DE1518109C - Process for the preparation of the new nonapeptide derivative N-carbobenzyloxy-Lseryl-L-tyrosyl-L-seryl-L-methionyl-Lglutamyl-L-histidyl-L-phenylalanyl-larginyl-L-tryptophan hydrazide - Google Patents

Description

It is well known that the hormones made up of polypeptides play very important roles in biological ones Processes of living organisms; find such hormones isolated from natural substances also widely used in therapy. Since those that can be produced from natural substances Products available in fairly limited quantities are the various preparative ones Methods of change lately. rapidly evolving peptide chemistry to an increasing extent Total synthesis of such hormones applied. The synthetic products are also more advantageous, because, as a result of their greater purity, they have less undesirable side effects show.

In the synthetic structure of such hormonal effects The polypeptides showing are mainly those constructed from approximately 7 to 12 amino acid units Peptides of importance, which are used as building blocks for the further formation of the natural products corresponding, made up of 25 to 30 units, complicated and synthetically only very difficult to obtain Polypeptide products can be used and, if necessary, also certain ones themselves Can exert hormonal effects.

From these points of view, particularly advantageous properties come from the new one in the literature hitherto not described nonapeptide of the following schematic formula (I) to:

Z - Ser - Tyr - Ser - Met - GIu - His - Phe - Arg - Try - NHNH ₂

(D

In the above formula as well as in the rest of this description, those in peptide chemistry common abbreviations and designations used:

Ser = L-serine residue
Tyr = L-tyrosine residue
Met = L-methionine residue
GIu = L-glutamic acid residue
His = L-histidine residue
Phe = L-phenylalanine residue
Arg = L-arginine residue
Try = L-trypsin residue

Z = carbobenzyloxy group
C ₆ H ₅ CH ₂ ■ COO -

OBz = benzyloxy group
C ₆ H ₅ CH ₂ O -

OMe = methoxy group CH _; jO
OR = alkoxy group

The above compound of formula (I), the N-carbobenzyloxy -L-seryl-L-tyrosyl-L-seryl-L-methionyl - α - L - glutamyl - L - histidyl -L- phenylalanyl-L-arginyl-L-tryptophan-hydrazide, is a nonapeptide in which the order of the amino acid units is the same as with the corticotropins (ACTH polypeptides) and with the amino acids 1 to 9 of the porcine α-melanophore stimulating hormone; the sequence of amino acids 4 to 9 Each new nonapeptide is identical to that of the four amino acids 7 to 12 of the melanophore stimulating agent Bovine hormone. The novel polypeptide according to the invention also contains structural parts, those with those of the natural polypeptides with CRF effects (Corticotropine Releasing Factor) are constructed in the same way. On the basis of these properties, the new nonapeptide of the formula (1) play a key role in the total synthesis of highly biologically active polypeptides, namely the more so as the hydrazide end group of the new polypeptide is easily converted into an azide group

can be, which is then capable of further couplings, while the amino end group is already from is protected from the start against unwanted further reactions, but if desired the protective Group can be easily removed.

The new nonapeptide derivative N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-L-methionyl-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophan-hydrazide is produced according to the invention in such a way that N-carbobexizyloxy-L-seryl-L-tyrosyU L-seryl-L-methionine hydrazide in the presence of dimethylformamide as a solvent in per se known Wise converted into the corresponding azide, the reaction solution obtained without the contained therein Remove water by drying immediately with an L-glutamyl-L-histidyl-L-phenylalanine alkyl ester converts, then the N-protected heptapeptide ester obtained in per se known Way into the corresponding hydrazide and this in a known manner in the presence of dimethylformamide as a solvent into the corresponding N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-L - methionyl - L - glutamyl - L - histidyl τ L - phenylalanine - azide transfers the contained reaction solution through without the water contained in it To remove drying, immediately reacted with an L-arginyl-L-tryptophan-alkyl ester and the obtained N-carbobenzylaoxy-nonapeptide-alkyl ester converted into the corresponding hydrazide in a manner known per se.

There has been a process of making similar peptides by coupling one in dimethylformamide produced azide in the same reaction mixture with an active ester of

ι ο L υ α

other component described (HeIv.Chim.Acta, 41, p. 18.66 [1958]). According to this method, the coupling of an azide with an ester (which, although are different from the reaction components according to the invention, but the reaction is analogous) also carried out in dimethylformamide and without intermediate isolation of the azide, but the The water content of the azide solution was removed with anhydrous sodium sulfate before adding the base, and thus the Reaction carried out in an anhydrous medium. According to the method according to the invention, however, the in In the form of 6η-hydrochloric acid, water added to the reaction mixture is not removed and as a result the reaction is carried out in a medium consisting of aqueous dimethylformamide. This Process difference brings with it significant advantages compared to those given in the present examples high yields (66 to 76%) makes clear.

Example 1 (column 9): 73%,
Example 3 (column 9): 66%,
Example 11, a) (column 12): 76%,
Example U, c) (column »): 76%.

This superiority becomes even more apparent when one considers that the procedure according to Invention itself provides a pure product that z. B. no longer needs to be dried, so that that of drying the heat-sensitive azide

ίο otherwise unavoidable losses are avoided, whereby a higher yield and a considerable saving of material and time can also be achieved. The total synthesis of the new nonapeptide of the formula (I) protected in the Ehdgruppen takes place

'5 in the steps shown schematically below, both the production of starting materials A, B and C, which is not claimed here, as also illustrate their subsequent condensation according to the method of the invention:

Ser ■

Tyr

Ser -

Mead

GIu

His

Phe

Arg

Try

Ser - Tyr - Ser - Met - GIu - His - Phe - Arg - Try

From this schematic representation of the synthesis process, it can be seen that the principle of the structure this new compound largely differs in its structure from that known from the literature, similar Higher polypeptides containing amino acid sequences differs (see R. A. Boissonnas, St. Guttmann, Waller, P. A. Jaquenoud: Experientia, 12, p. 446 [1956]; C. H. L i, J. Meienhofer, E. S c h η a b e 1, D. Chung, T. Lo, J. Ramachandran: J. Am. Chem. Soc, 82, P. 5760 [1960]; R. Schwyzer, W. Rittel, H. Capier, B. Iselin: Angew. Chem., 72, P. 915 [1960]; K. Hofmann, M. E. Woolner, H.'Ya jim a, G. Spühler, T. A. Thompson, E.T. Schwartz: J. Am. Chem. Soc, 80, p. 6458 [1958]).

For the total synthesis according to the invention of the new nonapeptide derivative of the formula (I), therefore, the the following operations are required, with steps a) to c) the production not claimed here of the starting materials, stages d) and e) explain the process of the invention:

a) Preparation of one containing amino acids 1 to 4 and protected on the free amino group Tetrapeptide hydrazides;

45

55

b) preparation of a tripeptide ester containing amino acids 5 to 7;

c) preparation of a dipeptide ester containing amino acids 8 and 9;

d) condensation of the protected tetrapeptide hydrazide 1 to 4 with the tripeptide ester 5 to 7 and conversion of the protected heptapeptide ester 1 to 7 formed into the hydrazide;

e) Condensation of the protected heptapeptide hydrazide with the dipeptide ester 8 and 9 and conversion of the protected nonapeptide ester 1 to 9 formed into the hydrazide.

The course of these synthesis steps is illustrated schematically by the table below.

the horizontal connecting line between the specified respective end groups represents the rest that amino acid or the chain of those amino acids, which over this horizontal Connecting line crossing vertical lines

never stand.

The method of the invention begins with step from I to J, while steps A to I illustrate how the starting materials are accessible.

s t-

tn

NN N N N N

N N

N ■. N

JO

JO

JO

JO

JO

73

73

JO

JO

JO

JO

SC-

■ ac

i-t

on

rs

-Γ

—Ο

—Ο

-ο

ac

r — o

03

JC

JO

N : -Χ

ο μ

CD I.

Γ "

X C. C.

X X

X X

X N

JC

JO

ac

JO

ac

O X

3 ■ ·

■ Χ

JC

JC

X - \ -

ΐ ·

-ι-

1 οίο ιυα

The individual steps of the above synthesis sequence can be carried out in the following way (the Letters and numbers following the structural symbols of the individual peptides refer to the corresponding rows or columns of the table above):

a) Production of the protected tetrapeptide <■
hydrazide Z - Ser - Tyr - Ser - Met - NHNH ₂ (I 1-4) (starting material)

The L-serine hydrazide (D 1) protected by a carbobenzoxy group (cf. J. S. F r u t ο η, J. Biol. Chem., 146, p. 463 [1942]) is in the corresponding Azide converted, and with this azide an L-tyrosine alkyl ester (C 2) is acylated, which immediately was released from an ester salt prior to this reaction. The dipeptide ester thus obtained Z - Ser - Tyr - OR (E 1-2) is then converted into the hydrazide (F 1-2).

Similarly, the dipeptide ester becomes H - Ser - Met -. OR (G 3-4) produced by Reaction of the azide prepared from the hydrazide Z - Ser - NHNH2 (D 3) with the ester H - Met - OR (C 2) and subsequent removal of group Z.

The dipeptide derivatives Z - Ser - Tyr - NHNH ₂ (F 1-2) and H - Ser - Met - OR (G 3-4) are then condensed using the azide method, and the tetrapeptide ester obtained

Z - Ser - Tyr - Ser - Met - OR

(H 1-4) is finally converted into the corresponding hydrazide (I 1-4).

b) Preparation of the tripeptide ester
H - GIu - His - Phe - OR

The hydrazide Z - His - NHNH ₂ (D 6) is converted into the azide, with this a phenylalanine alkyl ester (C 7) is then acylated, and the protected ester Z - His - Phe - OR (E 6- 7) the acyl protecting group of the terminal amino group is removed. .

The dipeptide ester freshly released from a salt H - His - Phe - OR (G 6-7) is mixed with carbobenzyloxy-L-glutamic acid y-benzyl ester (G 5) acylated from the protected tripeptide ester obtained

Z - GIu - His - Phe - Or
L_ OBz

(H 5-7) the protecting groups Z and Bz are removed, and in this way the becomes the further Tripeptide esters suitable for condensation

H - GIu - His - Phe - OR

? n ι ι

OH

(I 5-7) won.

η-

c) Production of the dipeptide ester
-Arg —Try —OR (H 8-9) (starting material)

(I 5-7) (starting material)

N-a-carbobenzyloxy-L-arginine (B 8) is condensed with a trypsin alkyl ester H - Try - OR (C 9), from the obtained dipeptide ester

Z —Arg —Try —OR

(D 8-9) the alkyl ester group becomes alkaline Removed saponification, then the carbobenzyloxy group is split off and so the free dipeptide Obtained H - Arg - Try - OH (F 8-9). This is then converted into the corresponding alkyl ester salt and the free H - Arg - Try - OR (H 8-9) base is released.

d) Preparation of the protected heptapeptide hydrazide Z - Ser - Tyr - Ser - Met - GIu - His - Phe - NHNH ₂

L-OH
(K 1-7) (method of the invention)

The hydrazide Z - Ser - Tyr - Ser - Met --NHNH ₂ (I 1-4) obtained under a) is converted into the amide and with the tripeptide ester obtained under b)

H - GIu - His - Phe - OR I - OH

(I 5-7) implemented. The heptapeptide ester (J 1-7) obtained is converted into the corresponding heptapeptide hydrazide (K 1-7) transferred. .

e) Preparation of the nonapeptide hydrazide Z - Ser - Tyr - Ser - Met - GIu - His - Phe - Arg - Try - NHNH ₂

L-OH
(M 1-9) (method of the invention)

The heptapeptide hydrazide (K 1-7) obtained under d) is converted into the corresponding azide and condensed with the dipeptide ester H - Arg - Try - OR (H 8-9), then the obtained crude Nonapeptide ester (L 1-9) converted directly into the corresponding hydrazide (M 1-9).

The process according to the invention is illustrated in more detail by the following examples. the Melting points given in the examples were determined by the capillary method indicated Values are uncorrected. The Rr values were assigned to Whatman no. 1-paper by ascending

009 «27/90

Determined by chromatography. The solvent systems used are indicated by in brackets Numbers indicated: (1) n-butanol — glacial acetic acid— Water 4: 1: 1; (2) Methyl ethyl ketone — pyridine — water 65:15:20. Fresh preparations were always made Mixed solvents used.

Examples 1 to 10 illustrate the preparation of the starting materials required for the process of the invention.

. ■■■. '■. ■■■'., ■ ■ ■ · · <ϊϊ ίο

For example

N-carbobenzyloxy-L-seryl-L-methionine methyl ester (E 3-4)

7.6 g of carbobenzyloxy-L-serine hydrazide are dissolved in 50 ml of dimethylformamide with stirring and cooling dissolved with salted ice and with the addition of 15 ml of 6N hydrochloric acid. The clear solution obtained 2.1 g of sodium nitrite are added and the mixture is stirred for a further 10 minutes.

6.0 g of L-methionine methyl ester hydrochloride are dissolved in 50 ml of dimethylformamide while cooling with salted ice and stirring, and 12 g of triethylamine are added, then the azide solution prepared in the manner described above is added dropwise to the mixture. The mixture is left to stand overnight, then the solvent is evaporated off in vacuo and the residue is dissolved in ethyl acetate, the solution is washed with sodium hydrogen carbonate solution and with dilute hydrochloric acid, dried and evaporated to dryness. It will be 8.4 g of crystalline product was obtained (73% of theory), mp. 101 ⁰ C.

Example 2

L-Seryl-L-methionine methyl ester hydrochloride
(F 3-4)

3.84 g of N-carbobenzyloxy-L-seryl-L-methionine methyl ester are dissolved in 100 ml of absolute methanol, and 2.0 ml of 6η hydrochloric acid are added to the solution and in the presence of 2.0 g of 10% palladium-activated carbon catalyst at atmospheric pressure hydrogenated. After the uptake of hydrogen has ceased, the catalyst is removed by filtration and the solution evaporated to dryness in vacuo. The residue is dissolved in 30 ml of methanol, the solution is saturated with hydrochloric acid gas and then evaporated to dryness again in vacuo. It will 2.6 g (88% of theory) resinous peptide ester hydrochloride were obtained. The purity of the product is sufficient for further synthesis, Rr (1) = 0.48.

B e i s ρ i e 1 3

N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl- '
L-methionine methyl ester (H 1-4),

3.8 g of N-carbobenzyloxy-L-seryl-L-tyrosine hydrochloride are dissolved in 25 ml of dimethylformamide and with stirring and cooling with 4.5 ml of 6N hydrochloric acid and 625 mg of sodium nitrite are added.

2.6 g of L-Seryl-L-methionine methyl ester (prepared according to Example 1) are dissolved in 25 ml of dimethylformamide, 5.2 ml of triethylamine are added to the solution, then the cold azide solution prepared in the above manner is stirred and Cooling added. The mixture is ^; Left to stand overnight, the solvent evaporated in vacuo, the residue dissolved in ethyl acetate and the solution washed with water, then with 10% hydrochloric acid and finally with saturated aqueous sodium hydrogen carbonate solution. After evaporation of the solvent, 3.8 g (66% of theory) of tripeptide ester are obtained as a solid residue, which can be used directly for the preparation of the azide without further purification. Mp. After recrystallisation from aqueous ethanol 160 to 170 ⁰ C (decomposition).

Analysis: C ₂ DH 1 Oi ₀ N ₄ S; Molecular weight 634.7. Calculated ... C 54.88%, H 6.04%, N 8.80%; Found ... C 55.14%, 7 H 6.40%, N 9.00%.

B e i s ρ i e I 4

N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-L-methionine hydrazide (I 1-4)

3.8 g of the carbobenzyloxy tripeptide ester prepared according to Example 3 are dissolved in 50 ml of hot methanol, the solution is made with 1.78 ml of 70% hydrazine hydrate solution added and slowly cooked for 30 minutes. By the end of boiling, imagination forms copious precipitate, this is filtered off and dried; 3.2 g (84% of theory) of tetrapeptide hydrazide are obtained receive. Melting point after recrystallization from aqueous dioxane: 237 ° C. (decomposition [α] ξ? = -15 ° (c = 3.3 in glacial acetic acid).

Analysis: C ₂₈ H ₃ SO ₉ N ₆ S; Molecular weight 634.7. Calculated ... C 52.98%, H 6.04%, N 13.23%; found ... C 58.58%, H 6.35%, N 12.77%.

35 B ei * s ρ ie 1 5

L-histidyl-phenylalanine methyl ester dihydrochloride • (F 6-7)

19.3 g of carbobenzyloxy-L-histidyl-L-phenylalanine methyl ester are dissolved in 350 ml of absolute methanol, the solution is mixed with 14.0 ml of 6N hydrochloric acid and added in the presence of 2 g of 10% palladium-activated carbon catalyst Hydrogenated at atmospheric pressure. After filtering off the catalyst, the solution is evaporated to dryness in vacuo, the residue is dissolved in 100 ml of absolute methanol, the solution is saturated with hydrochloric acid gas while cooling and evaporated to dryness again in vacuo. 14.9 g of dry dipeptide ester hydrochloride (91% of theory) are obtained; R _r (2) .. ■ = 0.6.

Example6

Carbobenzyloxy-a-L-glutamyl-L-histidyl-L-phenylalanine methyl ester-j'-benzyl ester (H 5-7)

9.6 g of carbobenzyloxy-L-glutamic acid-y-benzyl ester are dissolved in 150 ml of absolute dioxane, the solution is ^{cooled to +5 0} C, and the partially solidified mixture is mixed with 3.8 ml of absolute triethylamine, then dropwise with 2, 6 ml of ethyl chlorocarbonate are added. After stirring for 20 minutes, the mixture is cooled with salted ice until it has completely solidified.

10.0 g of the crude dipeptide ester hydrochloride obtained according to Example 5 are cold in 50 ml. Dissolved water, the solution is added drop by drop 6.7 ml of triethylamine are added, and the resulting mixture is mixed into the frozen solution of the above Anhydride added. The mixture is cooled first and then at room temperature.

stirs, then neutralized with glacial acetic acid to pH = 6 and evaporated in vacuo. The oily residue is triturated with water until the product solidifies and disintegrates into a powder. After filtering and drying in a desiccator, 14.0 g of the desired product (80% of theory) are obtained; M.p. 170 to 173 ⁰ C.

Analysis: C) ₆ H) HOsN ₅ ; Molecular weight 669.7. Calculated ... C 64.56%, H 5.88%, N 10.46%; found ... C 64.65%, H 6.0O ⁰ Vo, N 9.89%.

Example 7 ■ *

fi-L-Glutamyl-L-histidyl-L-phenylalanine methyl ester (I 5-7)

5.1 g of the protected tripeptide ester prepared according to Example 6 are dissolved in a mixture of 150 ml of methanol, 20 ml of water and 3 ml of glacial acetic acid and hydrogenated in the presence of 1.0 g of 10% palladium-activated carbon catalyst for 2 hours at atmospheric pressure . After filtering off the catalyst, the solution is evaporated to dryness, the residue is dissolved in 10 ml of dimethylformamide, and 50 ml of absolute ethyl acetate are added. The resulting precipitate is separated by decanting and reprecipitated again in a similar manner, then filtered and air-dried. 2.65 g of the desired product (78% of theory) are obtained; M.p. 110 to 120 ° C (decomposition). Rr (1) = 0.42. [«] ?? = -10.8 ° (c = 6.6 in water).

Example 8

N-d-carbobenzyloxy-L-arginyl-L-tryptophan methyl ester (D 8-9)

5.15 g of N-fi-carbobenzyloxy-L-arginine and 4.65 g of L-tryptophan methyl ester hydrochloride are dissolved in 20 ml of dimethylformamide, and 5.2 g of Ν, Ν'-dicyclohexylcarbodiimide are added to the solution while stirring . The further stirred solution warmed slowly to 4O ⁰ C, and N, N'-dicyclohexylurea precipitates. After standing overnight, it is filtered and the filtrate is evaporated in vacuo. The resinous residue is extracted with ether to remove the excess Ν, Ν'-dicyclohexyl-carbodiimide, then dissolved in chloroform and the solution washed with water, then with dilute hydrochloric acid and finally with saturated sodium hydrogen carbonate solution. After evaporation of the organic solution, the free dipeptide base is obtained as a dry residue, but it is still contaminated with N ^ '- dicyclohexylcarbamide. The 9 g of crude product obtained can be used for saponification (Example 9) without purification.

B e t s ρ i e 1 9

N-u-carbobenzyloxy-L-arginyl-L-tryptophan hydrochloride (E 8-9)

6.0 g of the crude, protected dipeptide ester prepared according to Example 8 are dissolved in 60 ml of methanol dissolved, and the solution is mixed with 22 ml of n-sodium hydroxide solution. That which is present as an impurity Ν, Ν'-dicyclohexyl-carbamide separates out in crystalline form and is filtered off

removed. The solution is left to stand for 2 hours at room temperature, then the methanol is distilled off in vacuo. The upper aqueous phase of the liquid residue is decanted from the lower yellowish oil, "boiled up with activated charcoal, then filtered hot and cooled. After several hours of ice-cooling, the precipitated crystal mass is filtered off and air-dried. 2.9 g of the desired product are obtained. Mp. 116 ίο to 117 ° C; Ia] I ⁰ = -15 ° (c = 2 in glacial acetic acid) or +2 "(c = 2.5 in dimethylformamide.)

Analysis: Ci ₅ HmO ₅ No · H ₂ O · HCl; Molecular weight 549.0. Calculated:

C 54.7%, H 6.1%, N 15.3%, Cl 6.45%; found:

C 55.0%, H 6.2%, N 15.0%, Cl 6.66%.

B e i s ρ i e 1 10

L-arginyl-L-tryptophan methyl ester dihydrochloride (G 8-9)

1.65 g of the carbobenzyloxydipeptide ester hydrochloride prepared according to Example 9 are added to a mixture of 45 ml of methanol, 2 ml of water and 1.5 ml of glacial acetic acid in the presence of 0.2 g of 10% palladium-activated carbon catalyst at atmospheric pressure hydrogenated. After filtering off the catalyst, the solution is evaporated to dryness, the residue is dissolved in 30 ml of absolute methanol and the solution is saturated with hydrochloric acid gas. Then the solvent is distilled off and the above operation is repeated. The residue obtained is 1.16 g of the very soluble dihydrochloride (76% of theory), R _r (1) = 0.36.

Example 11 illustrates the process of the invention.

Example 11

a) N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-

L-methionyl-ii-L-glutamyl-L-histidyl-L-phenylalanine-

methyl ester (J 1-7)

951 mg of the tetrapeptide hydrazide prepared according to Example 4 (I 1-4) are dissolved in 10 ml of dimethylformamide and the addition of 0.75 ml of 6 η-hydrochloric acid, and 104 mg of sodium nitrite are added to the solution, with vigorous cooling and constant stirring. After stirring for a further 10 minutes, the azide solution obtained becomes the likewise cooled solution of 735 mg of tripeptide ester (I 5-7) in 10 ml of dimethylformamide and 0.85 ml of triethylamine were added, and the mixture is left to stand overnight at room temperature. The one after distilling off the solvent The oily residue obtained is mixed with 20 ml of water, and the solidified thereby Product is triturated into a powder, filtered, washed thoroughly with water and left in the air dried. There are 1.19 g (76% of theory) of the

obtain the desired heptapeptide (J 1-7); M.p. 167 to 173 ° C. after recrystallization from aqueous methanol.

Analysis: C _1n HeIOt ₅ N ₉ S; Molecular weight 1,048.1. Calculated:

C 56.15%, H 5.9%, N 12.0%, S 3.1 «/ · .;" found:

C 55.7%, H 6.15%, N 11.7%, S'3.4%.

b) N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-

L-methionyl-u-L-glutamyte-L-histidyl-L-phenylalanine-

hydrazide (K 1-7)

12.2 g of that of Example 11; aj produced heptapeptide ester (J 1-7) are dissolved in 12 ml of methanol with heating, and the solution is made with 0.42 ml 70% hydrazine hydrate solution was added and the mixture was boiled for 2 hours. Then the mixture is cooled, Let stand for 24 hours, the deposited heptapeptide hydrazide is filtered, slurried with 6 ml of methanol, sucked off sharply and dried in a desiccator over concentrated sulfuric acid. It 0.82 g of hydrazide (73% of theory) are obtained; After recrystallization from water 214 bis 215 ° C, [α]? = -15 ° (c = 5.15 in glacial acetic acid).

Analysis: C ₄₈ H ₆₁ O ₁₄ N ₁₁ S 1 H ₂ O; Molecular weight 1120.2. Calculated ... C 51.48%, Ή 6.21%, N 13.75%; found ... C 51.86%, H 6.56%, N 13.57%.

ic) N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-

L-methionyl-a-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophan methyl ester (L 1-9)

210 mg of the heptapeptide hydrazide (K 1-7) prepared according to Example II, b) are dissolved in 2 ml of dimethylformamide solved; the solution solidifies like a gel when it is cooled with salted ice, but becomes after Add 0.10 ml of 6η hydrochloric acid to make it clear again. After adding 18 mg of sodium nitrite, the mixture was stirred for 10 minutes with strong cooling, then the obtained azide solution became a cooled one Solution of 90 mg of arginyltryptophan methyl ester hydrochloride (G 8-9) in 1 ml of dimethylformamide and 0.14 ml of triethylamine added. The mixture is left to stand overnight, then is the solvent is distilled off and the oil obtained as residue is triturated with 3 ml of water. The Solidified and ground product is filtered off, washed with water and left in the air dried. 230 mg (76% of theory) of the desired ester (L 1-9) are obtained; Decomposition point around 150 C.

Analysis: CmHkhOitNiöS: molecular weight 1389.
Calculated ... N 15.0%:
found ... N 15.2%.

d) N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-

L-methionyl-a-L-glutamyl-L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophan hydrazide (M 1-9)

120 mg of the nonapeptide ester (L 1-9) obtained according to Example 11, c) are dissolved in hot methanol, 0.10 ml of 70% hydrazine hydrate are added to the solution and the mixture is boiled for 4 hours. The mixture is left to stand overnight, then the product is precipitated by adding 15 ml of absolute ether. After filtering and drying in air, 112 mg of the desired nonapeptide hydrazide (M 1-9) are obtained (93% of theory); Decomposition point around 190 ⁰ C.

Analysis: Q ₅ H ₈₃ O ₁₆ N ₁₇ S; Molecular weight 1389.

Calculated ... N 17.0%;
found ... N 16.6%.

Claims (1)

Claim: Process for the preparation of the new nonapeptide derivative N-carbobenzyloxy-L-seryl-L-tyrosyl - L - seryl - L - methionyl - L - glutamyl - L - histidyl - L - phenylalanyl -L- arginyl - L - tryptophanhydrazide, characterized in that N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl-L-methionine hydrazide in the presence of dimethylformamide as solvent in a manner known per se into the corresponding azide transferred, the reaction solution obtained without drying the water contained therein remove immediately with an L-glutamyl-L-histidyl-L-phenylalanine alkyl ester converts, then the resulting N-protected heptapeptide ester in a manner known per se into the corresponding Hydrazide and this in a manner known per se in the presence of dimethylformamide as a solvent into the corresponding N-carbobenzyloxy-L-seryl-L-tyrosyl-L-seryl -ι- methionyl - L - glutamyl - l - histidyl-L-phenylalanine azide transferred, the reaction solution obtained without removing the water contained therein by drying, immediately with an L-Arginyi-L-tryptophan alkyl ester and the resulting N-carbobenzyloxy-nonapeptide alkyl ester converted into the corresponding hydrazide in a manner known per se.