at carbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline-tert-butyl-40 °; 20Oy on Whatman 1, staining with ninhydrin ester, dissolved in 50 ml of absolute ethyl acetate, while and Pauly: Rf (40) = 0.87; Rf (45) = 0.92; added dropwise after salt 5 minutes at -5 ° and with 15 ml acetic acid treatment: Rf (40) = 0.65; Rf (45) = 0.48. ester rinsed. The mixture is stirred for 30 minutes

ίο at 0 °, then lets the temperature rise to 20 ° and

Stage 5. Carbobenzoxy-N * -tert-butyloxycarbonyl- ^{agitates for a} further 1 hour at room temperature.

L-lysyl-L-valyl-L-tyrosyl-L-prohn-tert-butyl ester ^For working up, 170 ml of water and

extracted with 270 ml of ethyl acetate. The ethyl acetate solution

19.15 g of carbobenzoxy-N- ^e tert-butyloxycarbonyl are washed successively with 85 ml of tartaric acid

x-lysine and 21.8 g L-valyl-L-tyrosyl-L-proline-tert-bu- 15 10 ° / q (0 °), 85 ml water, twice with 100 ml sodium

The ethyl esters are diluted with bicarbonate solution in 200 ml of distilled acetonitrile, twice with 100 ml each

dissolves and to this at 20 ° all at once and with stirring sodium sulfate solution. After standing over magnetic

11.4 g of dicyclohexylcarboddimide in solid form, zinc sulfate, are evaporated to dryness and obtained

give. It dissolves immediately, with simultaneous pulverization and drying at 45 ° 29.98 g

crystallize from dicyclohexylurea. Initially 20 of an amorphous crude product. This one gets hot in

is briefly cooled with ice so that the temperature does not dissolve 300 ml of acetonitrile, seeded and stirring

rises above 25 °. The mixture is then stirred and allowed to cool for 3 hours. After standing at room

The crystalline precipitate is filtered off at room temperature and left at temperature overnight

stand. After adding 1 ml of glacial acetic acid, the mixture is stirred and dried at 45 ° in a vacuum. You get

30 minutes at room temperature, then 30 minutes 25 23.55 g carbobenzoxy-L-valyl-N ^s -tert.-butyloxycarbo-

at 0 ° and filtered the precipitated dicyclohexylnyl-L-lysyl-L-valyl-L-tyrosyl-L-proline tert-butyl ester

urea from. The filtrate is evaporated to a temperature of 159 to 1.64 °, which for further purification

Dry 44.3 g of an amorphous powder in 44.3 ml of corn for 3 minutes in 235 ml of boiling aceto

Ethyl acetate is dissolved warm and stirred by adding 221 ml of nitrile. After standing overnight

Petroleum ether is excreted again. After decant 30 room temperature is filtered, with 70 ml of acetonitrile

animals the solvent and drying the powder- washed and dried in a high vacuum at 45 °,

Sized precipitation at 45 ° in a high vacuum is obtained. 22.55 g of pure product with a melting point of 162 ° to 165 ° are obtained.

37.94 g of carbobenzoxy-N ^e -tert-butyloxycarbonyl-L-ly- [<x] _D = -71.4 ± 1 ° (c = 0.9 in methanol).

syl-L-valyl-L-tyrosyl-L-proline tert-butyl ester from F. Paper chromatography: after 1 hour at 40 ° in

about 100 °. The product is hydrogenated directly. [a \ _D 35 concentrated hydrochloric acid. 200 γ on Whatman I, An

= -62.3 ± 0.5 ° (c = 1.98 in methanol). staining with ninhydrin and Pauly, Rf (40) = 0.40;

Paper chromatography: after 1 hour at 40 ° with Rf (45) = 0.36.

concentrated hydrochloric acid. 200 γ on Whatman 1, An _o . . _O

staining with ninhydrin and Pauly: Rf (40) = 0.40; ^{Level 8} ;

Rf (45) = 0 28 40 L-valyl-L-t

47.98 g carbobenzoxy-L-valyl-N ^s -tert-butyloxy-

Level6. Νε-tert-butyloxycarbonyl-L-lysyl-L-valyl-carbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proHn-tert.-butyl-

L-tyrosyl-L-proline-tert-butyl ^{ester from F} · ^{161 to 163} ° are dissolved in 320 ml of methanol

suspended and after adding 3.2 g of palladium

37.37 g carbobenzoxy-N ^s -tert.-butyloxycarbonyl- 45 carbon 10% in the shaking duck under carbon dioxide-

L-lysyl-L-valyl-L-tyrosyl-L-proline tert-butyl ester is hydrogenated by absorption. The hydrogen uptake takes place

dissolved in 200 ml of methanol and after the addition of with simultaneous dissolution of the suspended

3 g palladium-carbon 10% in the shaking duck under pentapeptides and is finished in about 3 hours. To

Hydrogenated carbon dioxide absorption. The hydrogen- another 1 to 2 hours the catalyst is through

recording will end in about 6 hours. A G4 frit is filtered off, followed by adding methanol.

remove the catalyst through a G4 frit, rinse with wash and evaporate the filtrate to dryness. That

Methanol and the filtrate evaporated to dryness. powdered material is in a high vacuum at 45 °

After pulverization, drying is carried out in a high vacuum at constant weight. You get

45 ° and thus receives 29.68 g of chromatographically uniform 40.27 g (98.7% of theory) of amorphous L-valyl

lichen NMert.-Butyloxycarbonyl-L-lysyl-L-valyl-L-tyro- 55 N ^e -tert.-butyloxycarbonyl-L-lysyl-L -valyl-L-tyrosyl-

syl-L-proline tert-butyl ester from 95 to 100 °. [«] # L-proline tert-butyl ester with an indistinct melting point

= -61.8 ± 0.5 ° (c = 2.0 in methanol). at about 130 °. [tx] _D = -69.2 ± 0.6 ° (c = 2 in Me-

Paper chromatography: 200 γ on Whatman 1, ethanol).

staining with ninhydrin and Pauly: Rf (40) = 0.86. Paper chromatography: 200 γ on Whatman I, An

60 staining with ninhydrin and Pauly: Rf (40) = 0.92;

Step 7. Carbobenzoxy-L-valyl-Ne-tert-butyloxy- Rf ( ⁴⁵ ) = °> ⁹⁴

carbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline-tert.- _{grade 9} carbobenzoxy-L-arginyl-L-proline-methyl-

butyl ester hydrochloride

In a three-necked flask with a stirrer, drop 65 154 g (0.5 mol) carbobenzoxy-L-arginine and 83 g

funnel, thermometer and calcium chloride tube are (0.5 mol) L-proline methyl ester hydrochloride in

8.73 g of carbobenzoxy-L-valine and 5.09 ml of triethyl 2.51 chloroform suspended and under vigorous

amine dissolved in 85 ml of absolute ethyl acetate and stirred with 112 g (0.55 mol) of dicyclohexyl carbodiimide

9 XO

offset. The reaction mixture is extracted for 5 hours with 100 ml of chloroform each time. The aqueous solution is stirred at 50 °, then cooled to 20 ° and completely evaporated in vacuo and the stirring is treated with 5 ml of glacial acetic acid. After a further residue, dried in a high vacuum at 40 °: stirring for 2 hours at 0 to 5 °, the viscous becomes hydroscopic foam; 78 g (calculated crystalline mixture of dicyclohexylurea and 564.8 g); contains water, which can be filtered off with suction by further reaction products, cannot be removed with chloroform. Drying.
wash and dry in vacuo at 40 °. For further purification, 90 g of crude product are stirred in the mixture with 21 water for 30 minutes, dissolved 200 ml of methanol and, while stirring at 11 at 40 ° and filtered, precipitated from the undissolved dicyclohexyl urine ethyl acetate. The separated oily product material. The aqueous filtrate is solidified in vacuo at io, concentrated to a syrup on repeated trituration with vinegar at a bath temperature of from 40 to 50 °, and the ester to an amorphous powder. After drying, 150 ml of ethanol are added. After 2 hours it is 40 ° and 0.1 mm Hg: 86 g, m.p. 90 to 120 °. The precipitated crystalline product is filtered off and _{washed with precipitated product containing about 3 ° / 0} L-arginyl-L-proline ethanol. 192 g (= 84%) and about 3% carbobenzoxy-L ^ arginine are obtained; However, carbobenzoxy-LTarginyl-L-proline-methylester-hydro- 15 is sufficiently pure for further conversion,
chloride from F, 164 to 165 °.

From the mother liquor, after concentration step 12. Carbobenzoxy-L-arginyl-L-arginyl, another 5 g from F, 163 ° to 165 °, a total of 197 g (= 86% L-proline hydrochloride
the theory). After recrystallization from 1.5 l of absolute ethanol, the product melts at 165 to 167 °; ao 32.4 g (about 50 mmol) of crude carbobenzoxy - [#] f == -71.9 ° (c = 1.8 in water). -64.4 ° (e - 1.9 in L-arginyl-L-arginyl-L-proline methyl ester dihydrochloride, methanol) and -67.1 ° (c = 2 in methanol-water, while stirring, in 100 ml of 1 n-sodium hydroxide solution 1; 1), the product is dissolved by paper chromatography and stirred for a further 20 minutes at 20 °. Then mean Rf values in the systems 43; 0.79; 49: 0.90; 52 ml of 2η hydrochloric acid are added to 54: 0.86 with thorough stirring; 87: 0.82. Staining with Sakaguchi. 25 and the reaction solution evaporated in vacuo at 30 °

_. . _1A *. 1 1 ^ t. 1 * a. The viscous residue is up to the weight

Stage 10. L-Arginyl-L-proline-methylester- _{kongtanz bd 0> 1} ^ _{m Hg mw mm brittle)} ^

dinycirocrüond _{loose Schauni} dried; 34 g mixture containing

45.6 g (0.1 mole) carbobenzoxy-L-arginyl-L-proline * about 70% carbobenzoxy-L-arginyl-L-arginyl-L-proline methyl ester hydrochloride are in 400 ml of methanol 30 dihydrochloride in addition to sodium chloride and others Dissolved with heating and after cooling on room contamination.

temperature with a solution containing 0.15 mol For conversion into the carbobenzoxy tripeptide

Hydrogen chloride in methanol. After the addition of monohydrochloride, 34 g of the dihydrochloride are obtained of 2.0 g of palladium carbon (10%) is added to room in 150 ml of dimethylformamide, from hydrogenated at temperature and normal pressure; inside 35 insoluble sodium chloride filtered off and under 2180 ml of hydrogen are taken up for 30 minutes. Stirring at room temperature with 10.1 g of triethylamine The solution is filtered off from the catalyst and added. The precipitated triethylamine hydro vacuum evaporated at a maximum of 35 ° and the chloride is filtered off and the clear filtrate with the Dried residue at 0.1 mm Hg over potassium hydroxide monohydrochloride of carbobenzoxy tripeptide. The oil obtained gives 4 ° vaccinated when rubbed. The monohydrochloride slowly crystallizes with 150 ml of ethyl acetate 35 g (98%) of a colorless in fine-grain form. Leave for 20 to 24 hours amorphous powder, L-arginyl-L-proline-methylester- stand at room temperature, then sucks the crystalline dihydrochloride. The Substan? is paper phromato ^ sat off and washes with dimethylformamide and vinegar graphically uniform: Rf values in the systems 49: ester. Obtained after drying in vacuo at 50 ° 0.57; 54: 0.37; 56: 0.42; 87: 0.36 (staining with 45 man 19 g (= 66% of theory) carbobenzoxy-Sakaguchi reagent), L-arginyl-L-arginyl-L-proline monohydrochloride from F.

",, __ ,,,. ,. , 248 to 249 °. Recrystallized by dissolving in 100 ml

Stufell. Carbobenzoxy-L ^ rgmyl-L-argmyl-methanol, containing 1 equivalent of hydrogen chloride

L-prohn methyl ester dihydrochloride ^ _{Addition of 2} equivalents of triethylamine: 16.7 g.

30.8 g (0.1 mol) carbobenzQxy-L-arginine and 35.8 g 50 F, 259-262 °; [«] F = -64.8 ± 1 ° (c = 1.4 in (0.1 mol) L-arginyl-L-proline methyl ester-dihydrochlo-water); sparingly soluble in all organic solvents are suspended in 100 ml of dimethylformamide; easily soluble in water. The substance is stirred at room temperature for 30 minutes until it becomes uniform in the paper chromatogram (200 γ of an almost complete solution. After adding 100 ml of 5% solution in water): Rf values in the chloroform systems, the solution is cooled to -5 ° and with 55 49: 0.68; 54: 0.64; 56: 0.63; 87: 0.62; 22.7 g (0.11 mol) of dicyclohexylcarbodiimide were added to the dyeing process. Sakaguchi reagent.

Subsequently, while stirring at -5 ° within "" "" _,,,. ,. ,

about 30 minutes another 30QmI chloroform to stage 13. Carbobenzoxy-L-arginyl-L-arginyl-

dropped, and the reaction mixture, from which L-prolyl-L-valyl-tert-butyloxycarbonyl-L-lysyl-

dicyelohexylurea gradually separates out, becomes i-valyl-L-tyrosyl-L-prohn-tert-butyl ester sulfate via 5a
Stirred at 0 ° overnight. After adding 2 ml of glacial acetic acid, 9 g (15 mmol) of Carhobenzoxy-L-arginyl-L-arginyl-

and further stirring for 1 hour at 0 °, L-proline hydrochloride is dissolved in 75 ml of dimethylder precipitated dicyclohexylurea filtered off formamide and suspended by adding 7.5 ml and with dimethylformamide-chloroform 1: 4 for a 2 η solution of hydrogen chloride in ethyl acetate washed. The filtrate is dissolved in vacuo at 40 ° to 1/2 with stirring (conversion into the dihydrochloride). The syrup is evaporated and the residue in 300 ml. After cooling to -10 °, 1.8 g are added with stirring Water absorbed. The insoluble dicyclohexyl (15 mmol) freshly distilled pivaloyl chloride and anharnstojf is filtered off and the filtrate is added twice with a closing 1.5 g (15 mmol) of triethylamine,

11 12

and after 10 minutes the solution of the mixed stage 16. Carbobenzoxy-N ^e -tert.-butyloxycarbonyl anhydride with 9.5 g (12.5 mmol) of L-valyl-N ^e -tert.- L-lysyl-L- prolyl-L-valyl-glycyl-tert-butyloxycarbonylbutyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline-L-lysyl-tert-butyloxycarbonyl-L-lysine methyl ester

tert-butyl ester, dissolved in 20 ml of dimethylformamide,

offset. After stirring for a further 10 minutes, 23.3 g (36.8 mmol) of carbobenzoxy-N ^E -tert-butyl at -10 ° are added 100 ml of water and anoxycarbonyl-L-lysyl-L-prolyl-L-valyl -Glycine are then added dropwise in 75 ml of 2N sodium sulfate solution. 200 ml of absolute tetrahydrofuran and 5.65 ml of the precipitated crystalline sulfate of the octapeptide (1.1 equivalents) of absolute triethylamine dissolved. The derivative is filtered off for a further 15 minutes, water is added dropwise to the solution cooled to -15 °, washed under and dried in vacuo at 40 °: 3.63 ml of ethyl chloroformate (1.03 13.7 g (78 ° / ₀ )) are stirred. colorless powder with indistinct equivalents). The mixture is stirred for 20 minutes at -15 ° and melting point at about 220 °. The crude product is then dissolved in dropwise a solution of 17.5 g (35.9 mmol) 75 ml of 80 ° / ₀ methanol and tert by adding N ^e. -Butyloxycarbonyl-L-lysyl-N ^ tert-butyloxy precipitated from 250 ml of water: 11.8 g. In the thin-layer carbonyl-L-lysine methyl ester (shown according to the chromatogram on silica gel, the Rf values are 15 method of Belgian patent specification 594 338) in the systems 100: 0.30; 102: 0.42. Add 80 ml of absolute tetrahydrofuran to the substance. The reaction contains traces of a slower running compound, the mixture becomes 24 hours at room temperature but is sufficiently pure for the further conversion. permit. Then the excreted material

(Mixture of triethylamine hydrochloride and hexa-

Stage 14. ^{L-arginyl-L-arginyl-L-prolyl-L-valyl-20} peptide derivative) and suction filtered with tetrahydrofuran

tert-butyloxycarbonyl-i-lysyl-L-valyl-L-tyrosyl ^{and a} lot of ethyl acetate washed, the hexapeptide

L-proline tert-butyl ester triacetate derivative goes back into solution. One unites them

Tetrahydrofuran and ethyl acetate filtrates, evaporated to

14g (10mMol) carbobenzoxy-L-arginyl-L-arginyl dry and distribute the residue between L-prolyl-L-valyl-tert-butyloxycarbonyl-L-lysyl-L-valyl-25 water and a lot of ethyl acetate. The ethyl acetate solution is L - tyrosyl - L - proline - tert. - butyl - sulfate are suspended in ice-cooling with dilute Zitronensäurelösüng, 200 ml of 80 ° / ₀ methanol and geGegenwart in water, sodium bicarbonate and water of 2 g of palladium-carbon (10%) was hydrogenated at wash, dried with sodium sulfate and einZimmertemperatur and normal pressure. After steamed. The evaporation residue is stopped twice with uptake of the calculated amount of hydrogen, 30 each 500 ml of ether and three times with 11 tetrahydrofuran each time the hydrogenation comes to a standstill. The ether mixture from the catalyst (1: 4) was crushed. In this way, the filtered solution is obtained, in order to remove sulfate ions, a hexapeptide derivative is obtained as an insoluble gelatinous powder; through a column of 200 ml Amberlite IRA-400 the yield is 31.3 g. Nitrided for further purification (trade name) [acetate], and 80 ° / _o sodium, the crude product in 300 ml of acetonitrile is washed with methanol. The eluate is dissolved in a vacuum with heating and the solution is evaporated in a water bath and the residue is triturated to give a powder. from 60 ° (in the Dewar flask) allowed to cool slowly. The yield of L-arginyl-L-arginyl-L-prolyl-L-valyl- The hexapeptide derivative crystallized in the course of tert-butyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-pro several days Needles and lin-tert.-butyl ester triacetate also separates is almost quantitative. some gelatinous material emerges. The lower

In the thin-layer chromatogram on silica gel, 40 strokes are therefore repeated several times to about the Rf values in systems 54: 0.40; 101: 0.60; heated to 50 to 60 °, the crystals undissolved Aluminum oxide: 0.22 in system 102. The staining remains with and the gelatinous material goes into solution. Sakaguchi and Reindel-Hoppe reagents. Ultimately, this is how the hexapeptide derivative becomes

obtained completely crystalline; M.p. 121 to 126 °, from stage 15. Carbobenzoxy-Ns-tert-butyloxycarbonyl-45 yield 30.12 g (76 ° / ₀ of theory). For analysis, L-lysyl-L-prolyl-L-valyl-glycine is recrystallized from methanol and acetonitrile, F. 121

up to 126 °; [α] _Λ = -50.8 ° (c = 1.89 in methanol).

40.85 g (61.7 mmol) carbobenzoxy-N ^E -tert.-butyl- with thin layer chromatography on silica gel

oxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycine-ethyl ester plates shows the protected hexapeptide derivative only (R. Schwyzer and W. R i 11 e 1, HeIv., 44, p. 159 50 a spot, Rf value = 0.03 in ethyl acetate, Rf = 0.23 [1961]) are dissolved in 800 ml of ethanol, the solution in chloroform-methanol - 19: 1; Rf = 0.65 in cooled to 5 ° and added 80 ml of 1 η sodium hydroxide solution. Chloroform-methanol = 9: 1 (the plates are After 3 hours at 5 °, 11 water is added, which is developed with Reindel-Hoppe reagent), excess caustic soda by adding solid

Carbon dioxide is neutralized and the ethanol ^{is distilled off in step 17. Carbobenzoxy-N E} tert-butyloxycarbonyl vacuum. L-lysyl-L-prolyl-L-valyl-glycyyl-tert.-butyloxy-

remaining aqueous phase the sodium salt of carbonyl-L-lysyl-tert-butyloxycarbonyl-

Tetrapeptide derivatives as a gelatinous precipitate. L-lysine hydrazide

It is released from something by heating it to 45 °

The undissolved material is filtered off, the solution is cooled to 20.00 g (18.10 mmol) of carbobenzoxy-N ^s -tert. ~ butyl-cooled and 80 ml of cold 1 η hydrochloric acid are added. oxycarbonyl-L-lysyl-L-propyl-L-valyl-glycyl-tert-butyl-The carbobenzoxy-N ^ -tert-butyloxycarbooxycarbonyl-L-lysyl-tert-butyloxycarbonyl-L-lysinnyl-L falls -lysyl-L-prolyl-L-valyl-glycine as a finely powdered low methyl ester are dissolved in 200 ml of methanol and the precipitate is eliminated. It is filtered off and washed free of chloride with 10 ml of hydrazine hydrate with a solution cooled to 0 ° with ice water, then mixed in vacuo over sulfuric acid. The clear solution is left at 2 ° for 3 days and dried using acidic acid. The yield is 34.19 g then concentrated to a small volume and with <88 ^O / _O of theory); F. about 71 to 95 degrees. A lot of water is added to the product. The resulting product is sufficiently pure for further reactions. is thoroughly ground, sucked and with a lot

Washed ice water. The still moist material is again dissolved in a little methanol and again with Water pleases. Filtering off with suction and drying in vacuo at 40 ° gives 18.82 g (94% of theory) of crude hexapeptide hydrazide, F. 141 to 145 °.

After recrystallization from 500 ml of tert-butanol-water 2: 3, 37.0 g of crude product yields 34.30 g of crystalline hydrazide with a melting point of 156 to 158 °, [ocfi = -51.9 ± 0.5 ° (c = 1 , 91 in methanol).

In thin-layer chromatography on silica gel plates (6Oy of the hydrazide applied and with Reindel-Hoppe reagent developed) the following Rf values are obtained: 0.0 (benzene-ethyl acetate 1: 1); 0.26 (Chloroform-methanol 9: 1); 0.73 (dioxane-water 9: 1).

Stage 18. Carbobenzoxy-N ^ tert-butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycyl-tert-butyloxy-

carbonyl-L-lysyl-tert-butyloxycarbonyl-L-lysyl-

L-arginyl-L-arginyl-L-prolyl-L-valyl-tert.-butyloxy-

carbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline-tert.-

butyl ester

6.26 g (5.66 mmol) carbobenzoxy-N ^s -tert-butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycyl-tert-butyloxycarbonyl-l-lysyl-tert. - Butyloxycarbonyl - l - lysine hydrazide are dissolved in 50 ml of dimethylformamide, the solution is cooled to -20 ° and 15 ml of 1 η hydrochloric acid are added dropwise with stirring so that the temperature does not rise above -15 °. Then 5.7 ml of 1N sodium nitrite solution are added dropwise in the same way and the solution is stirred at -7 ° for 20 minutes. Then a solution of 6.12 g (4.53 mmol) of L-arginyl-L-arginyl-L-prolyl-L-valyl-tert. - Butyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline tert-butyl ester triacetate and 3.1 ml of triethylamine in 60 ml of dimethylformamide were added dropwise at -7 ° and the reaction mixture left at -2 ° for 65 hours . The solvent is then distilled off at 0.1 mm Hg and a bath temperature of 40 ° and the residue is washed several times with ether with trituration: 10.7 g of crude product. In thin-layer chromatography on silica gel, the product shows the tetradecapeptide derivative as the main stain with Rf 0.35 in system 100, 0.25 in system 53 (staining with Sakaguchi and Reindel-Hoppe reagent) and an additional unchanged octapeptide derivative as well as several by-products derived from the hexapeptide portion .

For the separation of the chlorine ions, the crude product is dissolved in 200 ml of 0.5 η-acetic acid in 50 ° / ₀ methanol, (weakly basic, acetate form) over a column of 50 ml of Merck ion exchanger II filtered and washed with 200 ml washed of the same solvent . The product contained in the eluate is further converted directly.

55 Level 19. N ^s -tert.-Butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycyl-tert.-butyloxycarbonyl-L-lysyl-tert.-

butyloxycarbonyl-L-lysyl-L-arginyl-L-arginyl-

L-prolyl-L-valyl-tert-butyloxycarbonyl-L-lysyl-

L-valyl-L-tyrosyl-L-proline tert-butyl ester tritosylate

The solution of the protected tetradecapeptide peptides ester is mixed with 10 ml glacial acetic acid and in the presence of Ig palladium carbon (10 ° / ₀ strength Pd). After 17 hours and uptake of 157 ml of hydrogen, the hydrogenation comes to a standstill. The catalyst is then filtered off and the filtrate is evaporated. The residue is washed with petroleum ether and dried for 18 hours at 40 ° and 0.01 mm Hg. 7.5 g of crude product are obtained. This is dissolved in 50 ml tert-butanol-water 1: 1 dissolved, applied to a column of 60 g of carboxymethylcellulose (suspended in the same solvent mixture) was poured and sodium using a Uvicords and a linear gradient from 11 to 50 ° / ₀ tert.-butanol and 11 50 ° / ₀ sodium tert-butanol, containing 10% acetic acid, acetate. After 560 ml of solvent have passed through, the elution of the tetradecapeptide derivative begins. This is collected in several fractions, which are evaporated separately. 4.1 g of pure N ⁸ -tert-butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycyl-tert-butyloxycarbonyl-L-lysyltertured. - butyloxycarbonyl - l - lysyl - l - arginyl - l - arginyl-L-prolyl-L-valyl-tert.-butyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline-tert. -butyl ester triacetate obtained. A further 2.8 g of impure substance is dissolved in 10 ml of water and again chromatographed on 16 g of carboxymethylcellulose, using a linear gradient between 500 mL of water and 500 ml of 10 ° / ₀ acetic acid. This gives a further 2.15 g of pure substance; a total of 6.25 g (= 60%, calculated on the octapeptide used). In the thin-layer chromatogram on silica gel, the Rf values are: 0.23 in system 100, 0.25 in system 52.

The resulting triacetate of the tetradecapeptide derivative is converted into the tritosylate as follows:

6.25 g (2.73 mmol) are dissolved in 100 ml of dry pyridine, treated with 1.70 g of p-toluenesulfonic acid, the solution evaporated to dryness, the residue for the removal of pyridine acetate for 18 hours 35 ° and 0.01 mm Hg dried and rubbed with acetone to remove pyridine tosylate and suction filtered: 7.26 g colorless powder with a melting point of 155 to 162 °.

The protected tetradecapeptide tert-butyl ester is in the manner described in the main patent with tert-butyloxycarbonyl-L-seryl-L-tyrosyl-L-seryl-L-methionyl-y-tert-butyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl -L-tryptophyl-glycine or condensed with the corresponding compound rtitylated on the histidine residue to give the protected tetracosapeptide tert-butyl ester and from this by means of trifluoroacetic acid, as described in the main patent, all protective groups cleaved.

Claims (2)

Patent claims: 1. Process for the preparation of a tetracosapeptide of the formula L-Seryl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-l-histidyl-l-phenylalanyl-L-arginyl-L-tryptophyl-glycyl-L-lysyl -L- prolyl-L-valyl-glycyl-L-lysyl-L-lysyl-L-arginyl-L-arginyl-L-prolyl-L-valyl-L-lysyl-L-valyl-L-tyrosyl-L-proline by condensation of tert-butyloxycarbonyl-L-seryl-L-tyrosyl-L-seryl-L-methionyl-y-tert.-butyl-L - glutamyl - l - histidyl - L - phenylalanyl - L - arginyl-L-tryptophyl -glycine with N ^e -tert.-Butyloxycarbonyl-L - lysyl -L- prolyl - L - valyl - glycyl - tert. - butyloxycarbonyl - L - lysyl - tert. - butyloxycarbonyl - L - lysyl-L-arginyl-L-arginyl-L-prolyl-L-valyl-tert.-butyloxycarbonyl-L-lysyl-L-valyl-L-tyrosyl-L-proline tert.-butyl ester according to patent 1196 666, characterized in that the tetradecapeptide derivative is prepared by condensing L-proline tert-butyl ester with carbobenzoxy-L-valyl-L-tyrosine azide, and the carbobenzoxy group is split off from the tripeptide derivative obtained. tet, condenses the free tripeptide ester with carbobenzoxy-N ^£ -tert-butyloxyearbonyl-L-lysine, splits off the carbobenzoxy group from the tetrapeptide ester obtained, condenses the free tetrapeptide ester with carbobenzoxy-L-valine, splits off the carbobenzoxy group from the pentapeptide ester obtained obtained L-valyl4ert.-butyloxy = carbonylrL-lysyl-L-valylrL-tyrosyl-L-proline-tert-butyl ester with carbobenzoxy-L-arginyl-l ^ arginyl-L-proline to form the protected octapeptide ester, from which the Carbobenzoxy group splits off and the resulting free octapeptide ester with carbobenzoxy-N ⁶ -tert. - [butyloxycarbonyk L-lysyl-L-prolyl-L-valyl-glycyl-tert-butyloxycarbo ^ nyl-L-lysyl-tert-butyloxycarbonyl-rL-lysine- ^ zid condensed to the tetradecapeptide ester and finally split off the carbobenzoxy group from this. 2. The method according to claim 1, characterized in that the hexapeptide derivative carbobenzoxy-N ^ -tert.-butyloxycarbonyl-Lyysyl-L-prolyl-L-valyl-glyeyl-tert.-butyloxycarbonyl-L-lysyl-tert.-butyloxycarbonyl L-lysine azide by condensation of carbobenzoxy-N ^ tert-butyloxycarbonyl-L-lysyl-L-prolyl-L-valyl-glycine with NHert-butyloxycarbonyl - l , lysyl - tert. - butyloxycarbonyl -. β-lysine methyl ester to carbo-benzoxy-N ^e -tert-butyloxycarbonylTL-.lysyl-L-prolylrLrvalyl-glycyl-tert-butyloxycarbonyl·Lτlysyl-tert-butyloxycarbonyl-L-lysine methyl ester and converting the ester into the hydrazide and prepares. 609 558/399 4.66 © Bundesdruckerei Berlin