DE1130817B - Process for the production of new hypertensitic octapeptides - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY KL.12q 6/01

ESTERNAT.KL. C 07 C

GERMAN

PATENT OFFICE

EXPLAINING EDITORIAL 1130 817

C19324IVb / 12q

REGISTRATION DATE: JULY 1, 1959

NOTICE
THE REGISTRATION
AND ISSUE OF
EDITORIAL: JUNE 7, 1962

The subject of the invention is the production of new octapeptides with hypertensin effects.

The decapeptide hypertensin I, which occurs in horse serum and has the amino acid sequence L-aspartic acid, L-arginine, L-VaHn, L-tyrosine, L-isoleucine, L-histidine, L-proline, L-phenylalanine, L-histidine, L- Leucine, known as Ileu ^B hypertensin I, and the Val ⁵ hypertensin I found in bovine serum, also a decapeptide that differs from the aforementioned only in the fifth amino acid, L-valine. Furthermore, an octapeptide, Ileu ^s -Hypertensin II, was isolated from horse serum, which differs from the corresponding hypertensin I in that it lacks the ninth and tenth amino acids and is formed from the decapeptide by means of an enzyme present in the serum. Obviously it would be of great advantage for the synthesis of hypertensin-active compounds if certain amino acids could be replaced by simpler amino acids which are more favorable in terms of process.

The present invention now relates to the preparation of octapeptides of the formula glycyl-L - "- (amino-lower alkyl) -" - amino-acetyl-L - " - lower alkyl - tx - amino - acetyl - L - tyrosyl - l - α - lower alkyl-α-amino-acetyl-L-histidyl-L-prolyl-L-phenylalanine and their salts.

The remainder of an L - «- (amino-lower alkyl) -« - aminoacetic acid is an L-arginyl, L-ornithyl or a

, NH Process for the production of new hypertensin-active octapeptides

Applicant: CIBA Aktiengesellschaft, Basel (Switzerland)

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

Claimed priority: Switzerland from June 8, 1958 (No. 61 490)

Dr. Robert Schwyzer, Riehen,

and Dr. Bernhard Riniker, Birsfelden (Switzerland), have been named as the inventor

L-lysyl residue. Residues of an L- <x-lower alkyl-a-aminoacetic acid are L-Valyl, L-Leucyl and L-Isoleucyl, also L-norvalyl, L-norleucyl and L-alanyl.

Surprisingly, the new octapeptides show a good hypertensin effect, especially that Glycyl-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine the formula

OH

NH

NH ₂

(CH ₂ ) ₃

CH ₃ CH ₃

CH ₃

CH ₂

CH

H-HN-CH ₂ - CO —NH-CH-CO —NH-CH-CO —NH- VaI Y -CH- CO - NH-CH-CO- GIy Arg CH ₂ Tyr VaI CH-N ^ - CO —NH-CH- C-NH CH, / \ II
NH-CH-CO-N COOH

His

Per Phe

209 608/350

The new octapeptides offer significant technical advantages over the known hypertensins, than they are much easier and cheaper to manufacture.

The new peptides are after those used for peptide production known methods, the amino acids individually in the order mentioned or linked after prior formation of smaller peptide units. One advantageous method exists in that one L-a-lower alkyl-a-amino-acetyl-L-tyrosyl-L-öi-lower alkyl-oi-amino-acetyl-L-histidyl-L-prolyl-L-phenylalanine ester with a glycyl-L-a- (amino-lower alkyl) -a-amino-acetic acid, in which the α-amino group and optionally also the α-amino-lower alkyl group are protected, condensed, for example according to the scheme I:

NO ₂

Z -

GIy

- OH H -

Arg | - O CH ₃ H -j 'VaI Tyr VaI

His Pro Phe! —OCH,

NO ₂

Z -

GIy

Arg

OCH,

NO ₂

GIy

Arg-OH

NO ₂

Z -I GIy

Arg

VaI Tyr VaI His Pro Phe - OCH ₃

H-GIy

Arg

VaI Tyr VaI His Pro Phe

-OCH ₃

H-GIy

Arg

VaI Tyr VaI His Pro Phe-OH

Fig. I.

The hexapeptide ester H-Val-Tyr-Val-His-Pro-Phe-OCHg mentioned as the starting material can be produced by the process of patent application C 15418 IVb / 12q (German Auslegeschrift 1125 942). Instead of this ester, the hexapeptide esters H-Val-Tyr-Leu-His-Pro-Phe-O CH ₃ or H-Leu-Tyr-Ileu-His-Pro-Phe-OCH _{3 with} glycyI-arginine, which are also described in the patent application mentioned, can also be used be condensed.

Free functional groups not involved in the reaction are expediently protected, in particular by means of residues which can be easily split off by hydrolysis or reduction, for example the amino group by the tosyl or trityl radical and in particular the carbobenzoxy group or colored protective groups, such as the p-phenylazo-benzyloxy-carbonyl group and the p- (p'-methoxyphenylazo) -benzyloxycarbonyl group. The hydroxyl group of the tyrosine residue does not necessarily need to be protected during the reaction.

The conversion of a protected amino group into a free amino group and the conversion a functionally modified carboxyl group into a free carboxyl group in the course of the process for the production of the octapeptides and intermediates 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. The bases can be obtained from the salts in a manner known per se. From the latter in turn can be achieved by reaction with acids which are suitable for the formation of therapeutically useful salts are to win salts, such as B. those with inorganic acids such as hydrohalic acids, e.g. B. Hydrochloric acid or hydrobromic acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, or with organic acids such as acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, Malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, Cinnamic acid, salicylic acid, 2-phenoxy- or 2-acetoxybenzoic acid, mandelic acid, methanesulphonic acid, ethanesulphonic acid, Hydroxyethanesulfonic acid, benzene or toluenesulfonic acid.

The octapeptides obtained according to the method can find use as blood pressure-increasing agents in the form of pharmaceutical preparations. These contain the peptides in a mixture with one suitable for enteral or parenteral administration pharmaceutical organic or inorganic carrier material.

In the examples below, the temperatures are given in degrees Celsius.

The following abbreviations are used:

H-Arg-OH
H-GIy-OH
H-VaI-OH
H-Tyr-OH
H-Phe-OH
H-His-OH
H-Pro-OH
Z

L-arginine.

Glycine.

L-valine.

L-tyrosine.

L-phenylalanine.

L-histidine.

L-proline.

Carbobenzoxy.

Example (see Fig. I)
a) Z-Gly-Arg (NOü) -OH

1.35 g (0.005 mol) of finely powdered H-Arg (NO ₂ > OCH ₃ · HCl is suspended in 7 ml of dimethylformamide and, after the addition of 1.21 ml (0.0051 mol) of tributylamine, stirred until it is completely dissolved 1.85 g (0.009 mol) of dicyclohexyl-carbodiimide and 1.46 g (0.007 mol) of Z-Gly-OH in solid form all at once and allowed to stand overnight at room temperature Allowed to stand for an hour, then the dicyclohexylurea (1.6 g) which has crystallized out is filtered off, evaporated to almost dryness in a high vacuum and taken up in ethyl acetate. It is then washed successively with 2N hydrochloric acid, 2N sodium carbonate (ice cold) and water, dried with sodium sulfate and evaporated 2.8 g of crude Z-GIy-A ^ (NOa) -OCH ₃ , which is saponified directly, are obtained.

2.8 g of methyl ester are dissolved in 10 ml of methanol and, in the course of 1 hour at 20 °, 10 ml of normal aqueous sodium hydroxide solution was added dropwise. After the end of the dropwise addition, stirring is continued for another V2 hour, then a little undissolved material (= dicyclohexylurea) is filtered off and the filtrate is added acidified by 10.5 ml of 1 η hydrochloric acid. The resulting oily substance is extracted with n-butanol, the butanol phase washed with water and evaporated to dryness. When rubbed with ethyl acetate crystallization occurs. 1.3 g of N-carbobenzyloxy-glycyl-nitro-L-arginine are obtained from 135 to 138 °. A single recrystallization from hot methanol gives 1.22 g of substance with a melting point of 142 to 144 °.

OS

b) Z-Gly-Arg (NO ₂ ) -Val-Tyr-Val-His-Pro-Phe-OCH ₃

775 mg (0.001 mol) of H-Val-Tyr-Val-His-Pro-Phe-OCH ₃ , prepared by the process of patent application C 15418 IVb / 12q (German Auslegeschrift 1125942), and 474 mg (0.0023 mol) of dicyclohexylcarbodiimide are dissolved in 2 ml of dimethylformamide and 10 ml of acetonitrile and 820 mg (0.002 mol) of Z-Gly-Arg (NOa) -OH, dissolved in 2 ml of dimethylformamide, are added. The mixture is left to stand at 20 ° overnight. After adding 0.2 ml of glacial acetic acid, the mixture is left to stand for a while and the dicyclohexylurea (450 mg) which has crystallized out is then filtered off. The filtrate is evaporated as much as possible in a high vacuum at 40 °, then taken up in n-butanol and the organic layer is washed successively with sodium carbonate solution (ice-cold) and water and, without drying, concentrated to about half at a bath temperature of 50 ° in vacuo . A fine-grain precipitate separates out, which is filtered off, washed with ethyl acetate and dried at 50 °. This gives 828 mg of Z-GIy-Arg (NO ₂ ) - Val-Tyr - VaI - His - Pro - Phe-O CH ₃ with a temperature of about 160 ° (decomposition), which is sufficiently pure for further processing. The butanol filtrate provides only a small amount of impure material when it is further concentrated.

c) H-Gly-Arg-Val-Tyr-Val-His-Pro-Phe-OCH ₃

828 mg of Z-Gly-Arg (NO ₂ ) -Val-Tyr-Val-His-Pro-Phe-O CH ₃ are dissolved in 10 ml of methanol and 4.2 ml of 1N hydrochloric acid (in methanol) and, after adding 300 mg of catalyst (palladium-carbon 10%) hydrogenated with absorption of hydrogen. After uptake of 5.1 mol of hydrogen, the hydrogenation comes to a standstill (duration 12 hours), and the catalyst is filtered off, evaporated in vacuo and dried in a high vacuum at 40 °. 763 mg of an amorphous powder (mixture of octapeptide methyl ester trihydrochloride and ammonium chloride), which is used directly for the hydrolysis, are obtained.

d) H-Gly-Arg-Val-Tyr-Val-His-Pro-Phe-OH

760 mg of crude methyl ester are dissolved in 5 ml of concentrated aqueous hydrochloric acid and kept at 40 ° for 1 hour. After evaporation to dryness in a high vacuum, 750 mg of a powdery hydrochloride are obtained, which is dissolved in a little water and filtered through a weakly basic ion exchanger. (Trade name Merck II as acetate; column with diameter = 12.5 mm, / = 12 cm). On lyophilization, the filtrate gives 675 mg of crude product, which is purified in a Craig distribution (system n-butanol — water — methanol 5: 5: 1; phase volume of 10 ml each) over 60 stages. 562 mg of pure H-Gly-Arg-Val-Tyr-Val-His-Pro-Phe-OH · CH ₃ COOH are isolated from tubes 6 to 19 (maximum at No. 12; G = 0.26). The compound is easily soluble in water, soluble in methanol, sparingly soluble in ethanol. It is precipitated from the aqueous solution by adding saturated sodium chloride solution. F. about 220 ° (decomposition). In the paper chromatogram (descending to Whatman No. 3) uniform spots are obtained which are positive for Pauly's reagent and ninhydrin. In the system tert-amyl alcohol — triethylamine — veronal — water — isopropanol (100: 0.8: 1.8: 50: 40), Rf = 0.48 is obtained; in sec-butanol — monochloroacetic acid — water — isopropanol (70 : 3:40:10) R _{ = 0.44.

Claims (2)

PATENT CLAIMS: 1. Process for the preparation of new hypertensitively active octapeptides of the general formula glycyl - L - «- (amino - lower alkyl) -« - amino - acetyl-La-mederalkyl-amino-acetyl-L-tyrosyl-L-Ä-lower alkyl-amino-acetyl -L-bistidyl-L-prolyl-L-phenylalanine, in which the L - "- (amino-lower alkyl) -" - aminoacetyl radical for L-arginyl, L-ornithyl or L-lysyl and the La-lower alkyl-a-amino- acetyl radical for L-valyl, L-isoleucyl, L-leucyl, L-norvalyl, L-norleucyl or L-alanyl, and their salts, characterized in that one L ~ # -lower alkyl - «- aminoacetyl - l - tyr osyl - l - α - lower alkyl - <x - amino-acetyl-L-histidyl-L-prolyl-L-phenylalanine ester with a glycyl-L - «- (amino-lower alkyl) -« - amino-acetic acid, in which the α-amino group of the glycine residue and optionally also the «- (amino-lower alkyl) group of the arginine residue are protected, condensed in the presence of a carbodiimide, the protective groups are split off and the ester group saponified and, if desired, basic compounds obtained in i Their salts or salts obtained are converted into bases. 2. The method according to claim 1, characterized in that one with a glycyl-nitro-L-arginine protected amino group with an L-valyl-L-tyrosyl-L-valyl - L-histidyl-L-prolyl-L-phenylalanine ester condensed in the presence of a carbodiimide, the amino protective group by a reductive route of the glycine residue and the nitro group of the arginine residue are removed and the ester group is saponified. © 209 60 & / 350 5.62