CH426862A - Process for the production of a previously unknown polypeptide - Google Patents

Description

  

  
 



  Process for the production of a previously unknown polypeptide
It has been found that a previously unknown polypeptide of the formula I (see formula sheet) can be obtained by converting the nonapeptide derivative of formula V (see formula sheet) into the end product I by oxidation in aqueous solution.



   The previously unknown nonapeptide derivative of the formula V can be obtained by using the new hexapeptide of the formula II (see formula sheet), in which R is a group used in peptide synthesis to protect the sulfhydryl group and R 'is a group in peptide synthesis to protect the amino group common group, with a reactive derivative of the free acid III (see formula sheet), in which R "is a group customary in peptide synthesis to protect the amino group and R" 'is a group customary in peptide synthesis to protect the sulfhydryl group, to the nonapeptide Derivative IV (see formula sheet) is condensed and this is converted into the nonapeptide derivative V by reduction with an alkali metal in liquid ammonia.



   For the temporary blocking of the amino groups, the groups customary for this purpose, such as the carbobenzoxy group, the carbo-p-chlorobenzyloxy, the p-toluenesulfonyl or the triphenylmethyl group, can be used. To protect the sulfhydryl group, a phenyl, a benzyl, a p-bromobenzyl, a p-chloro-benzyl, a p-nitrobenzyl or a p-xylyl group is preferably introduced. However, it is particularly advantageous to use the benzyl radical.



   It has been found that the compound I has a strong vasoconstrictor effect, like the natural vasopressins, from which the new compound differs in that it instead of lysine (vasopressin from pigs) resp. Arginine (bovine vasopressin) contains ornithine, instead of phenylalanine isoleucine and instead of tyrosine phenylalanine. The new compound differs from oxytocin in the presence of phenylalanine instead of tyrosine and that of ornithine instead of leucine (see formula sheet).



  In the present compound I, however, in contrast to the natural vasopressins, the antidiuretic active component is almost completely absent, and oxytocic active components largely, so that the present compound I can be introduced into therapy as a substance with a targeted vasoconstrictor effect. The specific vasoconstrictor effect is achieved by directly influencing the vascular muscles, which is why the present compound 1 - in contrast to adronalin and noradrenaline - does not cause any vegetative-nervous side effects.



   The main field of application of the new compound is the prophylaxis and therapy of parenchymal bleeding, the infiltration of the tissue with the compound I having a pronounced ischemic effect. The present compound I is used in particular in surgical interventions in ear, nose and throat medicine, gynecology and obstetrics, urology and dentistry. It is either added to the local anesthetic or, during operations under general anesthesia, diluted with physiological saline and used for infiltration.

   The process for the preparation of compounds of the formula V is carried out, for example, as follows:
A-N-carbobenzoxy-N-8-p-toluenesulfonyl-L-ornithine is condensed with glycine ethyl ester to give a-N carbobenzoxy-N-d-p-toluenesulfonyl-L-ornithyl-glycine ethyl ester. After splitting off the carbobenzoxy group, the N-8-p-toluenesulfonyl-lornithyl-glycine-ethyl ester is condensed with N-carbobenzoxy-Lproline to give N-carbobenzoxy-L-prolyl-N-8-p -toluenesulfonyl-L-ornithyl-glycine- ethyl ester, which is converted into the corresponding amide.

   After the carbobenzoxy group has been split off, the L-prolyl-Ndp-toluenesulfonyl-L-ornithyl-glycine amide with N-carbobenzoxy-L-glutamulyl-lasparaginyl-S-benzyl-L-cysteine-azide becomes N-carbobenzoxy-L glutaminyl-1-asparaginyl-S -benzyl-L-cysteinyl-1-prolyl-N-6-p -toluenesulfonyl-L-ornithyl-glycine amide condensed.

   After the carbobenzoxy group has been split off, the L-glutaminyl-L-aspara ginyl-S-benzyl-L-cysteinyl-L-prolyl-Ndp-toluenesulfonyl-L-ornithyl-glycinamide with carbobenzoxy-sbenzyl-L-cysteinyl-L- phenylalanyl-L-isoleucyl-azide to N-carbobenzoxy-S-benzyl-l-cysteinyl-L-phenyl-alanyl-l-isoleucyl-6, -lutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl -Ndp-toluenesulfonyl-L-omithyl-glycine amide condensed. This nonapeptide derivative is treated with an alkali metal, preferably with sodium or potassium in liquid ammonia, so that the linear nonapeptide V is formed. According to the invention, this is converted into the biologically active, cyclic polypeptide I, i.e. by oxidation, preferably with air, oxygen or hydrogen peroxide in aqueous solution. H.

   Phe2-Orn8-oxytocin, transferred. The previously unknown polypeptide I produced according to the process can be used as a free base or as a salt of an organic or inorganic acid, either as a medicament alone or in appropriate medicament forms for parenteral, enteral or intranasal administration.



  In order to produce suitable dosage forms, these are used with inorganic or organic pharmacologically indifferent auxiliaries. As auxiliaries are used, for. B. for tablets and dragees: lactose, starch, talc, stearic acid, etc .; for syrups: cane sugar, invert sugar, glucose solutions etc. a .; for injection preparations: water, alcohols, glycerol, vegetable oils and the like; for suppositories: natural or hydrogenated oils and waxes, etc. a. In addition, the preparations can contain suitable preservatives, stabilizers, wetting agents, solubilizers, sweeteners, colorants, flavorings, etc.



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



   Example 1: a) N-a-Carbobenzoxy-N-a-p-toluenesulfonyl-L-ornithyl-glycine-ethyl ester
104 g of N-α-carbobenzoxy-N-d-p-toluenesulfonyl-L-ornithine and 27 g of glycine ethyl ester are dissolved in 450 cc of acetonitrile, cooled at 0, 51 g of dicyclohexyl carbodiimide are added and shaken for 4 hours at room temperature. The precipitated dicyclohexylurea is filtered off and washed with acetonitrile. The entire filtrate is evaporated in vacuo. The residue crystallizes after the addition of petroleum ether. After recrystallization from n-propanol, 93 g of N-α-carbobenzoxy-N-p4eluolsulfonyl-L-ornithyl-glycine-ethyl ester are obtained; M.p. 136 ".



      La] 2 = -7 "(960 /% ethanol). B) N-carbobenzoxy-L-prolyl-N-o-p-toluenesulfonyl-L-ornithyl-glycine amide
90 g of N-α-carbobenzoxy-N-d-p-toluenesulfonyl-L-ornithyl-glycine ethyl ester are dissolved in 800 cc of anhydrous acetic acid saturated with hydrogen bromide. The mixture is left to stand at 20 for one hour, evaporated in a vacuum below 40 and the residue is carefully washed with diethyl ether.

   The residue is dissolved in 500 cc of acetonitrile, 25 cc of triethylamine and 43 g of N-carbobenzoxy-L-proline are added, the mixture is cooled at 0, 35.5 g of dicyclohexylcarbodiimide are added and shaken overnight at 20. After filtering off the dicyclohexylurea, the filtrate is evaporated in vacuo at 300, the residue is dissolved in ethyl acetate and this solution is washed with dilute sulfuric acid and aqueous ammonia. After drying over sodium sulfate, the ethyl acetate is evaporated off in vacuo and the residue is dissolved in 1 liter of absolute ethanol. The solution is cooled to 0, saturated with ammonia and left to stand at 20 overnight.

   After evaporation in vacuo at 30, the residue is dissolved in 100 ml of dimethylformamide and crystallized by adding 1500 ml of ethyl acetate. 66 g of N-carbobenzoxy-L-prolyl-N-a-p-toluenesulfonyl-L-ornithyl-glycidamide are obtained; 122 "(dec).



     [a] D22 = -46 "(950% glacial acetic acid). c) N-carbobenzoxy-L-glutaminyl-lasparaginyl-
S-benzyl-L-cysteinyl-L-prolyl-N-d-p-toluenesulfonyl-L-ornithyl-glycine amide
100 g of N-carbobenzoxy-L-prolyl-Ndp-toluenesulfonyl-L-ornithyl-glycine amide are dissolved in 500 cc of anhydrous acetic acid saturated with hydrogen bromide, left to stand for 1 hour at 200 and evaporated in a vacuum below 40 ". The residue is with Diethyl ether was carefully washed and then added to a solution of 100 g of N-carbobenzoxy-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl azide [Boissonnas & coll., Helv. Chim. Acta, 38, 1491 (1955 )] and 26 cc of triethylamine in 1000 cc of dimethylformamide were added.

   It is left to stand overnight at 20, 3000 cc of ethyl acetate are added, the precipitate is filtered off and washed with ethyl acetate. 105 g of N-carbobenzoxy-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N-o-p-toluenesulfonyl-lornithyl-glycine amide are obtained; M.p. 188 (dec.).



      [a] = 330 (dimethylformamide). d) N-carbobenzoxy-S-benzyl-L-cysteinyl-L-phenylalanyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl N-d-p-toluenesulfonyl-L-ornithyl-glycine amide
Dissolve 50 g of N-carbobenzoxy-L-glutaminyl-L asparaginyl-S-benzyl-1-cysteinyl-L-prolyl-N-8-p-toluenesulfonyl-L-ornithyl-glycine amide in 250 cc of anhydrous acetic acid saturated with hydrogen bromide and let stand at 20 for 1 hour.

   After evaporation of the solvent in vacuo below 40, the residue is carefully washed with diethyl ether and with a solution of 31.1 g of N-carbobenzoxy-S-benzyl-1-cysteinyl-1-phenylalanyl-L-isoleucyl azide and 7.5 ccm of triethylamine in 250 ccm of dimethylformamide. It is left to stand for 2 days at 200, 1000 cc of ethyl acetate are then added, and the precipitate is washed with ethyl acetate. After drying in vacuo at 300, the product is washed with warm methanol.



  45 g of N-carbobenzoxy-S-benzyl-L-cysteinyl-L-phenylalanyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-S -benzyl-L-cysteinyl-L-prolyl-N-8-p-toluenesulfonyl are obtained -L-omithyl-glycine amide. M.p. 242 ". A] 21 = (dimethylformamide).

   e) L-cysteinyl-L-phenylalanyl-L-isoleucyl-L glutaminyl-L-asparaginyl-L-cysteinyl-L-prolyl-
L-ornithyl-glycinamide
A solution of 5 g of N-carbobenzoxy-S-benzyl-L-cysteinyl-L-phenylalanyl-L-isoleucyl L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-Ndp-toluenesulfonyl- L-ornithyl-glycine amide in 1200 ccm of dry liquid ammonia with stirring at the boiling point of the solution with enough sodium or potassium metal until a constant blue color has occurred. After adding 3 g of ammonium chloride, the solution is evaporated to dryness.

   The residue contains L-cysteinyl-L phenylalanyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-L-cysteinyl-L-prolyl-L-ornithyl-glycine amide. f) Polypeptide Compound I.
The residue containing L-cysteinyl-L-phenylalany-1-isoleucyl-L-glutaminyl-L-asparaginyl-1-cysteinyl-L-prolyl-L-ornithyl-glycinamide is dissolved in five liters of 0.01 N acetic acid and oxidized at a pH of 6.5-8.0 by passing in air or oxygen for 1 hour at 0-40. The solution containing the substance is brought to a pH of 4.0-5.0 and, after the addition of 50 g of sodium chloride or 0.64 g of methanesulfonic acid, is evaporated to dryness, a dry powder being obtained which can be kept well is.

   It can be stored and cleared when in use. However, the solution can also be used directly, possibly after diluting with water or a salt solution.



   Example 2:
The procedure is as in Example 1. At the end, however, the oxidation is carried out at 0-10 by adding 7.5 cc of a solution of hydrogen peroxide in water at a pH of 4.0-9.0 (instead of oxidation by introducing air or Oxygen).



   Example 3:
The procedure is as in Example 1. At the end, however, the oxidation is carried out at 0-350 by adding 6.7 ccm of 1N solution of potassium ferricyanide in water at a pH of 5.5-7.5.
EMI4.1
   

 

Claims (1)

PATENT CLAIM Process for the preparation of a previously unknown polypeptide of the formula I (see formula sheet), characterized in that the nonapeptide derivative of formula V (see formula sheet) is converted into the end product I by oxidation in aqueous solution. SUBCLAIMS 1. The method according to claim, characterized in that one carries out the oxidation of V to I by introducing air. 2. The method according to claim, characterized in that the oxidation of V to I is carried out by introducing oxygen. 3. The method according to claim, characterized in that the oxidation of V to I is carried out by adding hydrogen peroxide. 4. The method according to claim, characterized in that the oxidation of V to I is carried out by adding potassium ferricyanide.