DE1126397B - Process for the production of new MSH-active octadecapeptides - Google Patents

Description

GERMAN

PATENT OFFICE

C 21514 IVb / 12q

REGISTRATION DATE: MAY 24, 1960

NOTICE THE REGISTRATION AND ISSUE OF THE EDITORIAL: MARCH 29, 1962

The invention relates to the production of a new octadecapeptide of the formula L-asparaginyl-L-seryl-glycyl-L-prolyl-L-tyrosyl-L-lysyl-L-methionyl-L-glutaminyl -L- histidyl - L- phenylalanyl - L - arginyl - L-tryptophyl-glycyl-L-seryl-L-prolyl-L-prolyl-L-lysyl- L-aspartic acid and the corresponding compound, which instead of the glutaminyl radical is the remainder of the Glutamic acid, and its derivatives and acid addition salts. Have the new connections the effect of the natural melanocyte-stimulating pituitary hormone /? - MSH, however this has the advantage that they can be more easily synthesized. The new octadecapeptides Can be used in place of natural MSH as a remedy or as an intermediate in the manufacture of /? - MSH can be used.

There are various possibilities with regard to the structure of the octadecapeptides from the individual ones Amino acids or smaller peptide units.

For example (see Fig. 1) the Hep tapeptide H-Asp-Ser-Gly-Pro-Tyr-Lys-Met-O H with protected amino groups or its reactive _% derivatives with the undekapeptide H-GIu (NH ₂ ) -His-Phe-Arg-Try-Gly-Ser-Pro-Pro-Lys-AspO H, whose carboxyl groups are functionally modified and whose e-amino group of the lysyl residue is protected, or the corresponding compound which instead of the glutaminyl residue is the remainder of glutamic acid contains, or link their reactive derivatives to form a peptide bond between the L-methionyl and the L-glutaminyl or glutamyl radical. The abbreviations Asp, Ser, Pro, Tyr etc. denote the amino acid residues of aspartic acid, serine, proline, tyrosine etc. in the L-form.

The heptapeptide used as the starting material is, as shown in Fig. 1, by condensation of the Tripeptide H-Asp-Ser-Gly-0 H, its sc-amino group is protected, with an ester of the tetrapeptide H-Pro-Tyr-Lys-Met-OH, whose ε-amino group is protected is made. It is also obtained if the hexapeptide H-Asp-Ser-Gly-Pro-Tyr-Lys-OH, whose \ - and ε-amino groups are protected, condensed with a methionine ester.

The undekapeptide can be obtained by condensation as described in German Patent 1,108,232 Hexapeptide H-GIu-His-Phe-Arg-Try-Gly-OH with a diester of the pentapeptide H-Ser-Pro-Pro-Lys-Asp-OH, whose e-amino group is protected. Figure 1 illustrates the synthesis.

Z means the carbobenzoxy group, Tos the tosyl group, BOC is the tert-butyloxycarbonyl group and PNBz the para-nitrobenzyl radical.

Process for the production of new MSH-active octadecapeptides

Applicant: CIBA Aktiengesellschaft, Basel (Switzerland)

Representative: Dipl.-Ing. E. Splanemann, patent attorney, Hamburg 36, Neuer Wall 10

Claimed priority: Switzerland of May 28, 1959 (No. 73 685)

Dr. Robert Schwyzer, Dr. Beat Iselin, Riehen, Dr. Heinikapier, Birsfelden,

Dr. Werner Rittel, Basel,

and Dr. Herbert Zuber, Riehen (Switzerland),

have been named as inventors

The peptide bond is preferably produced in the presence of a carbodiimide as a condensing agent.

Free functional groups present in the starting materials that are not involved in the reaction are expediently protected, in particular by means of those which can easily be split off by hydrolysis or reduction Residues, the carboxyl group preferably by esterification, z. B. with methanol, benzyl alcohol, p-nitrobenzyl alcohol, or "by amidation, the Amino groups, for example, by introducing the tosyl or trityl radical or, in particular, the carbobenzoxy group or colored protecting groups, such as the p-phenylazo-benzyloxycarbonyl group. For protection of the ε-amino group of the lysyl radicals, the tosyl radical is preferably used.

209 557-424

-OH Z- Ser z- Ser H- Ser GIy H- GIy 1 126 397 Tos -OCH ₃ H- Tos
I. -OCH ₃ Tos -OH Tos Mead -OCH ₃ Z - Asp -OPNBz H- Per Tyr Lys Per Tyr Lys Per Tyr Lys Per Tyr Lys Ser GIy Tos -OPNBz BOC- Per Tyr Lys Ser GIy Tos -OPNBz BOC- Per Tyr Lys Ser GIy Tos Mead -OCH ₃ -OPNBz BOC- Per Tyr Lys GIy Mead -OH Asp -OPNBz BOC- GIy Mead -OH Asp -OH H- Mead -OH Asp

Asp 1 Asp Ser GIy Tos
I. Mead NH ₂
I. Z- 1
Per Tyr Lys I.
GIu Ser GIy Mead NH _S Per Tyr Lys CL

Phe Arg Try GIy

OH Z-

OH Z

Per

OHZ-

Per

H-

Per

Per

Per

Per

OHZ-

Tos

I.

OCH _S

Lys

OH H

Tos

Lys

Per

Per

Per

Per

Tos

_J

Lys

Tos

Tos

I.

ι Lys

Tos

Lys

Tos

Lys

Tos

Lys

Tos

Asp

OCH ₃

OCH ₃

Asp

OCH ₃

OCH ₃

Asp

OCH,

OCH,

Per I. I. Z- I.
Lys Asp Per Tos
I. OCH
i H- I.
Lys Asp

OCH ₃

OCH ₃

OCH ₃

Asp

OCH ₃

OCH ₃

Asp

OCH ₃

OCH ₃

Asp

-OCH ₃

OCH ₃

Asp

- OCH ₃

OCH ₃

Phe Arg Try GIy

Per

Per

Lys

Asp

OCH.

Tos

OCH ₃

Phe Arg Try GIy

Per

Per

Lys

Asp

OCH ₃

Tos

OCH ₈

J

Phe Arg Try GIy

Per

Per

Lys

Asp

OCH ₃

OH

OH

Phe Arg Try GIy

Per

Per

Lys

Asp

The conversion of a protected NH ₂ group into a free group and the conversion of a functionally modified carboxyl group into a free carboxyl group takes place according to methods known per se by treatment with hydrolyzing or reducing agents.

Depending on the method of operation, the new compounds are obtained in the form of bases or their salts. From the salts can in a manner known per se

To convert to the free ester, 58 g (0.2 mol) of the hydrobromide are dissolved in 100 ml of water, covered with 300 ml of ethyl acetate and mixed with 50 ml of cold, saturated potassium carbonate solution at 0 ° while shaking. The ethyl acetate phase is separated off and, after saturation with solid potassium carbonate, the aqueous solution is extracted twice more with ethyl acetate. The combined ethyl acetate phases are dried and, after careful, the bases are obtained. Of the latter, in turn, evaporation in vacuo at room temperature, 36.4 g can be converted into therapeutically usable salts by reaction with acids which form therapeutically useful salts (87%) H-GIy-OPNBz as a crystalline residue with a melting point of 50 to 52 °, which can be further processed directly are to win salts, such as B. those with inorganic can. After two recrystallization from a lot of local acids, such as hydrohalic acids, e.g. B. ether, the melting point of the analytically pure hydrochloric acid or hydrobromic acid, nitric acid, 15 material is unchanged.
Thiocyanic acid, sulfuric acid, phosphoric acid, or 1 ν q π πηκτή

with organic acids such as acetic acid, propionic acid, '· Z-Ser-Gly-OPNBz

Glycolic acid, lactic acid, pyruvic acid, oxalic 12 g (0.05 mol) Z-Ser-OH and 10.5 g (0.05 mol)

acid, malonic acid, succinic acid, maleic acid, Fu-H-GIy-OPNBz are in 120 ml of methylene chloride marsic acid, tartaric acid, citric acid, benzoic acid, dissolved and cooled to 0 °, whereby a hydrochloric acid, Salicylic acid, 2-phenoxy or 2-acetoxy-like complex of the two reactants is eliminated (this reaction occurs to a greater extent when acetonitrile is used as a solvent, a little slower in dimethylformamide). The mixture 25 is mixed with a solution of 11.3 g (0.055 mol) of dicyclohexylcarbodiimide added in 20 ml of methylene chloride and shaken overnight at 0 °; this is possible Salt gradually dissolves, and at the same time a mixture of the reaction product and dicyclo compound separates, which instead of the glutaminyl residue from the residue 30 hexylurea, which after the addition of 0.5 ml of Eisder Contains glutamic acid, because of its vinegar (to destroy excess carbodi-MSH activity in the form of pharmaceutical imide) filtered off and washed with methylene chloride Find preparations use. These will contain the. For the separation of dicyclohexylurea Peptides mixed with one for enteral or the material is parenteral with 150 ml of tetrahydrofuran Application suitable pharmaceutical 35 stirred, the undissolved urea filtered off and the

benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, benzene or Toluenesulfonic acid, naphthalenesulfonic acids, sulfanilic acid.

The octadecapeptides obtained according to the method and also the undekapeptide H-GIu (NH ₂ ) -His-Phe-Arg-Try-Gly-Ser-Pro-Pro-Lys-Asp-O H or the corresponding intermediate product

organic or inorganic carrier material.

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

The filtrate was evaporated in vacuo. When ethyl acetate is added to the residue, the reaction product crystallizes and is filtered off after standing overnight at 0 °. After recrystallization from ethanol, 15.0 g (70%) of pure material with a melting point of 121 to 123 ° are obtained, [«] ^s ₀ ° = -8.5 ° (c = 2.01 in glacial acetic acid); the mother liquor gives only amorphous, partially racemized product.

If the reaction is carried out in dimethylformamide, (0.6 mol) p-nitrobenzyl chloride and 85 ml (0.6 mol) 45, about 15% carbobenz-triethylamine is formed as a by-product in 1000 ml of ethyl acetate is during oxy-i - seryl-N, N'-dicyclohexylurea from F. 170 to Heated under reflux for 15 hours, then cooled to 171 ° (from methanol), which is difficult to remove from the main and can be separated from the precipitated triethylamine hydrochloride product.

filtered off. The filtrate is cooled with ice. A sample is used to check the optical purity

2 n sodium carbonate solution and water washed, 50 of the Z-Ser-Gly-Q P N Bz in the presence of palladium

example
Z-GIy-OPNBz

A solution of 105 g (0.5 mol) Z-GIy-OH, 103 g

dried and concentrated in vacuo to about 300 ml, the reaction product being in crystalline form separates; a second fraction is produced by further concentration of the mother liquor and the addition of ether won, total 146 g. After recrystallization from alcohol: 139 g (81%), melting point 108 ° to 110 °.

coal hydrogenated. The H-Ser-GIy-OH obtained after taking up the calculated amount of hydrogen (5 equivalents) shows [*] jf = + 31.3 ° (c = 3.17 in 1N hydrochloric acid).

4. H-Ser-Gly-OPNBz

2. H-GIy-OPNBz

103 g (0.3 mol) of Z-GIy-OPNBz are in 600 ml dissolved in warm glacial acetic acid and at 10 to 15 ° with 215 ml 4.2 N hydrogen bromide in glacial acetic acid (0.9 mol) was added. This separates when standing at room temperature crystalline hydrobromide of H-GIy-OPNBz from; after 2 hours it is filtered off and the crystals are washed with glacial acetic acid and alcohol; Yield 80.5g (92%), mp 197-200 °. The product is recrystallized from methanol: mp 202 ° to 205 °.

A solution of 43.1 g (0.1 mol) of Z-Ser-Gly-OPNBz in 100 ml of ethyl acetate is mixed with 200 ml of a 2 η solution of hydrogen bromide freshly prepared at 0 ° added to ethyl acetate. After about 10 minutes, crystals begin to separate The solvent is decanted off for 2 hours, the crystalline material is washed with ethyl acetate and filtered and dried in vacuo. The crude hydrobromide, 30.0 g (79%), melts at about 140 ° and is for the subsequent conversion into the free dipeptide ester enough pure.

Analytically pure material with a melting point of 148 ° to 150 ° is obtained by recrystallization from alcohol; [<x]% = + 12 ° (c = 2.02 in water).

The reaction can also be carried out with hydrogen bromide in dioxane, but this crystallizes in the process Hydrobromide does not come from the reaction solution and is accordingly more difficult to isolate; with hydrogen bromide in glacial acetic acid or nitromethane only oily material is produced.

To convert into the free ester, 29.0 g (0.077 mol) of the hydrobromide are suspended in 450 ml of dry chloroform and 40 ml of a 2.5 η solution of ammonia in methanol (about 1.3 equivalent) are added at 0 °. When the mixture is stirred at 0 °, the starting material quickly goes into solution with simultaneous precipitation of ammonium bromide. After 15 minutes, the mixture is filtered, the solvent is distilled off in vacuo at room temperature and the crystalline residue is washed with cold ethyl acetate and ether. The crude ester, 16.9 g (74 ⁰ I ₀ ), mp about 85 °, can be used directly for further reactions.

Recrystallization twice from acetonitrile gives the analytically pure dipeptide ester with a melting point of 94 ° to 96 °.

5. Z - (/? - NH ₂ ) -Asp-Ser-Gly-OPNBz * ⁵

26.6 g (0.1 mol) of Z - (^ - NH ₂ ) -Asp-0 H and 19.3 g (0.065 mol) of freshly prepared H-Ser-Gly-0 PN Bz are dissolved in 100 ml of dimethylformamide and with stirring at -10 ° with a precooled solution of 20.6 g (0.1 mol) of dicyclohexylcarbodiimide in 200 ml of acetonitrile. The reaction mixture is admixed with a further 300 ml of cold acetonitrile over the course of 10 minutes and stirred for 1 hour at -10 ° and overnight at 0 °. After adding 0.5 ml of glacial acetic acid, the precipitated mixture of amorphous reaction product and dicyclohexylurea is filtered off, washed with dimethylformamide-acetonitrile 1: 5, acetonitrile and ether and dried in vacuo. To separate any starting material present, the finely powdered mixture is stirred with 1N hydrochloric acid and then with cold 1N sodium bicarbonate, filtered and washed with water and cold alcohol. The dry material is stirred with 100 ml of dimethylformamide, the insoluble dicyclohexylurea is filtered off, washed with dimethylformamide and the filtrate is concentrated to about 50 ml in vacuo. When 150 ml of hot methanol are added, the substance crystallizes in the form of fine needles, mp 210 to 212 °, [*]% = -12.2 ° (c = 2.38 in glacial acetic acid); Yield 15.8g (45%).

6. Z - (, 9-NH ₂ ) - Asp-Ser-Gly-OH

A suspension of 8.2 g (15 mmol) of Z - (^ - NH ₂ ) -Asp-Ser-Gly-OPNBz in 75 ml of dioxane is mixed with 36 ml of 0.5 η sodium hydroxide solution (ph Π to 11.2). After a further 15 minutes, the clear solution is adjusted to pH 7 with 1 η hydrochloric acid and concentrated to about 10 ml in vacuo. The precipitated crystalline material is dissolved by adding 20 ml of water and the solution is extracted twice with ethyl acetate to remove p-nitrobenzyl alcohol and acidified at 40 ° with 15 ml of 2η hydrochloric acid. With rapid cooling, the reaction product separates out in the form of matted needles, which are filtered off after 1 hour at 0 ° and washed neutral with cold water (5.0 g, mp 176 ° to 178 °). After recrystallization from water: 4.1 g (67%), F. 185 to 186 °, [x]% ° = -8.4 ° (c = 1.90 in glacial acetic acid).

7. Z-Pro-Tyr-OCH ₃

A solution of 100 g (0.4 mol) of Z-Pro-OH and 78 g of H-Tyr-OCH ₃ in 1000 ml of methylene chloride is added at 0 ° with 86.5 g (0.42 mol) of dicyclohexylcarbodiimide and overnight at 0 ° left. After adding 3 ml of glacial acetic acid, the precipitated dicyclohexylurea is filtered off and the filtrate is washed, while cooling, with 1 η hydrochloric acid, 1 n sodium bicarbonate solution and water, dried and evaporated. When ethyl acetate is added, the residue gives 160 g (91%) of crystalline material with a melting point of 72 to 74 °. After recrystallizing twice from acetone-ether, the melting point of the analytically pure material is unchanged, [λ] % = -24 ° (c = 4.08 in alcohol).

8. Z-Pro-Tyr-OH

149 g (0.35MoI) of Z-Pro-Tyr-OCH ₃ are dissolved in 750 ml of methanol, cooled to 0 °, and 900 ml of pre-cooled 1η sodium hydroxide solution are added. The solution is left to stand for 2 hours at 0 °, adjusted to pH 7 with 2N hydrochloric acid, freed from methanol in vacuo and acidified at 0 ° with 2N hydrochloric acid. The precipitated oily material is taken up in ethyl acetate, extracted with 1N sodium bicarbonate solution, precipitated from the alkaline aqueous solution as an oil by adding 2η hydrochloric acid at 0 ° and again taken up in ethyl acetate. The dried ethyl acetate solution, after evaporation in vacuo, gives 135 g (94%) of the substance as a colorless foam, which can be used directly for the further reactions.

9. Z-Pro-Tyr- (N ^e -Tos) -Lys-OCH ₃

A solution of 135 g (0.33 mol) Z-Pro-Tyr-OH and 109 g (0.35 mol) (N ⁶ -Tos) -Lys-OCH ₃ in 1000 ml acetonitrile is at 0 ° with 72 g ( 0.35 mol) of dicyclohexylcarbodiimide are added, the mixture is left to stand overnight at 0 ° and, after addition of 2.5 ml of glacial acetic acid, the dicyclohexylurea which has separated out is filtered off. The filtrate is evaporated in vacuo, the residue is taken up in 1000 ml of ethyl acetate, the solution is washed while cooling with 1N hydrochloric acid, 1 n sodium bicarbonate solution and water and concentrated in vacuo to about 500 ml. When standing at 0 °, the reaction product slowly separates out in amorphous form (163 g, melting point 131 to 133 °). After recrystallization from methanol, 139.5 g (60%) of finely crystalline material with a melting point of 134 ° to 136 ° are obtained, [λ] | ⁸ = -47 ° (c = 4.01 in alcohol).

Acetylation with acetic anhydride in pyridine gives Z-Pro- (OC OCH ₃ ) -Tyr- (N ^e -Tos) -Lys-O CH _{3 with a} melting point of 109 to 111 ° (from ethyl acetate), [x] * _D ° = - 42.3 ° (c = 1.80 in ethanol).

10. Z-Pro-Tyr- (N ^e -Tos) -Lys-OH

7.1 g (10 mmol) of Z-Pro-Tyr- (N ⁶ -Tos) -Lys-OCH ₃ are dissolved in 30 ml of methanol, mixed with 30 ml of 1N sodium hydroxide solution and left to stand for 2 hours at room temperature. The reaction solution is worked up in the manner indicated for Z-Pro-Tyr and results after the crude product has been triturated

209 557/424

with ether 6.9 g (98%) substance as a colorless, amorphous powder, [*] _D = -33.2 ° (c = 4.76 in alcohol).

11. H-Pro-Tyr- (N ⁶ -Tos) -Lys-OCH ₃

50 g (0.07MoI) of Z-Pro-Tyr- (N ^e -Tos) -Lys-OCH ₃ are dissolved in 200 ml of methanol, 1 equivalent of a solution of hydrogen chloride in methanol is added and in the presence of 1.5 g of palladium carbon (10%) hydrogenated at room temperature and normal pressure, the carbon dioxide formed being absorbed in sodium hydroxide solution. After the calculated amount of hydrogen has been absorbed, the hydrogenation comes to a standstill. The solution filtered off from the catalyst is evaporated in vacuo and the partially crystalline residue is triturated with ethyl acetate. Twice recrystallization from methanol gives the analytically pure tripeptide ester hydrochloride with a melting point of 178 to 180 ^c (with rapid heating) or 198 to 200 ° (with slow heating), [x] d = -19.4 ^: (c = 2.16 in water ); Yield 37.0g (87%).

To convert into the free ester, 33.5 g (0.055 mol) of the hydrochloride are suspended in 350 ml of ethyl acetate, 30 ml of a 2.5 η solution of ammonia in methanol are added and the mixture is stirred at room temperature for 30 minutes, the starting material being completely dissolved in Solution works. The precipitated ammonium bromide is filtered off and the filtrate is evaporated in vacuo and the residue is taken up in 100 ml of ethyl acetate. When standing overnight at 0 ^; the tripeptide ester separates out in crystalline form: 31.3g (99%). F. 82 to 84 °. After two recrystallization of a sample F. 85 to 87 "; [rv] f? = -15.8 ^C (c = 5.44 in alcohol).

Both the hydrochloride and the free ester are uniform according to paper chromatography.

12. BOC-Pro-Tyr- (N ^e -Tos) -Lys-OH

A solution of 31.3 g (54 mmol) of freshly prepared H-Pro-Tyr- (N'-Tos) -Lys-OCH ₃ in 60 ml of dry pyridine is mixed with 13.1 g (55 mmol) of tert-butyl-p Nitrophenyl carbonate is added and the reaction solution is left to stand overnight at 20 ". The reaction solution is concentrated to a small volume in vacuo, ethyl acetate is added and, while cooling with ice, it is washed three times with 2N hydrochloric acid, twice with 1N sodium bicarbonate solution and with water, dried and in vacuo After repeated trituration with ether, the residue yields 35.5 g (98%) of a pale yellowish foam. The substance is found by paper chromatography (after hydrolysis of the tert-butyloxycarbonyl and ester groups with concentrated hydrochloric acid, 60 minutes at 40 ° ) and with the countercurrent distribution according to Craig (system methanol — water — chloroform — carbon tetrachloride 8: 2: 5: 5) as uniform.

13, H-Pro-Tyr- (N ^e -Tos) -Lys-OH

a) A solution of 6.11 g (10 mmol) of H-Pro-Tyr- (N ⁶ -Tos) -Lys-OCH ₃ , HCl in 40 ml of ^{1N sodium hydroxide solution is left to stand for 1 hour at O 3} and then with 1 η-hydrochloric acid set to pH 5. The precipitated material is filtered off and washed with water and alcohol: 4.67 g (83%), mp 222-224 °. For further purification, the substance is dissolved in 20 ml of hot dimethylformamide, the little insoluble material is filtered off and 50 ml of hot methanol are added, the N ^s -tosyl tripeptide being deposited in the form of fine needles with a melting point of 224 to 226 °: 4 , 40 g (79o / o); [« ^{Ss 7} = -16.6 ° (c = 3.99 in dimethylformamide), -36.4 ° (c = 2.36 in 0.5 N potassium bicarbonate solution); uniform in terms of paper chromatography.

b) 3.47 g (5 mmol) of Z-Pro-Tyr- (N ^e -Tos) -Lys-OH are dissolved in 35 ml of methanol and hydrogenated (carbon dioxide in Caustic soda adsorbed). After the calculated amount of hydrogen has been taken up, the precipitated reaction product is dissolved by adding 20 ml of hot water, the catalyst is filtered off while hot, the filtrate is evaporated in vacuo and the crystalline residue is washed with alcohol: 1.90 g (68%), mp 219 bis 222 °. After recrystallization from dimethylformamide — methanol, identical to the material prepared according to a).

14. BO C-Pro-Tyr- (N ^f -Tos) -Lys-OH

34.0 g (50mMoI) BO C-Pro-Tyr- (N ^E -Tos) -Lys-OCH ₃ are dissolved in a mixture of 50 ml of methanol and 150 ml of ^{1N sodium hydroxide solution, cooled to 10 =.} The reaction mixture is left to stand for 1 hour at room temperature, adjusted to pH 7 with 2N hydrochloric acid, freed from methanol in vacuo, covered with ethyl acetate and acidified at 0 ° with 2N hydrochloric acid while shaking. The separated ethyl acetate phase is washed with water and extracted three times with 0.5 N potassium bicarbonate solution. After the combined aqueous extracts are covered with ethyl acetate, the mixture is again weakly acidified at 0 ° with 2η hydrochloric acid (Congo), and the ethyl acetate solution is washed with water (until neutral), dried and evaporated in vacuo. The foam obtained as residue gives, after trituration with ether, 32.2 g (96%) of a colorless powder which is found to be uniform by paper chromatography (after hydrolysis of the protective groups).

For analysis, a sample is dissolved in a little ethyl acetate at 30 °, cooled to 0 °, and the oil which has separated out is passed through Decant from the supernatant solution separately and triturated with ether. Titration with 0.1 N sodium hydroxide solution in methanol gives the expected equivalent weight of 660.

15. BO C-Pro-Tyr- (N ^e -Tos) -Lys-Met-O CH ₃

A solution of 33.0 g (50 mmol) of BOC-Pro-Tyr- (N ^e -Tos) -Lys-OH and 9.0 g (55 mmol) of freshly prepared H-Met-OCH-j in 250 ml of acetonitrile is made up - 10 ^c cooled and mixed with 11.4 g (55 mmol) of dicyclohexylcarbodiimide. After 15 hours at -5 ° and 24 hours at 0 °, 1 ml of glacial acetic acid is added, the precipitated dicyclohexylurea is filtered off and the filtrate is evaporated in vacuo. The residue is taken up in ethyl acetate, washed with 1N hydrochloric acid, 0.5N potassium bicarbonate solution and water while cooling with ice, dried and evaporated in vacuo and the material obtained as an oil is triturated with ether. The yellowish, amorphous powder (38.3 g, 95%) shows in the paper chromatographic test (after hydrolysis of the protective groups) about 10% impurities, which are caused by countercurrent distribution according to Craig (system methanol-water-chloroform-carbon tetrachloride 12: 3: 4 : 8) do not disconnect.

In the same way, when reacting with H-Met-OPNBz [made from Z-Met-OPNBz, mp 62 to 64 °, [<x] _D = -15 ° (c = 2.02 in acetone) by decarbobenzoxylation with hydrogen bromide in ethyl acetate and conversion of the H-Met-OPNBz,

HBr, m.p. 149 to 151 °, [x] f = +4 ^C (c = 1.97 in methanol) in the free ester] BOC-Pro-Tyr- (N ⁶ -Tos) -Lys-Met-OPNBz in 96 ° / _o yield as a yellowish foam, which has a corresponding to the above-described Methylester purity.

16. BO C-Pro-Tyr- (N ^e -Tos) -Lys-Met-OH

20.1 g (25 mmol) of BOC-Pro-Tyr- (N ⁶ -Tos) -Lys-Met-OCH ₃ are dissolved in 40 ml of methanol, 75 ml of 1 η sodium hydroxide solution are added at about 10 ° and the mixture is 45 minutes at room temperature ditched. After working up in the manner indicated for BO C-Pro-Tyr- (N ^€ -Tos) -Lys-OH, the crude product obtained as a foam, when triturated with ether, gives 15.4 g (78%) of a pale yellowish powder, which is further processed directly will. Equivalent weight 824 (calculated 791).

The same product is formed when the corresponding p-nitrobenzyl ester is saponified (yield 83%).

17. H-Pro-Tyr- (N ^f -Tos) -Lys-Met-OH

15.0 g (19 mmol) of BOC-Pro-Tyr- (N ⁶ -Tos) -Lys-Met-O H are dissolved in 75 ml of warm ethyl acetate, cooled to room temperature and shaken in 300 ml of a 2.5N solution of hydrochloric acid poured into ethyl acetate. After 1 minute, the separation of an initially oily, then crystallizing material begins, which is filtered off after 30 minutes and washed with ethyl acetate and ether. The somewhat hygroscopic hydrochloride (12.6 g) is dissolved in 50 ml of water to convert it into the free tetrapeptide derivative and adjusted to pH 6 with 1N ammonium hydroxide. The excreted oily material solidifies when rubbed at 0 ° and is separated off by centrifugation. After washing several times with water, the moist, amorphous product is triturated with 50 ml of hot alcohol and the finely crystalline material is filtered off after 2 hours and washed with alcohol. 8.9 g (75%), m.p. 208 to 21Γ. For further purification, the substance is dissolved in 150 ml of hot dimethylformamide and mixed with 300 ml of methanol; the tetrapeptide derivative quickly separates out in the form of fine needles with a temperature of 229 ° to 231 °, yield after isolation of a second fraction from the mother liquor: 8.65 g (66%); [*] « = -42.0 ° ± 0.2 ° (c = 2.02 in 0.5N potassium bicarbonate). The purified substance shows the expected analytical values, but contains about 5% of a by-product that can be detected by paper chromatography.

The same substance is obtained by treating BOC-Pro-Tyr-iN ^ -Tos ^ Lys-Met-OPNBz with hydrochloric acid in ethyl acetate (yield 71%) and saponification of the H-Pro-Tyr- (N ^S -Tos) - Lys-Met-OPNBz, HCl (yield 60%).

18. Z-Q3-N H ₂₎ -Asp-Ser-Gly-Pro-Tyr (N ^f -Tos) -Lys-Met-OH

A solution of 1.03 g (2.5 mmol) of Z-QO-NH ₂ ) -Asp-Ser-Gly-OH (see section 6) and 0.38 ml (0.28 mmol) of triethylamine in 5 ml of dimethylformamide is used cooled to 10 ° with exclusion of moisture and, while stirring, a solution of 0.35 g (2.5 mmol) of isobutyl chlorocarbonate in 2 ml of tetrahydrofuran was added dropwise over the course of 5 minutes. The solution is stirred for a further 10 minutes at -10 ° and 20 minutes at 20 °, cooled to 0 ° and a solution of 1.73 g (2.5 mmol) of Pro-Tyr ~ (N ^e -Tos) is added dropwise over the course of 10 minutes. -Lys-Met-O dog 0.41 ml (3 mmol) of triethylamine in 15 ml of dimethylformamide (solution prepared by heating in a closed vessel to about 120 ° and rapid cooling) was added. The reaction mixture is left to stand for 1 hour at 0 ° and overnight at room temperature, then concentrated to about 4 ml at 0.1 mm and treated with 20 ml of 1η-acetic acid. The precipitated oily material is washed several times with 1 η-acetic acid and water

ίο washed, whereby it partially solidifies, and dried in vacuo at room temperature. Twice recrystallization from alcohol gives 1.25 g (46%) of amorphous substance with a melting point of 167 to 172 °, which contains about 10% of an impurity that can be detected by paper chromatography. For further purification, the product is distributed in a Craig apparatus in the system methanol — water — chloroform — carbon tetrachloride (3: 1: 3: 1) over 60 steps with 10 ml lower and upper phase each (maximum at G = 0.63 ). After recrystallization from methanol, the combined top fractions yield 0.54 g of substance vom F which is uniform according to paper chromatography. 175 to 177 ³ (light sintering at 171 °); [<%] | ⁶ = -30.1 ° (c = 2.06 in dimethylformamide).

In the same way, when Z-Q9-NH ₂ ) -Asp-Ser-Gly-OH is reacted with H-Pro-Tyr- (N> Tos) -Lys-OH (see item 13), the Z-QS-N is formed H ₂ ) -Asp-Ser-Gly-Pro-Tyr- (N ^f -Tos) -Lys-OH, which, after two recrystallization from alcohol, gives pure substance with a temperature of 138 ° to 143 ° according to paper chromatography (slight sintering at 133 °); [x] d = -31.1 ° (c = 2.16 in dimethylformamide). Hydrogenolytic decarbobenzoxylation yields H-Q3-N H ₂ ) -Asp-Ser-Gly-Pro-Tyr- (N-Tos) -Lys-OH. After recrystallization from dimethylformamide-methanol, mp 192 ° to 196 ° (brown color from 180 °).

19. (N «-Z) - (N" -Tos) -Lys-QS-O CH ₃ ) -Asp-0 CH ₃

45.0 g (0.28 mol) of H-Q3-OCH ₃ ) -Asp-OCH ₃ and 126 g (0.29 mol) of (N "-Z) - (N ^E -Tos) -Lys-O H dissolved in 1.5 l of acetonitrile. 60.0 g (0.29 mol) of dicyclohexylcarbodiimide are added to the solution, which has been cooled to -15 ° and then left at -15 ° for 30 minutes and at 3 ° for 48 hours of the dipeptide dimethyl ester with dicyclohexylurea) is suction filtered (203.5 g, further processing see below). The filtrate is mixed with 2 ml of glacial acetic acid and left at 3 ° for a further 2 hours, then evaporated and the residue taken up in ethyl acetate. After washing with dilute hydrochloric acid , Water, sodium bicarbonate solution and water, drying and evaporation give 35.0 g of residue.Crystallization from ethyl acetate gives 11.8 g of the above compound with a temperature of (130 °) 137 to 140 ° ^{By-product obtained with a} temperature of 122 to 124 °; it is l- (N a -carbobenzyloxy-Nf-tosyl-L-lysyl) -1,3-di cyclohexylurea.

The mixture of the above dipeptide ester and dicyclohexylurea (203.5 g) which has separated out from the reaction solution is triturated with cold chloroform and the undissolved urea is sucked off (57.5 g, melting point 222 to 225 °). The filtrate is evaporated and the residue is crystallized from methanol, whereby 136.8 g of (N ^a -Z) - (N ⁱ -Tos) -Lys - (^ - OCH ₃ ) -Asp-OCH ₃ are obtained, F. (140) 144 to 146 °. Total yield: 148.6 g (92% of theory).

15 16

Recrystallization from ethyl acetate, then methanol. A total of 42.0 g (83%) of crystalline Trier yields analytically pure product with a melting point of 144 ° to 146 °, a peptide derivative. Mn = + 27 ° ± 0.6 ° (c = 1.11 in chloroform). ^ _z .p _ro _ _(Ne _ _Tos) _ _Lys _ ₍ ^ _N H ₂ ) _Asp-NH ₂

20. (N ⁶ -Tos) -Lys - (^ - OCH ₃ ) -Asp-OCH ₃ 5 The tripeptide derivative of number 21 becomes with a lot

excess ammonia in methanol for 48 hours

Leave 50.0 g (0.087 mol) (N ^a -Z) - (N ⁶ -Tos) -Lys- at room temperature. Evaporation and (/? - OCH ₃ ) -Asp-OCH ₃ are dissolved with moisture crystallization from methanol in 85% yield exclusion in 200 ml of absolute glacial acetic acid, with Z- ^{Pro- (N e} -Tos) -Lys- ( / SN H ₂ ) -ASp-NH ₂ , m.p. 196 to 90 ml of 4,2N hydrogen bromide solution in glacial acetic acid, added at io 202 °, [x] _D = -20.8 ° ± 0.7 ° (c = 1.30 in dimethyl and at room temperature until the formamide ceases). Leave evolution of gas (2V ₂ hours). The reaction solution is then evaporated to dryness at 0.1 Torr and 35 ° Bad- ²³ · H-Pro- (N <-Tos) -Lys- (£ -O CHg) -ASp-OCH ₃ temperature and the return 2, 00 g (2.97 mmol) of Z-Pro- (N ⁶ -Tos) -Lys - (/ 3-O CH ₃ ) stand while cooling with ice with ethyl acetate for so long as 15 Asp-OCH ₃ are dissolved in 25 ml of methanol , Rub 2 ml until it crystallizes through. 44.37 g of 2N hydrochloric acid solution in methanol are added and the (97% of theory) crude crystalline (N ⁶ -Tos) -Lys solution in the presence of 100 mg palladium-carbon (/ 3-OCHa) -ASp-OCH ₃ , HBr obtained from m.p. (140 °) 142 to (10% Pd) at room temperature and normal pressure hy-146 °. The product is turned over for the rest of the world. (The carbon dioxide evolved becomes sufficiently pure at the same time _{: <} Two recrystallization is absorbed in sodium hydroxide solution.) The hydrogenation comes from methanol - ether gives analytically pure uptake of slightly less than the calculated material with a temperature of 144 to 146 °; [x] _D = + 7.0 ° ± 0.3 ° Amount of hydrogen after 5 hours of standstill. (c = 2.14 in methanol). Filter off the catalyst and evaporate

44.0 g (0.084 mol) (N ⁶ -Tos) -Lys - (/ J-OCH ₃ ) -Asp- the hydrochloride of the above tripeptide dimethyl ester OCH ₃ , HBr are dissolved in 120 ml of water, the 25 (1.66 g ) as a colorless resin. For conversion into the solution, filtered clear, mixed with a lot of chloroform free base, the hydrochloride is dissolved in a little water and while cooling with ice with cold, concentrated solution, the aqueous, clear solution is made strongly alkaline with a lot of ethyl acetate potassium carbonate solution. The free dipeptide ester, which is overlaid and excreted as an oil with cooling to about 5 °, is taken up by adding cold, concentrated Potash solution of chloroform, and the chloroform solution is made strongly alkaline. The reaction product separates twice with a little cold sodium sulphate solution in the process in oily form and dissolves during washing, drying and evaporating. This is done by shaking in ethyl acetate. The ethyl acetate solution is 33.0 g (89%) (N ⁶ -Tos) -Lys - (/ 3-OCH ₃ ) -Asp-OCH ₃ washed with cold sodium sulfate solution, dried as a viscous, clear oil and immediately evaporated as below and below . The described as a colorless resin obtained, processed further. 35 H- ^{Pro- (N E} -Tos) -Lys - (/ 5-OCH ₃ ) -Asp-OCH ₃ (l, 27g)

will be implemented immediately.

21. Z-Pro-eN-Tos) -Lys - (/ 3-OCH ₃ ) -Asp-0 CH ₃

24. H-Pro- (N * -Tos) -Lys - (/ 3-NH ₂ ) -Asp-NH ₂

In a solution of 33, Og

(0.075 mol) (N ^f -Tos) -Lys - (/? - OCH ₃ ) -Asp-OCH ₃ 40 The Z- ^{Pro- (N E} -Tos) - described in section 22 and 20.4 g (0.082 mol ) Z-Pro-0 H in 500 ml Aceto- Lys - (/ 3-NH ₂ ) -Asp-NH ₂ gives under the same nitrile 17.0 g (0.082MoI) dicyclohexylcarbo- hydrogenation conditions as stated above, H- Given prodiimide. After 30 minutes at -15 ° and (N ^e -Tos) -Lys - (/? - NH ₂ ) -Asp-NH ₂ , NCl, F. 236 to 20 hours at 3 °, the dicyclohexylurea is from-238 ° (from Water — acetone), [«) b = -37.9 ° ± 1.2 ° sucked (16.4 g, mp 223 to 226 °) and the filtrate after 45 (c = 1.08 in water). The addition of 2 ml of glacial acetic acid is prepared from the hydrochloride and left for a further 2 hours at 3 ° free base by partitioning between n-butanol. 0.98 g of dicyclo and 2N soda solution separate. The butanol solution emits hexylurea. The filtrate is evaporated, evaporation and trituration of the residue with the residue dissolved in ethyl acetate and the solution with ether H- ^{Pro- (N e} -Tos) -Lys - (/ 3-NH ₂ ) -Asp-NH ₂ as dilute hydrochloric acid, Water, sodium bicarbonate 50 colorless powder with a melting point of 117 to 135 °, [x] d = -40.4 ° solution and washed with water, dried and at ± 0.6 ° (c = 1.09 in water), evaporated . The residue (50.0 g) is several times with
much ether rubbed. (Processing of the ether-soluble 25.
Proportions [8.0 g resin] see below.) The ether-insoluble
Material (41.7 g) gives after prolonged standing in ^{55.0 g of Z-Pro- (N e} -Tos) -Lys- ( N ⁶ -Tos) -Lys - (/? - OCH ₃ ) -Asp-OCH ₃ and 11.35 g (^ -OCH ₃ ) -Asp-OCH ₃ in needle form, F. (96 °) 110 to (0, 0455 mol) Z-Pro-OH in 300 ml acetonitrile at 113 °. When recrystallizing from acetone-ether and -15 °, 9.40 g of dicyclohexylcarbodiimide (0.0455 methanol gives an analytically pure product by the mole). The reaction solution is then left at temperature 126 to 129 °, [x) _D = -22.6 ° ± 0.8 ° (c = 1.02 in 60 1 hour at -15 ° and 24 hours at 3 °. Chloroform) . After filtering off the dicyclohexylurea (7.77 g,

The mother liquors of the crude crystals (35.0 g) 90% of theory) the filtrate with 1 ml of glacial acetic acid are combined with the above (8.0 g) ether-soluble and left at 3 ° for 1 hour. Divorce in the process Material, a total of 13.2 g and 50 times its weight - a further 100 mg of urea. The filtrate will amount of silica gel chromatographed. The evaporated with chloro-65, the residue taken up in ethyl acetate — methanol Eluted fractions give men and washed neutral as usual. The vinegar 7.0 g of crystalline tripeptide ester from F. about 120 to ester solution gives 28.44 g of residue after evaporation 126 °. (Resin). The raw product is made several times with a lot

17 18

Ether grinded and the ether-insoluble part used to prepare the free pentapeptide ester

(24.29 g) subjected to countercurrent distribution. 5.00 g (6.58 mmol) of the above Hydrochloride under

The 78-stage distribution between 80% methanol and ice cooling between 500 ml of chloroform and 50 ml

Chloroform - carbon tetrachloride = 1: 1 results in 2N soda solution distributed. The reaction product dissolves

17.0 g of pure tetrapeptide derivative (distribution number G 5 in chloroform. The chloroform extract is

= 0.351), as a colorless resin; [<%] d = - 70.6 ° ± 1 ° washed twice with cold sodium sulfate solution,

(c = 2.09 in methanol). the soda and sodium sulfate solution with others

xj ρ ρ 200 ml of chloroform rewashed; the United

_aii _ _{Λ T} ²⁶ γ- * 7 ^ nu \ ° ~ _nru chloroform solutions, after drying and

(N * -Tos) -Lys- (£ -OCH ₃ ) -Asp-OCH _{3 10 steam 4j00g (84} o _{/ o)} H-Ser-Pro-Pro- (N'-Tos) -

10.00 g (12.95 mmol) Z-Pro- ^{Pro- (N E} -Tos) -Lys- Lys - (^ - OCH ₃ ) -Asp-OCH ₃ as a colorless resin, which ((S-OCH ₃ ) -Asp-OCH ₃ can be further reacted directly in 200 ml of methanol,
dissolved, 10 ml of 2 normal methanolic hydrochloric acid were added and the solution was added in the presence of 500 mg of 29. Z- (y-NH ₂ ) -Glu-His-Phe-Arg-Try-Gly-Ser-palladium-carbon (10% Pd) Room temperature 15 Pro-Pro-iN ^ -Tos ^ Lys-dS-OCH ^ -Asp-OCHg, HCl and normal pressure hydrogenated (using a

second hydrogenation vessel filled with sodium hydroxide solution (1.36 g (1.4 mmol) of Z- (γ-NH ₂ ) -Glu-His-Phe-Arg for absorbing the carbon dioxide evolved). Try-Gly-OH (cf. German patent specification 1 108 232) After the calculated amount of hydrogen has been absorbed, the solution is filtered off from the catalyst in 16 ml of freshly distilled dimethyl form and one amide is dissolved in the heat and re-evaporated to room. The hydrochloride is cooled to the above temperature, and 1.6 g (2.1 mmol) of H-Ser tetrapeptide ester are obtained as a colorless resin. Pro- ^{Pro- (N e} -Tos) -Lys - (/? - OCH ₃ ) -Asp-OCH ₃ , HCl as described in section 23, is added to the free H-Pro-Pro- and then at room temperature ge (N ^e -Tos) -Lys - (/? - OCH ₃ ) -Asp-OCH _{3 transferred} . stirs. After stirring for 2 hours, there is a clear yield of 6.86 g (83%). This is a colorless resin solution. 590 mg (2.9 mmol) of dicyclohexyl-containing product are added and the reaction is continued directly. carbodiimide in 3.5 ml of dimethylformamide and

lets react for 3 days at room temperature.

27. Z-Ser-Pro-Pro- The dicyclohexylurea freed solution

(N ^E -Tos) -Lys - (/ S-OCH ₃ ) -Asp-OCH _{3 are} mixed with 500 ml of ethyl acetate, the filtered off

30 precipitated material through a fine glass frit and

6.86 g (10.8 mmol) of H-Pro- ^{Pro- (N e} -Tos) -Lys- are obtained after drying in a high vacuum at 40 °

(ß-OCH ₃ ) -Asp-OCH ₃ are dissolved in 150 ml of 2.3 g of crude product, which is still with starting material

Acetonitrile and 2.61g (12, OmMoI) Z-Ser-OH is too contaminated.

given. The solution is cooled to -15 °, with For cleaning, the 2.3 g of a multiplier

2.47 g of dicyclohexylcarbodiimide are added and then 35 tive distribution over 60 stages between the phases

Leave for 1 hour at -15 ° and 60 hours at 3 °. n-Butanol - 1% acetic acid = 1: 1, subjected (G.

Then the deposited dicyclohexyl- = 0.8).

urea sucked off (2.33 g corresponds to 91% of the The main amount of the protected paper chromatography

Theory) and the filtrate evaporated. The residue graphically pure carbobenzoxyundekapeptide-dimethyl-

is dissolved in ethyl acetate and, as usual, neutral 40 ester hydrochloride is found in the elements

washed. The ethyl acetate solution gives 9.06 g of back 15 to 30. These fractions are combined for

was standing; this is evaporated to dryness with a lot of petroleum ether, then ether, and twice more from a little

grated. The ether-insoluble crude product (8.35 g) dimethylformamide precipitated with a lot of ethyl acetate,

is used to purify a countercurrent distribution. The yield is 1.34 g (= 60% of theory)

threw. The 48-stage distribution between 80% 45 amorphous, white powder. [a]% ⁵ = -53.8 ° ± 1.8 ° (c

Methanol and chloroform — carbon tetrachloride = 0.9475 in dimethylformamide).
= 1: 1 gives 84% pure pentapeptide ester as colorless

Resin ^ (distribution number G - 0.507). After rubbing in the three systems

with ether, the analytically pure product shows tert-amyl alcohol — isopropanol — water

Melting point from about 70 to 105 °, [<x] _D = -92.0 ° 50 = 100: 40: 55,

± 0.6 ° (c = 2.16 in methanol). sec-butanol — isopropanol — monochloroacetic acid

28 H Ser Pro Pro acid water = 70: 10: 3 g: 40,

,. _{I (T} w ^ TSTT nru _sec. Butanol — Isopropanol — 5% Veronal Na—

_(N * _Tos) -Lys - (/? - OCH3) -Asp-OCH _{3 water = m} T ₁₅ ¹ T ₁₀ . ₆₀

8.00 g (9.33 mmol) Z-Ser-Pro- ^{Pro- (N f} -Tos) - 55

Lys - ((S-OCH ₃ ) -Asp-OCH ₃ are dissolved in 200 ml, the protected ester shows the following R _f values (DurchMethanol, 4 ml 3.3 normal methanolic hydrochloric acid average values): 0.55 and 0.62 or 0.67.
added and the solution in the presence of 500 mg The quantitative amino acid determination according to palladium-carbon (10% Pd) at room temperature Stein and Moore on an Amberlite XE-69 column and normal pressure (absorption of carbon dioxide in 60 (trade name) gives the expected amino Lye) hydrogenated. The hydrogenation is after 4V ₂ hour acid ratio,
the and uptake of slightly more than the calculated amount of hydrogen ended. Filtering off the 30. H-iy-NH ^ -Glu-His-Phe-Arg-Try-Gly-Ser catalyst and evaporation gives the hydrochloride Pro- ^{Pro- (N 6} -Tos) -Lys - ((8-OCH ₃ ) -Asp-OCH ₃ ,
of the above pentapeptide ester as a colorless resin. More- 65 3 HCl
there are repeated precipitation from acetone solution with ether

6.53 gamorphic powder, m.p. HO to 150 °; [<x] b - -86.7 ° 1.25 g (0.72 mmol) carbobenzoxyundekapeptide di-

± 0.7 ° (c = 2.41 in methanol). methyl ester hydrochloride in 70 ml of methanol,

Claims (5)

19 20 which contain 2.2 equivalents of hydrochloric acid gas, which is to be expected in a counter-paper chromatographic separation of 200 mg of 10% palladium-carbon catalyzing ninhydrin-positive amino acid spots, shaken with hydrogen. For absorption of the carbon dioxide formed is a second with ³² · H-OS-NH ^ -Asp-Ser-Gly-Pro-Tyr-Lys-Met- concentrated potassium hydroxide filled hydrogenation duck da- 5 (y-NH ₂ ) -Glu-His-Phe-Arg-Try-Gly-Ser-Pro- interposed. After 6 hours the water is Pro-Lys-Asp-OH, CH ₃ COOH Substance uptake ended. The methanolic solution 400 mg (0.15 mmol) protected octadecapeptide is freed from the catalyst, the solvent in the case of dimethyl ester hydrochloride in 8 ml of 75% 40 ° evaporated in vacuo and the evaporation dioxane dissolved and under nitrogen with 0.4 ml 0.46n- backlog once from abs. Methanol ether and barium hydroxide solution for 14 minutes with cinnamon pleases. offset in temperature (ph 11.4). The saponification After drying, 1.19 g of amorphous solution are obtained; 1.84 ml of VwH sulfuric acid are added to neutralize the solution. slightly pink colored trihydrochloride des undekapep- siert and the solvent at 40 ° under reduced tiddiesters. Pressure evaporated. The compound shows in the three in number 29 are 15 The evaporation residue is taken in 10 ml Chromatography systems recorded only a 50% dioxane, separated by a fine glass Pauly and ninhydrin positive stain; Remove the insoluble barium sulfate with the frit and wash the following R ₁ values (average values) 0.57 or two more times with 5 ml of 50% dioxane. To 0.68 and 0.71, respectively. evaporation in vacuo at 40 ° and drying 20 of the residue over phosphorus pentoxide in the high 31. Z ~ 03-NHo) -Asp-Ser-Gly-Pro-Tyr- (N ^c -Tos) - vacuum gives 385 mg of saponification product. Lys-Met- (y-NH ₂ ) -Glu-His-Phe-Arg-Try-Gly-Ser- Above 385 mg are in 80 ml over sodium Pro- ^{Pro- (N f} -Tos) -Lys - (^ - OCH ₃ ) -Asp-OCH ₃ , HCl dissolved in dried liquid ammonia and with Calcium at the boiling point of ammonia 210 mg (0.128 mmol) of undecapeptide dimethyl ester-25 reduced. After adding 110 mg of calcium trihydrochloride are freshly distilled in 2 ml, the solution remains dark blue for about 10 minutes. Dissolved dimethylformamide, 0.28 ml at 0 °. Then the reaction mixture is mixed with In a solution of triethylamine in dimethylformamide, much ammonium bicarbonate is added, whereupon the blue coloration sets and left at 0 ° for 2 hours. Slowly separates slowly disappears and the reaction solution becomes milky the triethylamine hydrochloride from. After about 30 remains. The ammonia is vaporized at room The salt deposition is completed in 90 minutes. Then let off the temperature and remove the last remains in the 152 mg (0.14 mmol) of Z - (/ 3-NH ₂ ) -Asp-Ser are given in high vacuum over sulfuric acid. Gly-Pro-Tyr- (N ^e -Tos) -Lys-Met-O H (see section 18) The residue is repeated with 1 n-ammonium in addition and after a further 15 minutes 0.21 ml of a bicarbonate solution (three times 10 ml) and twice with In solution of dicyclohexylcarbodiimide in dimethyl 35 5 ml of water extracted, filtered through cellite, the clear formamide. The mixture is left for 3 days at 0 ° and 1 day at aqueous solution (p _H = 8.4) with glacial acetic acid to p _H 4.2 React room temperature, then filtered and the water is removed at 40 ° in a vacuum. from the separated dicyclohexylurea and evaporates. The residue is evaporated five more times falls with 60 ml of ethyl acetate. _e j with 10 ml water and finally the sublimated After drying in a high vacuum at 40 ° 40 ammonium acetate in a high vacuum at 40 °. 290 mg of amorphous white powder are obtained. For further cleaning, the raw octadeca The Papierchromatogramm shows in addition to the peptide from sec-butanol and 0.5 ° / ₀ aqueous Z-03-NH2) -Asp-Ser-Gly-Pro-Tyr- (N ^E -Tos) -Lys- Trichloroacetic acid 1: 1 distributed over 160 levels. Met- (yN H ₂ ) -Glu-His-Phe-Arg-Try-Gly-Ser-Pro- The determination of the optimal density of each Pro- (N ^f -Tos) -LyS-OS-OCHs) -ASp-OCH ₃ , HCl ₄₅ fractions at 277m _/ tt shows the majority of the still considerable amounts of starting material. Octadecapeptide in elements 49 to 65. The For purification, 360 mg of raw material (from the distribution coefficient is 0.53. two similar approaches) on a cellulose- The phases of the distribution elements 49 to 54, column (diameter 2.5 cm, length 90 cm) with n-But-55 to 60 and 61 to 65 are combined and under anol, saturated with 1% acetic acid, chromatographed. 50 reduced pressure at 40 ° to a small volume The elution rate is 3 to 4 ml each time evaporated. To remove the trichloroacetic acid 20 minutes. Fractions 81 to 175 show Pauly - the three fractions are each passed through a column positive reaction. of 10 ml of Amberlite IR-4B (acetate form) and washes There are the fractions 82 to 84, 85 to 93, 94 to remove the peptide with 25 ml of water. The three factions 122, 123 to 132 and 133 to 175 combined. The fraction 55 gives 100 mg of octadecapeptide acetate.
functions 85 to 93 consist of a mixture of Carbobenzoxyheptapeptide in addition to carbobenzoxyocta- PATENT CLAIMS: decapeptide esters, on the other hand the fractions contain 1. Process for the production of new MSH- 94 to 122 the pure protected octadecapeptide- active octadecapeptides of the formula L-Aspara- ester, while in the remaining fractions the main- 60 ginyl-L- seryl-glycyl-L- prolyl-L-tyrosyl-l-lysyl- thing undekapeptide ester is included. l-methionyl- L- glutaminyl-l- histidyl -L- phenyl- The combined fractions 94 to 122 give alanyl-L-arginyl-L-tryptophyl-glycyl-L-seryl-L-pro- 113 mg of white, amorphous powder, which is contained in the paper lyl-L-prolyl-L-lysyl-L-aspartic acid and the chromatogram only one Pauly and one honest-speaking compound, which instead of the glutaminyl shows positive stain. 65 remainder has the remainder of the glutamic acid, and A sample of the purified, protected peptide salts thereof, characterized in that the esters, with 6N hydrochloric acid for 15 hours with heptapeptide L-asparaginyl-L-seryl-glycyl-L-prolyl- 105 ° hydrolyzed, results after electrophoretic and L-tyrosyl-L-lysyl-L-methionine with protected ami- no groups obtained by condensation of the tripeptide L-asparaginyl-L-seryl-L-glycine with protected a-amino group, with an ester of the tetrapeptide L-prolyl-L-tyrosyl-L-lysyl-L-methionine with protected ε-amino group and saponification of the ester group, or by condensation of the hexapeptide L-asparaginyl-L-seryl-glycyl-L-prolyl-L-tyrosyl-L-lysine with protected «- and ε-amino group, with an L-methionine ester and saponification the ester group, condensed with a diester of the undecapeptide L-glutaminyl or L-glutaniyl-L-histidyl-L-phenylalanyl-l-arginyl-L-tryptophyl-glycyl-L-seryl-L-prolyl-L-prolyl-L-lysyl-L-aspartic acid with protected ε-amino group, obtained by condensation of the hexapeptide L-glutaminyl- or L-glutamyl-L-histidyl-L - phenylalanyl - l - arginyl - l - tryptophyl - glycine with protected «amino group with a diester des Pentapeptide s L-Seryl-L-prolyl-L-prolyl-L-lysyl-L-aspartic acid with protected ε-amino group and splitting off of the -amino protecting group, and, if desired, the carboxyl groups of the aspartic acid residue and the amino groups of the asparaginyl residue and which sets lysyl residues free. 2. The method according to claim 1, characterized in that the linkage of the heptapeptide with the undekapeptide in the presence of a carbodiimide. 3. The method according to claim 1 and 2, characterized in that the free amino group of the asparagine residue is protected by a residue that can be split off reductively. 4. The method according to claim 1 to 3, characterized in that the ε-amino group of the Protects lysine residues through the tosyl group. 5. The method according to claim 1 to 4, characterized in that the diester of the pentapeptide with protected ε-amino group through gradual build-up starting from the carboxyl end with the aspartic acid diester and condensation of a further amino acid, manufactures. © 209 557/424 3.62