CH358431A - Process for the production of new octapeptides - Google Patents

Description

  

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 Process for the production of new octapeptides The subject of the invention is a process for the production of octapeptides with hypertensin effect. As is well known, hypertensin is a hormone with a blood pressure-increasing effect that was first obtained from bovine blood.

   The constitutional investigation showed that hypertensin is a linear decapeptide with the amino acid sequence L-asparagic acid, L-arginine, L-valine, L-tyrosine, L-valine, L-histidine, L-proline, L-phenylalanine, L-histide 'in, hLeucine. As a result, two more hypertensins were found: Hypertensin I, which occurs in horse blood and instead of the 5th

   Amino acid residue (valine) contains the remainder of the isoleucine, and hypertensin 1I, an octapeptide which corresponds in structure to hypertensin I, but does not contain the last two amino acid residues (cf. e.g. Skeggs et al., J. Exp. Med. , Volume 100 [1954], 363; ibid., Volume 103 [1956], 301; WS Peart, Bioch. J., Volume 62 [1956], 520).



  It has now been found that octapeptides with the amino acid sequence L-asparagine, La- (amino-lower alkyl) -amino-acetic acid, La-amino-lower alkyl-acetic acid, L-tyrosine, La-amino-lower alkyl-acetic acid, L = histidine, L-proline, L-phenylalanine and their salts also have a very good hypertensive effect.

   Residues of α-amino-lower alkylene-acetic acid are mainly valyl, leucyl, iso-leucyl, and also norvalyl, norleucyl and alanyl. As a- (amino-lower alkyl) -amino-acetic acids arginine, lysine, omithine, a, #, "-diaminobutyric acid or citrulline come into consideration. It has been shown that octapeptides, in which those in the natural hypertensins occurring amino acids arginine,

        Valine and isoleucine are replaced by other amino acids, for example lysine or leucine, also have a hypertensin effect. This is of technical importance because the octapeptides mentioned are easier and therefore cheaper to obtain compared to the already known peptides. Likewise, the use of an octapeptide which contains the aspartic acid residue instead of the aspartic acid residue means sheared. procedurally a step forward.

   The octapeptides mentioned can also be used as intermediates for the preparation of other active compounds. For example, the amide residue of asparagine can be converted into the carboxyl group; if you start from L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine, then you get hypertensin II, which is the first time the synthesis of this hormone is described.



  The conversion of the peptides containing the asparagine residue into corresponding peptides with the aspartic acid residue can be carried out by treatment with aqueous or alcoholic alkalis, e.g. B. caustic soda in methanol, or with aqueous or alcoholic mineral acids.



  There are a large number of ways to produce the new peptides. The process according to the invention is characterized in that an L-asparaginyl-La- (amino-lower alkyl) -a-aminoacetic acid, a La-amino-lower alkyl acetic acid, L-tyrosine, a La-amino-lower alkyl acetic acid, L -Histidine, L-proline and L-phenylalanine combined with one another by condensation with intermediate protection of amino or carboxyl groups.

   If desired, the carbonyl amide group of the asparagine residue can be converted to a free carboxyl group.



  If the octapeptide to be synthesized contains arginine as the 2nd amino acid, it is advantageous to start from L-asparaginyl-nitro-L-a ginine and condense

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    ss this with an I,

  - a-amino-lower alkyl-acetyl-L-tyrosyl-L-a-amino-lower alkyl-acetyl-L-histidyl-L-prolyl-L-phenylalanyl ester to the desired octapeptide. But you can also condense an L-asparaginyl-nitro-L-arginine with La-amino-lower alkyl-acetyl-L-tyrosine to give an asparaginyl-nitro-L-arginyl-La-amino-lower alkyl-acetyl-L-tyrosine and convert this with a La-amino-lower-alkyl-acetyl-L-histidyl-L-prolyl-L-phenylalanine ester to form the octapeptide.

   It is also possible to acyher La-amino-lower-alkyl-acetyl-L-tyrosyl-La-amino-lower-alkyl-acetyl-histidine with an L-asparaginyl-nitro-L-arginine and the L-asparaginyl-nitro-L- to convert arginyl-La-amino-lower-alkyl-acetyl-L-tyrosyl-L-a-amino-lower-alkyl-acetyl-histidine with L-prolyl-L-phenylalanine to the octapeptide. The intermediates mentioned for the acylation can be obtained in various ways.

   Thus, for the time being, La-amino-lower-alkyl-acetic acid can be converted into La-amino-lower-alkyl-acetyl-L-tyrosine with L-tyrosine, with L-histidine to form La-amino-lower-alkyl-acetyl-L-histidine, and finally Condense L-proline with L-phenylalanine ester to form L-prolyl-L-phenylalanine ester.

   The resulting La-amino-lower-alkyl-acetyl-L-histidine is then combined with La-amino-lower-alkyl-acetyl-L-tyrosine to form La-amino-lower-alkyl-acetyl-L-tyrosyl-La-amino-lower-alkyl-acetyl L-histidine acylated. But you can also use La-amino-lower-alkyl-acetic acid with La-amino-lower-alkyl-acetyl-L-tyrosine to give La-amino-lower-alkyl-acetyl-L-tyrosyl-L-cc-amino-lower-alkyl-acetic acid and then condense them with L-histidine. As amino protective groups, residues which can be split off by means of hydrolysis or reduction, such as, for.

   B. the carbobenzyloxy radical in question. The acylation can, for example, by reacting an acid with the corresponding phosphite amide, such as. B. with the corresponding diethylphosphitamide, by reaction of an acid with the amine in the presence of a carbodiimide, such as in the presence of 1- cyclohexyl-3- (2-morpholinyl- [4] -ethyl) -carbodiimids- and especially by reacting an amine with an activated ester in which the cyano or nitro group is advantageously present as the activating substituent in the alcohol component.



  The conversion of a protected amino group into a free amino group and the conversion of a functionally modified carboxyl group into a free carboxyl group in the course of the process for the preparation of the various polypeptides and in particular in the octapeptides obtained according to the process can be carried out according to methods known per se by treatment with hydrolyzing or done with hydrogenating agents.



  In the process products obtained, free carboxyl groups can be functionally modified, in particular converted into esters or amides. It is also possible to substitute free amino groups, such as. B. to alkylate or acylate. The products of the process can also be obtained or used in the form of their salts.



  A special embodiment of the method according to the invention, as described in the first example, is shown in the following equation under 3 to 7. 1. N-carbobenzyloxy-L-asparaginyl-nitro-L-arginine-zethyiester (11).
 EMI2.91
 

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 EMI3.1
 2. N-C'arbobenzyloxy-L-asparaginyl-nitro-L-arginine (IV).
 EMI3.2
 
 EMI3.3
 3. N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-val) "1-L-tyrosine methyl ester (V1).
 EMI3.4
 
 EMI3.5
 4. N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosine (VII).
 EMI3.6
 

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 EMI4.1
 S. N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-hi, stidyl-L-prolyl-L-phenylalanine methyl ester (IX).
 EMI4.2
 
 EMI4.3
 6th

   L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (X).
 EMI4.4
 

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 7. L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanil (XI).
 EMI5.2
 The octapeptides obtained according to the process can be used as intermediate products for the production of medicaments or also as medicinal products.



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



  Example 1 a) N-carbobenzyloxy-L-asparaginyl-nitro-L-argifyl-L-valyl-L-tyrosine methyl ester (V1).



  3.72 g (13 mmol) of freshly prepared L-valyl-L-tyrosine methyl ester (V) and 5.47 g (11.8 mmol) of carbobenzyloxy-L-asparaginyl-nitro-L-arginine (IV ) (cf. Patent No. 356122) are dissolved in 35 cm3 of dimethylformamide and 2.52 g (12.3 mmol) of 1,3-dicyclohexylcarbodiimide are added.

   After 24 hours at 21, the precipitated dicyclohexylurea is filtered off with suction and the filtrate is completely freed from dimethylformamide at 0.1 mm Hg and a bath temperature. The remaining oil is washed first with petroleum ether, then with ice cooling with dilute bicarbonate solution, water, dilute hydrochloric acid and water, the initially oily product slowly becoming granular.

   The crude product (8.79 g) is reprecipitated from hot methanol for further purification and the methanol-insoluble powder is triturated with hot acetone. This gives 2.91 g (33%) carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosine methyl ester (V1) of melting point 202 to 206 [a] D = -4 4 ( c = 0.94 in dimethylformamide).



  b) N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosine (V11).



  2.58 g (2.5 mmol) of N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosine methyl ester (V1) are dissolved in 30 cubic meters of dimethylformamide and within 15 minutes in total 100 cm3 1; 1 () - n. Sodium hydroxide solution was added in several portions, taking care that the pH of the solution does not exceed 11.

   After adding the alkali, the mixture is left at pH 11 for a further 15 minutes, then brought to pH = 8 by adding solid CO 2 and the solution is freed from water first at 11 mm and 45 bath temperature, then from dimethylformamide at 0.1 mm. The residue is dissolved in 30 cm3 of water, a trace of flakes is filtered off and the clear filtrate is washed with 2-n. Acidified hydrochloric acid. The excreted, viscous product solidifies when rubbed (with ice cooling).

   The crude product (2.14 g) is dissolved in hot methanol and precipitated by adding acetonitrile. 1.77 g (70%) of carbobenzoxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosine (VII) are obtained as a powder, m.p. about 175-183 (foaming); [a] 23 = 0 4 (c = 0.4 in methanol).



  c) N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (IX).



  1.88 g (2.58 mmoles) of carbobenzyloxy-L-asparagiryl-nitro-L-arginyl-L-valyl-L-tyrosine (VII) and 1.31 g (2.50 mmoles) of freshly prepared L-isoleucyl -L-histidyl-L-prolyl-L-phenyl-alanine methyl ester (VIII) are dissolved in 15 cm3 of dimethylformamide and a solution of 0.62 g (2.62 mmol) of 1-cyclohexyl-3-morpholinyl-ethyl-carbodiimide admitted.

   The solution is left at 20 for 21 hours, the dimethylformamide is then removed in a high vacuum and the oil that remains is triturated with water while cooling with ice, where it gradually becomes granular. The powder is washed several times with water, then with dilute bicarbonate solution and water and dried.

   The crude product (3.70 g) is washed with acetone and methanol for further purification and yields 0.68 g of N-carbobenzyloxy-L-asparaginyl-nitro-L-a gginyl-L-

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    valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (IX), m.p. = 190-205; [a] D__ -29 4 (c = 0.52 in dimethylformamide). The product is sparingly soluble in common organic solvents, with the exception of dimethylformamide; soluble in very dilute hydrochloric acid.



     d) L-asparcaginyl-L-argi: zyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (x).



  370 mg (0.3 mmol) carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (X) are suspended in 15 cm3 of methanol and by adding 1.0 cm3 of 1.24-n. (4 eq.) Hydrochloric acid dissolved in methanol.

   A trace of flakes is removed by filtration and the solution is hydrogenated in the presence of 100 mg palladium carbon (10% Pd) at room temperature and normal pressure (using a second hydrogenation vessel filled with dilute sodium hydroxide solution to absorb the CO2 evolved). The hydrogenation is complete after 13 hours and the uptake of slightly more than the calculated amount of hydrogen. The solution is then filtered off from the catalyst and evaporated to dryness in vacuo.

   The residue (resin) weighs 310 mg (calculated for a mixture of equimolar amounts of trihydrochloride and ammonium chloride = 360 mg). The product (X) is processed further without purification. It is easily soluble in water, but sparingly soluble in aqueous alkali; soluble in methanol, insoluble in ether.



  e) L-asparaginyl-L-caz-ginyl-L-valyl-L-tyrosyl-L-isoleiicyl-L-histidyl-L-prolyl-L-phenylalanine. For the solution of 256 mg (25 mol) L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester in 12.0 cm, '66 % Methanol becomes 0.1-n in several proportions. Sodium hydroxide solution was added until the p11 value of the solution remained at 10.5-11.0 for 20 minutes (total 18 cm3 0.1-n.

   After a total of 30 minutes, the pH is brought to 8 by adding some solid CO2, a flaky precipitate (25 mg) is removed by filtration and the filtrate is freed from methanol in vacuo. The aqueous solution (volume 15 cm3) is mixed with a few cubic meters of dilute soda solution and extracted four times at pH = 9 with 100 cm3 of water-saturated n-butanol each time. The butanol extracts are washed once with 8 cm dilute sodium sulfate solution, dried with sodium sulfate and give 195 mg of crude product on evaporation.

   This is washed three times with 5 cm3 each of dry n-butanol, whereby 30 mg of more easily soluble components can be removed. The main amount (135 mg) of L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine is sparingly soluble in dry butanol and is in the form of a colorless, fine-grain fittings. The product is soluble in water and methanol. The crude product (135 mg) is subjected to a 32-stage multiplicative distribution (Craig basic process) in the n-butanol-water system. The majority (75 mg) of the active material is found in fractions 9-21 with a maximum at fraction 15 (G = 0.89).

   Fractions 0-8 contain 45 mg of a slower moving, inactive impurity; fractions 22-32 contain approximately 20 mg of inactive material. A sample of the substance from fraction 15 acts on the rat in the experimental arrangement of Peart3) twice as strongly as noradrenaline (0.25 "/ kg in 0.1 cm 'physiological saline solution intravenously corresponds to 0.5" noradrenaline ).



  The material from fractions 9-21 is combined and distributed again in the same way as above. Activity is again found in fractions 9-22, which contain a total of 32 mg of material (maximum in fraction 16). Fractions 0-8 still contain 20 mg of inactive material, fractions 23-32 still contain about 15 mg of inactive material.



  A sample of the substance from fraction 16 proves in the above test to be five to ten times more effective than noradrenaline.



  The material from fractions 9-22 is combined again and distributed in the n-butanol-4% acetic acid system in 30 steps (Craig basic process). After the end of the 30th

   In the partitioning step, the pH of all fractions is brought to 5.5-6.0 by adding a little methanolic ammonia solution, the solutions are evaporated to dryness at 40 o / a and reduced pressure and the evaporation residues at 50 for 8 hours to remove ammonium acetate and dried 0.01 mm Hg. Fractions 0-6 contain a total of 30 mg of substance (maximum for fraction 1, K == 0.051); fractions 7-30 do not contain any weighable parts. The experimentally found weight distribution curve in fractions 0-6 agrees with the theoretically calculated one.

   A sample of the substance from fraction 1 shows in the above-mentioned test by PeartO) a five times greater effectiveness than norepinephrine. The L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine purified in this way is obtained in the form of a colorless powder; M.p. (after drying in a high vacuum) 195-205 (decomp.). In the paper chromatogram it shows in the systems ethanol-n-butanol-H, O-diethylamine (100: 100 - 50: 20), n-butanol-acetone-H., 0-diethylamine (100:

   100: 50: 20), ethanol-n-butanol-H = O (100: 100: 50), n-butanol-glacial acetic acid-H2O (100: 10, total with H. = 0) and sec. Butanol -3% NH3 (120-44) on Whatmann No. 1 the RF values 0.37; 0.31; 0.18; 0.15 and 0.24. The stains show the Pauly reaction: no ninhydrin-positive substances are detectable on the paper.



  The ss-carbonamide group of the asparaginyl residue can be saponified to the carboxyl group as follows, for example: 5 mg L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine (XI )

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 are concentrated in 0.1 ml. HCl dissolved and left to stand at 39 for 100 minutes. The solution is then evaporated to dryness in a high vacuum, the residue is pulverized and dried at 40 in a high vacuum.

   The resulting L-asparagyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine (Hypertensin II) can be carried out by Craig partition or chromatography on a cellulose column with suitable solvent spectra, e.g. B. butanol-methanol- 0.33 m NH4COOH (3: 1: 4; pH = 6.5) or sec. Butanol-3 Oh, NH3 (120:44) can be separated from unsaponified starting material.

   Water-soluble, colorless powder, R, = 0.2 in the system sec. Butanol- 3% NH3 (130:44).



  The L-valyl-L-tyrosine methyl ester used as the starting material can be produced as follows: 1. N-carbobenzyloxy-L-valyl-L-tyrosine methyl ester.



  A solution of 68.1 g (0.275 mol) of carbobenzyloxy-L-valine and 50.9 g (0.261 mol) of L-tyrosine methyl ester in 1 liter of tetrahydrofuran are mixed with 59.2 g of 1,3-dicyclohexyl-carbodlimide and leave the solution at 2111 for 16 hours. The dicyclohexylurea which has separated out is then suction filtered (46.0 g, corresponding to 77% of theory) (melting point 218-224) and the filtrate is evaporated to dryness.

   The residue (viscous oil) is triturated with 300 cm3 of hot petroleum ether until crystallization occurs. The crystals are filtered off with suction and washed with plenty of hot petroleum ether. Recrystallization from acetone petroleum ether gives a total of 91 g (81%) carbobenzyloxy-L-valyl-L-tyrosine methyl ester (V) mp. 144-147, [u] D = + 19 4 (c = 1, 06 in chloroform).



  A total of 17 g of a by-product are obtained from the petroleum ether solution and from the mother liquor of the above crystals, mp 128-130 (from petroleum ether). It is 1- (carbobenzoxy-I, valyl) -1,3-dicyclohexyl urea. 2. L-valyl-L-ty; -osifa-methyl ester (V). a) L-valyl-I, - tyrosine methyl ester hydrobromide: 20.0 g (0.0468 mol) carbobenzoxy-L-valyl-L-tyrosine methyl ester are dissolved in 200 cm3 of a 1,1-n. Solution of hydrogen bromide in glacial acetic acid.

   After one hour at 21, the mixture is evaporated to dryness in vacuo at 45 bath temperature and the oil which remains is triturated with a lot of ether, whereupon it solidifies. The residue is washed with ether; 17.26 g (98%) of L-valyl-L-tyrosine methyl ester hydrobromide are obtained; 206-208. When recrystallizing from methanol-ether, needles with a melting point of 208-209 are obtained; [ab = -I-31 4 (c = 1.08 in methanol).



     r,) L-valyl-L-tyrosine methyl ester: 5.63 g (15 mmoles) of the hydrobromide obtained under a) are added, suspended in 80 cm 3 of ethyl acetate and 2.1 cm 3 (15 mmoles) of triethylamine. The precipitated crystals of triethylamine hydrobromide are filtered off with suction after 10 minutes (2.68 g, corresponding to 99% of theory) and the filtrate is evaporated to dryness.

   The remaining L-valyl-L-tyrosine methyl ester (resin 4.61 g) is immediately processed further as indicated.



  The L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (VIII) used as starting material under c) can be prepared as follows: 3. N-carbobenzyloxy-L-isoleucyl-L-histidine-me - ethyl ester hydrochloride.



  55.5 g (0.27 mol) of dicyclohexylcarbodiimide, dissolved in 100 ml of ethyl acetate, and 71.5 g are added to a solution of 44.0 g (0.26 mol) of L-histidine methyl ester in 50 ml of acetonitrile g (0.27 mol) of N-carbobenzyloxy-L-isoieucine, dissolved in 700 ml of ethyl acetate, were added. Immediately slight heating and soon afterwards precipitation of crystalline dicyclohexylurea occurs. It is cooled with ice for a short time,

     so that the temperature does not rise above about +30. After a few minutes, the mixture turns into a gelatinous mass, which is left at room temperature overnight.



  The mixture is filtered, the residue is triturated twice more with 50 ml of ethyl acetate each time, filtered off with suction and dried at 90 ° in vacuo. From the mixture of dipeptide and dicyclohexyl urea obtained in this way, the former is concentrated with a solution of 25 ml. Hydrochloric acid dissolved out as the hydrochloride in 20 ml of water and 80 ml of methanol and the residue twice with 2 ml of conc. Hydrochloric acid was washed in 10 ml of water and 20 ml of methanol.

   The combined filtrates are evaporated to dryness in vacuo and crystallized from methanol-ethyl acetate. 52.6 g of dipeptide hydrochloride (= 44.6% of theory) of melting point 170-172 are obtained, with decomposition.

   39.1 g of hydrochloride (33.1% of theory) with a melting point of 160-165 (decomp.) Are obtained from the mother liquors by further evaporation and dilution with ethyl acetate. 4. N - carbobenzyloxy - L - isoleucyl - L - histidine hydrazide.



  2.5 g (0.0053 mol) of N-carbobenzyloxy-L-isoleu- cyl-L-histidine methyl ester hydrochloride monohydrate are dissolved in 10 ml of abs. Methanol was refluxed with 1.5 ml of hydrazine hydrate for 1 hour. Most of the solvent is evaporated off in vacuo and 100 ml of ice water are added to the evaporation residue. The initially oily precipitate crystallizes after standing briefly at 0. The carbobenzyloxy-dipeptide hydrazide is filtered off, washed with plenty of water and dried in vacuo over phosphorus pentoxide and sulfuric acid.

   The crude yield is 1.82 g (83%), single recrystallization from boiling water gives 1.6 g of analytically pure product; F. = 186 to 187; [a] D = -51 4 (c = 1.327 in 1N hydrochloric acid) _ - 22 4 (c = 0.558 in methanol).



  The hydrazide is easily soluble in methanol, ethanol, acetone, ethyl acetate and 1-n. Hydrochloric acid, heavy

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 soluble in hot water and insoluble in benzene, ether and petroleum ether.



  5. N-carbobenzyloxy-L-prolyl-L-phenylalanine methoxide.



  To the solution of 1 g (0.004 mol) of crystalline N-carbobenzyloxy-L-proline in 15 ml of absolute tetrahydrofuran is added 0.61 ml (0.0044 mol) of triethylamine at -10 and, after 5 minutes at the same temperature, 0, 42 ml (0.0044 mol) of ethyl chloroformate. The triethylamine hydrochloride falls out immediately as a white crystalline precipitate.



  The solution of L-phenylalanine methyl ester, made from 1.1 g (0.0051 mol) of phenylalanine methyl ester hydrochloride and 0.71 ml (0.0055 mol) of triethylamine, was also prepared and filtered from triethylamine hydrochloride -10 cooled and added to the solution of the mixed anhydride.

   After standing for two hours at room temperature, the precipitate is filtered off and the solvent is evaporated off in vacuo at 40-45. The water-clear residue is taken up in ethyl acetate, washed four times with 5 ml of 1-n each time. HCl solution, twice with water, four times with 2 ml of 2-n. Sodium hydrogen carbonate solution and finally neutral with water. After evaporation and drying, the ethyl acetate solution leaves 1.8 g of yellowish oil which still contains little solvent.

   The crude carbobenzyloxy-L-prolyl-L-phenylalanine methyl ester is immediately decarbo-benzoxylated to give L-prolyl-L-phenylalanine methyl ester.



  6. L-Prolyl-L-phenylalanine methyl ester.



  2.46 g (0.006 mol) of carbobenzyloxy-L-prolyl-L-phenylalanine methyl ester are dissolved in 40 ml of methanol, 6 equivalents of methanolic hydrochloric acid and 0.6 g of palladium carbon (10% Pd) are added and the mixture is hydrogenated at room temperature and normal pressure ( formed CO, absorbed in NaOH). After the uptake of a little more than the calculated amount of hydrogen, the hydrogenation comes to a standstill.

   The solution filtered off from the catalyst is evaporated in vacuo and the residue is taken up in a little acetone. When ether is added, the hydrochloride of L-prolyl-L-phenylalanine methyl ester separates out in crystalline form; Yield: 1.62 g (86 0/0). After recrystallizing twice from acetone-ether, the substance melts at 157-158; [a] D = -410 L (c = 4.15 in H2O).



  To convert it into the free ester, a solution of 1.10 g (0.0035 mol) of the hydrochloride in 1 ml of water is covered with a layer of ethyl acetate and saturated potassium carbonate solution is added at 0 while shaking vigorously. The ethyl acetate extract is dried and evaporated in vacuo and the residue is dried at 0.1 mm Hg. The L-prolyl-L-phenylalanine methyl ester obtained (0.98 g) is an oil which is readily soluble in water and ether and which is directly suitable for further conversions. 7. N-carboben-1yloxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine-methyl ester.



  The solution of 1.49 g (0.035 mol) of n-carbobenzyloxy-L-isoleucyl-L-histidine hydrazide in 15 ml of 1-n. Hydrochloric acid is extracted once with a little ethyl acetate, the ethyl acetate phase again with 3 ml 1-n. Washed hydrochloric acid and the combined hydrochloric acid solutions in a separating funnel covered with ethyl acetate and cooled to 0 with ice.

   275 mg (0.004 mol) of sodium mitrite in 5 ml of ice water are slowly added dropwise, the mixture is left to react for 3 minutes and the hydrochloric acid solution is made alkaline to phenolphthalein with 3 ml of saturated potassium carbonate solution. The aqueous solution is extracted two more times with a lot of ethyl acetate while cooling with ice, and the organic phases are washed neutral with water.



  The ice-cold azide solution, dried over sodium sulfate, is filtered into the freshly prepared solution of L-prolyl-L-phenylalanine methyl ester, which has been cooled to 0 and freed from triethylamine hydrochloride; prepared from 1.24 g (0.004 mol) of L-prolyl-L-phenylalanine methyl ester hydrochloride and 0.55 ml (0.004 mol) of triethylamine in 15 ml of abs. Ethyl acetate.



  The mixture is left to react for 18 hours at 0-5 and 2 hours at room temperature, the solvent is evaporated to half at 40 in vacuo and washed with 1-n. Hydrochloric acid, ice-cold 2-n. Soda solution and neutral with water; dries over sodium sulfate and evaporates the ethyl acetate in vacuo.



     The yield is 2 g (85%) of yellowish amorphous carbobenzyloxy tetrapeptide ester. The 36-stage distribution between 80% methanol: chloroform carbon tetrachloride = 1: 1: 1 gives 1.6 g of pure N-carbobenzyloxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester. After a single reprecipitation from methanol-water, the analysis fraction shows F = 105-110;

   [a] D = -56 4 (c = 0.971 in methanol).



  The N-carbobenzyloxy tetrapeptide ester is readily soluble in most organic solvents, with the exception of ether and petroleum ether.



  B. L-isoleucyl-L-histidyl-L-pi-olyl-L-phenylalanine methyl ester.



  a) Dihydrobromide: 1.72 g (2.61 mmol) of N-carbobenzyloxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are mixed with 8.3 em3 1,1-n. Hydrogen bromide in glacial acetic acid, whereupon solution soon occurs. After standing for two hours at 21, the glacial acetic acid is completely distilled off at 0.1 mm Hg and 25 and the remaining resin is triturated with ether, whereby it becomes granular.

   The crude L-isoleucyl-L-histidyl-L-prolyl-L-phenyl-alanine methyl ester dihydrobromide weighs 1.79 g, m.p. 130-140.



     @) Free ester: 1.96 g (2.85 mmol) of L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester dihydrobromide is dissolved in a little water, a slight turbidity is removed by washing with a little ethyl acetate , the aqueous solution with 50 cm3 chloroform

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    and saturated potash solution is added while cooling with ice until the pH of the aqueous phase is = 10.

   The ester that has separated out dissolves in the chloroform phase when shaken. The chloroform solution is washed once with a little saturated sodium sulfate solution; Potash and sodium sulfate solutions are washed with a further 30 cm3 of chloroform. After drying with sodium sulfate and evaporation, the chloroform solutions leave 1.31 g of L-isoleu- cyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (VIII) as a resin which is immediately processed further. Yield 88 0/0.



  Example 2 a) A'-Ca-bobenzyloxy-L-asparaginyl-nitro-L-argittyl-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-hhenylalanine-inethyl yter.



  A solution of 467 mg (1 mmol) N-carboben-zyloxy-L-asparaginyl-nitro-L-arginine and 272 mg (0.35 mmol) I-- valyl-L-tyrosyl-L-valyl-L-histidyl- L-prolyl-L-phenylalanine methyl ester in 2.5 ml of dimethylformamide is mixed with 237 mg (1 mmol) of 1-cyclohexyl-3-morpholinylethylcarbodiimide. After standing for 24 hours at room temperature, the reaction solution is freed from dimethylformamide at 0.1 mm Hg and 35.

   The residue is triturated with ice water and the deposited solid material is washed with water and dried in vacuo at 30. The crude product is triturated with acetone, then extracted with boiling acetone and finally washed with ice-cold methanol. The material purified in this way (289 mg, 67%) is a pale yellowish powder of F. 207-210.



  For further purification, the substance is distributed in countercurrent between n-butanol and 0.1 l acetic acid over 30 steps (K = 4.17). Fractions 23-28 yield 132 mg of pure product, and fractions 17-22 (74 mg) containing a by-product, after redistribution, yield a further 34 mg of pure material; total 166 mg (39 0/0). After reprecipitating twice from methanol, the substance melts at 214-216; [a] D = -27 2 (c = 2.22 in dimethylformamide). b) L-asparaginyl-L-argiiiyl-L-valyl-L-tyrosyl-L-vcrlyl-L-histi, -lyl-L-prolyl-L-phenylalanine-diethyl ester.



  85 mg (0.07 mmol) N-carbobenzyloxy-L-asparaginyl-nitro-I: arginyl-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine- methyl esters are suspended in 10 ml of methanol, brought into solution by adding 0.3 M equivalents of methanolic hydrochloric acid and shaken in a hydrogen atmosphere in the presence of 0.3 g of palladium carbon (100g0 Pd) at room temperature and normal pressure for 15 hours. The neutral solution is filtered off from the catalyst and evaporated in vacuo.

   The semi-solid reaction product is washed with acetone and, after reprecipitation from methanol-ether, gives 51 mg (63%) tri-hydrochloride of the octapeptide methyl ester. c) L-_. 4sparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-val yl-L-liistid yl-L-prolyl-L-phenylalanine.



  a) 48 mg (0.042 mmol) of L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester tri-hydrochloride are added in 0 , 5 ml of methanol suspended and gradually increased over 1 hour with 0.3 ml of 1-n. Sodium hydroxide solution (about 7 equivalents) is added so that the solution always has a pH between 10.5 and 11.5.

   After a further 30 minutes, the solution is freed from methanol in vacuo at room temperature, with 1-n. Acetic acid adjusted to pH 7.4 and lyophilized. The mixture of free peptide and inorganic salts (79 mg) obtained as residue is distributed by countercurrent distribution in the butanol / 0.1-n system. Fractionated ammonium hydroxide. The pure octapeptide is obtained as a colorless powder that is soluble in water and methanol, more difficult in ethanol and insoluble in acetone.



     13) 400 mg of crude octapeptide methyl ester (containing about 3 mol of HCl + 1 time NH4C1) are dissolved in 4 ml of methanol (clear, brown-yellow solution) and, after the addition of 8 ml of 1-n. K "CO3 in H, 0 at room temperature for 1 hour. Finally, the solution, which contains a little brown flakes as a sediment, is neutralized to pH = 6 with 4N HCl, frozen with acetone-CO, and im High vacuum lyophilized, giving 930 mg of a brownish powder which, in addition to the peptide, contains about 590 mg KCl.



  The total amount is distributed over 30 stages in the system n-butanol-0.33 m NH4COOH (ammonium formate) in H.0 (pH 6.5). Phase volume per I 0 ml.



  The free octapeptide shows G = 0.17 and is isolated from the combined tube contents 0-8 by extracting three times with 400 ml of a butanol-methanol mixture (3: 1) each time and washing the organic solution twice with 30 ml of 5 0 / 0 NH4COOH (ammonium formate) in H20. After evaporation at 45 (11 mm Hg) and drying in a high vacuum (8 hours at 40), a total of 190 mg of a white powder which is readily soluble in water is obtained, which in a biological test on kidneyless rats shows an effect 20 times stronger than norepinephrine.

   In the paper chroinatogram the substance proves to be uniform, with an R, value = 0.19 in the system sec. Butanol-3% NH3 (120:44).



  As a second fraction, 140 mg of unsaponified starting material are obtained back from pipes 18-30 (G = 42) during distribution (-octapeptide methyl ester). R, value in the above system = 0.30.



  y) 500 mg octapeptide methyl ester [same starting material as in a)] are concentrated in 2 ml of HCl. dissolved and left to stand at 39 for 100 minutes. The solution is then evaporated to a Troclme in a high vacuum. the residue is finely pulverized and dried again for a few hours at 40 in a high vacuum

 <Desc / Clms Page number 10>

    net. This gives 510 mg of a light brown crude product which already has 20 times the activity of noradrenaline on the kidneyless rat.



  For cleaning, 300 mg of it are dissolved in 2 ml of water, with 2-n. NaOH neutralized to pH 6 and distributed over 30 levels in the system n-butanol-methanol-0.33 m NH4COOH - 3: 1: 4 (pH 6.5), each with 10 ml phase volume. From the tubes 5 to 14, 170 mg of hydrolyzate can be obtained as a colorless, water-soluble substance by working up as under a), which in the kidneyless rat is 20 to 25 times more active than noradrenaline. In the paper chromatogram, sec. Butanol - 3 0 / a NH3 (120: 44) splits into two compounds, with Rf values 0.13 and 0.19.

   The second spot shows the greater color intensity with ninhydrin.



  The compound with the Rf value 0.13 represents L-asparagyl-L-arginyl-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine, while the Compound with the Rf value 0.19 is the octapeptide with the asparaginyl residue.



  Tubes 15-19 of the Craig distribution contain 55 mg of mixed fraction, while 22 mg of starting material can be regenerated from tubes 20-29 (R, = 0.30).



  In addition to the Craig distribution over many stages, the two hydrolysis products (asparagyl and asparagyl compounds) can also be chromatographed on a column of paper powder with suitable solvent systems, e.g. B. BuOH-MeOH NH4COOH, completely separate from each other.

   The L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester used as starting material can be prepared as follows: 1. N-curbobenzyloxy-L-valyl-L-tyrosine ethyl ester . A solution of 50.2 g (0.2 mol) of carbobenzyl oxy-L-valine and 41.8 g (0.2 mol) of L-tyrosine ethyl ester in 400 ml of acetonitrile is mixed with 45.3 g (0.22 mol) 1,3-Di-cyclohexyl-carbodiimide was added and the mixture was left to stand for 5 hours at room temperature.

   The precipitated mixture of di-cyclohexylurea and the reaction product is filtered off and the dipeptide derivative is extracted from this mixture with ethyl acetate. The original reaction solution is evaporated in vacuo, the residue is dissolved in ethyl acetate, this solution with 2-n. Hydrochloric acid, 2-n. Washed sodium hydrogen carbonate solution and water, dried and combined with the above ethyl acetate solution of the originally separated reaction product.

   The residue obtained on evaporation in vacuo crystallizes on addition of ether and gives 74.5 g (84%) of carbobenzyloxy-L-valyl-L-tyrosine ethyl ester with a melting point of 142-145. Recrystallization from acetone-ether gives 68.6 g of pure material with a melting point of 148-149; [a] 24 - -f- 33 1 (c = 3.96 in chloroform).



  2. N-carbobenzyloxy-L-valyl-L-tyr-osine.



  70.7 g (0.16 mol) of carbobenzyloxy-L-valyl-L-tyrosine-ethyl ester are dissolved in 300 ml of methanol and, while stirring, within 15 minutes at about 10 with 400 ml of 1-n. Sodium hydroxide solution added. The clear reaction solution is left to stand for 2 hours at room temperature, then freed from methanol in vacuo and a little solid material is filtered off. When the filtrate is acidified with dilute hydrochloric acid, the carbobenzyloxy-L-valyl-L-tyrosine separates out in crystalline form and is filtered off, washed with water and dried in vacuo at 50;

   F. = 161-163. [a] D = +26 1 (c = 4.01 in pyridine). The yield is 64.01 Q (97 0/0). 3. N-CarbobenZylo.xy-L-valyl-L-histidine-tnetlayl-ester. A solution of 25.1 g (0.1 mol) of carbobenzyl-bxy-L-valine and 16.7 g (0.1 mol) of L-histidine methyl ester in 100 ml of acetonitrile is about 10 with 26.1 g of 1-cyclohexyl-3-morpholinylethyl-carbodiimide (0.11 mol) are added and the mixture is left to stand at room temperature.

   After about 1 hour, a gelatinous product begins to separate out, which is filtered off after 15 hours and washed with acetonitrile: 30.8 g (77% / a); F. = 152-156. The carbobenzyioxy-L-valyl-L-histidine methyl ester recrystallized from acetone-ether melts at 161-163; [a] 21 = -22 2o (c = 2.04 in ethanol).



  4. L-valyl-L-histidine methyl ester.



  10 g (0.025 mol) of carbobenzyloxy-L-valyl-L-histi- dine methyl esters are dissolved in 100 ml of methanol, mixed with a solution of 0.03 mol of hydrochloric acid gas in methanol and in the presence of 2 g of palladium carbon ( 10% Pd) hydrogenated at room temperature and normal pressure (CO formed = simultaneously absorbed in sodium hydroxide solution). After the calculated amount of hydrogen has been absorbed, the hydrogenation comes to a standstill after 1 hour.

   The catalyst is filtered off and the reaction solution is evaporated in vacuo. The hydrochloride of the base obtained as an oil (8.7 g) is dissolved in a little water and, after the solution has been covered with ethyl acetate, while shaking, is made strongly alkaline with saturated potash solution. The ethyl acetate phase containing the reaction product is dried and evaporated in vacuo, and the L-valyl-L-histidine methyl ester (6.5 g) obtained as an oily residue is reacted further directly. 5. N-Carbobertzyloxy-L-valyl-L-tyrosyl-L-valyl-L-histidine-nzethyl ester.



  A solution of 9.94 g (0.024 mol) of carbobenzyloxy-L-valyl-L-tyrosine in 100 ml of acetonitrile is mixed with 6.64 g (0.28 mol) of 1-cyclohexyl-3-morpholinyl-ethyl-carbodiimide and then with 6.5 g (0.024 mol)

 <Desc / Clms Page number 11>

    L-valyl-histidine-methyl ester added. After 15 hours, the precipitated reaction product is filtered off and washed with acetonitrile.

   The crude product (11.2 g = 70%) with a temperature of 208-211 is dissolved in 500 ml of hot methanol, the solution is concentrated to 100 ml and the same volume of ether is added. The excreted pure carbobenzyl-oxy-L-valyl-L-tyrosyl-L-valyl-L-histidine methyl ester melts at 222-224.

   After countercurrent distribution in the methanol-water 4: 1 / chloroform-carbon tetrachloride 1: 1 system, the substance shows the F. 224 to 226e and [ri] D - # -31 "-_ @ - 2 (c - = 2.09 in glacial acetic acid).



  6. N-carbobenzyloxy-L-valyl-L-tyrosyl-L-valyl-L-hi.StIChil. A suspension of 3.32 g (0.005 M01) of carbobenzyloxy-L-valyl-L-tyrosyl-L-valyl-L-histidine methyl ester in 30 ml of methanol is mixed with 15 ml of 1-n. Sodium hydroxide solution added, whereupon the starting material quickly goes into solution. The reaction solution is left to stand for 2 hours at room temperature, then freed from methanol in vacuo and - after some flakes have been separated off by filtration - with 20 ml of 2-n. Acetic acid acidified.

   The precipitated solid material is centrifuged off, suspended several times in water and centrifuged off again and finally washed with acetone and methanol and dried at 40 in a vacuum. The carbobenzyl-oxy-L-valyl-L-tyrosyl-L-valyl-L-histidine is obtained in a yield of 2.74 g (840 / a) and, after washing with boiling methanol, melts at 236 to 238; [rX] D = --- 10 2 (c - 2.10 in glacial acetic acid).

   The substance. is insoluble in all common organic solvents except in dimethylformamide and glacial acetic acid. 7. N-carbobenzyloxy-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanih methyl ester.



  1.95 g (0.003 mol) of carbobenzyloxy-L-valyl-I, - tyrosyl-L-valyl-L-histidine are suspended in 10 ml of dimethylformamide and mixed with 0.83 g (0.003 mol) of I_ - Prolyl - L - - phenylalanine methyl ester and 0.84 g (0.0035 mol) of 1 - cyclohexyl - 3 - morpholinyl - ethyl carbodiimide added. After 1 hour the starting material is completely dissolved.

   The reaction solution is left to stand overnight at room temperature, then freed from dimethylformamide at 0.1 mm Hu, and ice water is added to the residue. The precipitated solid material is twice with 2-n. Hydrochloric acid washed (the reaction product forming a hydrochloride which is soluble in water but not in 2-n. Hydrochloric acid) by treatment with 0.5-n. Potassium carbonate solution converted back into the basic form, washed several times with water and dried in vacuo at 35. The crude product obtained (2.40 g) is taken up in 100 ml of acetone, insoluble material (0.30 g) is filtered off and evaporated in vacuo.

   After trituration with ethyl acetate, the residue gives 1.57 g (58% / a) of solid material from F. 151-156. .



  For further purification, 1 g of this substance is distributed over 48 steps by countercurrent distribution in the system methanol-water 4: 1 / chloroform-carbon tetrachloride 1: 1. The majority of the material (0.84 g) is in fractions 15-28 with a maximum weight in fraction 21 (K = 0.78). After reprecipitation from acetone, these fractions give 0.66 g of amorphous material with a temperature of 158-162. After repeated reprecipitation from a lot of acetone, the substance shows F. 161-163 and [a] D = -56 2 (c 1.38 in ethanol).



  B. L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester.



  635 mg (0.7 mmol) of carbobenzyloxy-L-valyl-L-tyrosyl-L-valyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are triturated with 1 ml of glacial acetic acid until a uniform solution is obtained, and with 1.9 ml 1.6-n. Hydrogen bromide (3 mmol) was added in glacial acetic acid. When standing at room temperature, the reaction product begins to separate out as an oil after 10 minutes. The mixture is shaken for 2.5 hours and then completely evaporated at 0.1 mm Hg and 30%. After washing with ether and acetone, the residue gives 648 mg of dihydrobromide of the hexapeptide methyl ester as an almost colorless powder.



  To convert into the free ester, a solution of 562 mg (0.6 mmol) of this substance in 2 ml of water is made strongly alkaline with saturated potassium carbonate solution and extracted four times with 20 ml of cold chloroform each time. The chloroform extracts are washed with a little water, dried and evaporated in vacuo. When rubbed with ether, the oily residue yields 272 mg (59%) of the peptide ester as a pale yellow powder, which is further reacted directly.



  Example 3 a) N-f'arbobenZyloxy-L-asparaginyl-nitro-L-argiizyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-pl7eti.ylalaniya-methvlester .



  760 mg (0.96 mmol) L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester and 1.35 g (2.88 mmol) N-carbobenzyloxy-L- Asparaginyl-nitro-L-arginine in 10 ml of dimethylfprmamide are mixed with 690 mg (2.9 mmol) of 1-cyclohexyl-3- (morpholinylethyl) -carbodiimide and left to react for 2 days at room temperature. The dimethylformamide is then removed in a high vacuum and the remaining oil is triturated five times with 5 ml of water each time while cooling with ice, during which it becomes partially flaky.



  The crude product, 2.4 g, is washed repeatedly with a lot of ice-cold acetone and methanol for further purification and gives 250 mg of carbobenzyl'oxy octapeptide ester. Another 180 mg of carbobenzyloxy octapeptide ester are obtained from the acetone and methanol extracts.

 <Desc / Clms Page number 12>

 The crude carbobenzyloxy-L-asparaginyl-nitro-L-ar, inyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-1 = phenylalanine methyl ester (430 mg) shows F. = 175 up to 190 "and is used directly for further processing.



  b) L-asparaginyl-L-ar, -ihyl-L-valyl-L-tyrosyl-L-leiicyl-L-histidyl-L-poly l-L-phenylalanine methyl ester. 410 mg (0.33 mmol) of carbobenzyloxy-asparaginyl-nitro-L-arginyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are suspended in 10 ml of methanol and by adding 1 ml of 1.39-n. (4 equivalents) hydrochloric acid in methanol, brought into solution. The flaky impurities are filtered off and the solution is hydrogenated in the presence of 250 mg of Pd-charcoal (10% Pd) under normal conditions.

   (The CO z formed is absorbed in a 2-hydrogenation vessel with dilute sodium hydroxide solution.) After hours and uptake of slightly more than the calculated amount of hydrogen, the hydrogenation is complete. The catalyst is filtered off and the solution is evaporated to dryness in vacuo at 40 '. 360 mg of resin are obtained (406 mg calculated for trihydrochloride and the equimolar amount of ammonium chloride).



  The product is processed further without further purification. It is easily soluble in water and methanol. c) L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-leiieyl-L-histidyl-L-prolyl-L-phenylalanine.



  360 mg of crude L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine ester trihydrochloride are dissolved in 10 ml 66 "/ aigem methanol and in Portions with 1/10 N sodium hydroxide solution are added so that the pH value of the solution remains at 10.5-11 for 20 minutes (total consumption of 1.1.0 N NaOH = 18.5 ml) the solution is adjusted to pH 7-8 with solid CO, and the impurities which have separated out are filtered off (10 to 20 mg), the methanol is largely evaporated off in vacuo at 351 'and the solution is made with 2-n.

   Soda solution to pH 9.



  The aqueous solution is extracted four times with 100 ml of water-saturated n-butanol each time, the butanol extracts are washed once with 10 ml of sodium sulphate solution and dried over sodium sulphate. The evaporation residue is washed three times with 3 ml each of dry butanol, whereby 50 mg of easily soluble components can be removed.



  190 mg of L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine are obtained as a fine, slightly gray-colored powder which is dissolved in water and methanol is readily soluble.



  A sample of the raw product shows an effect on the rat that is as strong as that of noradrenaline.



  The carbonamide group of the asparaginyl residue can be split off as follows: 2.08 g (1.75 mmol) of L-asparaginyl-L-arginyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl- L-prolyl-L-phenylalanine methyl ester trihydrochloride is heated in conc. HCl to 37 'for 85 minutes. After about 10-15 minutes a clear solution appears. The hydrochloric acid is quickly evaporated off under reduced pressure at 25 and the residue is dried over potassium hydroxide and phosphorus pentoxide in a high vacuum.



  The yield is 2 g of amorphous L-asparagyl-L-ar, ynyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine trihydrochloride, which is still contaminated with ammonium chloride .



  1.26 g of the crude product are dissolved in 20 ml of ethanol and the peptide is reprecipitated with 120 ml of acetone. Two further reprecipitations give 1.08 g of white, granular octapeptide trihydrochloride. In animal experiments, the reprecipitated product shows the same activity as noradrenaline (Peart test) and the double effect on the kidneyless rat.



  A sample (200 mg) of the reprecipitated L-asparagyl-L-arginyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl - L - phenylalanine - trihydric chloride is, according to Craig, over 30 Levels between n-butanol and 0.3 cm-3 ammonium acetate 1: 1 (pH = 6.5-7) distributed. G = 0.67. The solutions are evaporated in vacuo at 40 and the ammonium acetate is sublimed off in a high vacuum at 40 for 30 minutes.



  Fractions 7-16 (maximum fraction 12) show the same activity in the Peart test as the undistributed product.



  The ammonia and total nitrogen determinations show that the asparagine was mainly saponified to aspartic acid during the acidic saponification. Qualitative halogen samples show that the octapeptide is present as a mono- or dihydrochloride.



  The L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester used as starting material can be prepared as follows: 1. N-Carboben @ yloxy-L-leucyl-L-histidine -methyl- ester.



  To a solution of 16.9 g (0.1 mol) of L-histidine methyl ester and 21.6 g (0.105 mol) of dicyclohexyl carbodiimide in 100 ml of abs. Acetate and 20 ml of acetonitrile are 26.5 g (0.1 mol) of N-carbobenzyloxy-L-leucine, dissolved in 200 ml of abs. Ethyl acetate, added all at once and soon too. a jelly solidifying mixture shaken for 30 minutes at room temperature.

   After leaving to stand overnight, the solvent is suctioned off from the crumbly mass as much as possible (while the filter residue is repeatedly triturated with a little fresh ethyl acetate) and dried in vacuo at 90 °. 47.8 g of a mixture of dicyclohexylurea and dipeptide are obtained, from which the latter is dissolved out with 3 portions of 50 ml each of cold methanol. The combined extracts still contain small amounts of dicyclohexyl urea and are purified by dissolving them in methanol and reprecipitating them with ethyl acetate / petroleum ether.

   29.3 g of N-carbobenzyloxy-L-leucyl-L-histidine methyl ester (70.5% of theory) of melting point 131-132 are obtained.

 <Desc / Clms Page number 13>

 From the filtrate of the reaction mixture are extracted with 2-n. Hydrochloric acid still 4.0 g of dipeptide (9.5% of theory) obtained with a melting point of about 120-125, which can be further purified by reprecipitation from methanol-ethyl acetate-petroleum ether.



  2. N-carbobenzyloxy-L-leucyl-L-histidine hydrazide. 10 g (0.024 mol) of N-carbobenzyloxy-L-leucyl-L-histidine methyl ester, in 40 ml of abs. Dissolved ethanol, 3 ml of hydrazine hydrate are added and the mixture is left to react for 2 days at room temperature. The solvent is evaporated off in vacuo at 40 and the residue is triturated with 100 ml of water. After three hours of standing at 0, the crystalline precipitate is filtered off, washed in portions with 20 ml of water and the crude hydrazide is dried over sulfuric acid and P, 0.



  Crystallization from 100 ml of boiling water gives 6.8 g (68%) of pure carbobenzyloxy-L-leucyl-L-histidine hydrazide, m.p. 168-169, [a122 = -27 4 (c = 0.808 in methanol) and [a] D = -52 4 (c = 0.733 in 1-n. HCl).



  The hydrazide is in cold ethanol and methanol as well as in cold 1-n. Hydrochloric acid is readily soluble, but only in warm acetone; it is insoluble in ethyl acetate.



  3. N-carbobezzwyloxy-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine benzethyl ester.



  The solution of 6.6 g (0.0158 mol) of N-carbobenzyloxy-L-leucyl-L-histidine hydrazide in 50 ml of 1-n. Hydrochloric acid is extracted once with a little ethyl acetate, the ethyl acetate solution again with 5 ml 1-n. Washed hydrochloric acid and the combined hydrochloric acid solutions covered with fresh ethyl acetate and cooled to 0.

   A solution, cooled to 0, of 1.14 g (0.0165 mol) of sodium nitrite in 5 ml of water is then slowly added, the mixture is left for 5 minutes and the hydrochloric acid azide solution is made alkaline to phenolphthalein with 9 ml of saturated sodium carbonate solution. The aqueous phase is extracted two more times with a lot of ethyl acetate while cooling with ice, then the ethyl acetate solutions are washed neutral with water and dried over sodium sulfate.



  From 5 g (0.016 mol) of L-prolyl-L-phenylalanine methyl ester hydrochloride, suspended in 50 ml of dry ethyl acetate, the solution of the free 1.- is obtained by adding 2.25 ml (0.0162 mol) of triethylamine. Prolyl-I: phenylalanine methyl ester obtained. This solution is cooled to 0 and mixed with the azide solution prepared according to the above procedure.



  After standing at 0-5 for 18 hours and at room temperature for 2 hours, the solvent is evaporated off in vacuo at 40 l -, and washed with 1-n. Hydrochloric acid, ice-cold 2-n. Soda solution and neutral with water. The hydrochloric acid, soda and water extracts are washed again with plenty of ethyl acetate in two further separating funnels.



  After evaporation of the ethyl acetate, 9.17 g (87%) of N-carbobenzyloxy-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are obtained as an amorphous powder.



  The multiplicative distribution according to Craig over 36 steps of 1.32 g of carbobenzyloxytetrapeptide ester between 80% methanol and chloroform: carbon tetrachloride 1: 1 gives 1.2 g of pure product. After one recrystallization from methanol-water, the analysis fraction shows F. = 110-115 [a] D_ -57-4 (c = 1.031 in methanol).



  The N-carbobenzyloxy-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester is readily soluble in alcohol, methanol, acetone, ethyl acetate and very dilute hydrochloric acid; insoluble in petroleum ether and ether.



  4. L-Leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester.



  3.4 g (0.005 mol) of carbobenzyloxy-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester in 30 ml of abs. Methanol are hydrogenated to a standstill at room temperature with the addition of 2 equivalents of methanolic hydrochloric acid in the presence of 600 mg of palladium carbon (10 0 / a0 Pd) at room temperature. After about 13/4 hours a little more than the calculated amount has been absorbed. The catalyst is filtered off and the solution is evaporated at 40 in a vacuum.

   The residue is dissolved in 5 ml of ice-cold water, covered with 50 ml of chilled chloroform and made alkaline with phenolphthalein with saturated potassium carbonate solution. The aqueous solution is extracted two more times with 20 ml of chloroform each time, the organic phases with conc. Washed sodium sulfate solution and dried over sodium sulfate. After evaporation of the solvent, 2.68 g of amorphous tetrapeptide ester remain, which are used directly for the further reaction.



  5. N-carbobenzyloxy-L-valyl-L-tyroYyl-L-leucyl-L-hi.stidyl-L-prolyl-L-phenylalanine methyl ester.



  The solution of 2.1 g (0.005 mol) of N-carbobenzyloxy-L-valyl-L-tyrosine in 10 ml of dry acetonitrile is mixed with 1.16 g (50/0 excess) of dicyclohexylcarbodiimide and the solution of 2.68 g (0.005 mol) of L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester in 10 ml of acetonitrile. The dicyclohexylurea is currently eliminated. After about 5 hours, the N-carbobenzyloxy-hexapeptide ester begins to separate out as a gelatinous mass.

   The mixture is left to stand for 20 hours at room temperature and the acetonitrile is then evaporated at 40 under reduced pressure. The evaporation residue is digested with 10 ml of ice-cold methanol and washed five more times with 1 ml of cooled methanol each time. The methanol is evaporated off in vacuo and the residue in chloroform three times with 2-n. Hydrochloric acid (five and two times 2 ml), washed five times with 2 ml of ice-cold sodium bicarbonate solution each time and with water until neutral.

   The solution, dried over sodium sulfate, is concentrated to half and once: briefly boiled with activated charcoal.

 <Desc / Clms Page number 14>

 Complete evaporation of the solvent gives 4.2 g of crude N-carbobenzyloxy-hexapeptide ester, which is still contaminated with a little dicyclohexylurea. The crude carbobenzoxy ester is dissolved in a little cold acetone, the remaining dicyclohexylurea is filtered off and precipitated again with a lot of ether.



  3.7 g (79 0! 0) of N-carbobenzyloxy-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are obtained.



  After two further purifications from acetone / ether, the analysis preparation shows F. = 142-147 / 149 "[a] D ---- -600 i- 40 (c = 1.291 in methanol). 6. L-valyl-L-tyrosyl -L-leucyl-L-izistidyl-L-prolyl- I -phenylalanine methyl ester.



  To 970 mg (- 0.001 mol) of N-carbobenzyloxy-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are added 6 ml of an approx. 2-n. Solution of hydrogen bromide in glacial acetic acid. The substance clumps together to form a sticky mass. After stirring for one hour with a magnetic stirrer, a clear solution appears; it is left to react for a further hour at room temperature, the glacial acetic acid is then evaporated in vacuo at 400 and the residue is triturated twice with abs. Ether.

   The granular dihydrobromide of L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester is dissolved in 10 ml of water, the aqueous solution is extracted once with a little ethyl acetate, the washes Ethyl acetate phase once more with 2 ml of water and freed the combined water solutions from dissolved ethyl acetate in vacuo. With 2-n. Na bicarbonate solution, the cooled solution is adjusted to pH 8, the hexapeptide ester which has separated out is filtered off and washed neutral with ice water.

   The dried product (730 mg =: 89 0l0) shows F. = 130-1400 and can be purified from chloroform / ether. [a] D = -530 40 (c = 0.636 in methanol).



  Example 4 a) N-carbobenzyloxy-L-asparaginyl- (Ne-carbo-benzyloxy) -L-lysyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine- methyl ester.



  330 mg (0.42 mmol) of L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (see Example 3) and 445 mg (0.84 mmol) of N- Carbobenzyl-oxy-L-asparaginyl- (NE-carbobenzyloxy) -L-lysine are dissolved in 3 cm3 of dimethylformamide, 200 mg (0.84 ml / vol) of 1-cyclohexyl-3-morph @ olinylethyl-carbodiimide are added and the reaction mixture 21 Leave at 220 hours.

      Then it is evaporated to dryness in a high vacuum at 450 and the remaining oil is washed with small portions of water, very dilute ammonia solution and water, whereupon it gradually solidifies. The powder is suction filtered, dried (520 mg) and further washed with acetone and finally with methanol, N-carbobenzyloxy-L-asparaginyl- (N ± -carbobenzyloxy) -L-lysyl-L-valyl-L-tyrosyl-L -leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester can be obtained in the form of a gray, methanol-insoluble powder. About 190-205.

   The powder is dissolved in dimethylformamide and precipitated with ether, 130 mg of flakes with a melting point of 210-215 being obtained. Yield 24 (1 / o of theory. The protected octapeptide is sparingly soluble in most common solvents, but easily soluble in dimethylformamide.



  b) L-asparaginyl-L-lysyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester trihydrobromide.



  53 mg (41 icMol) N-carbobenzyloxy-L-asparaginyl- (N = -carbobenzyloxy) -L-lysyl-L-valyl-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester of m.p. 210-215 are mixed with 0.3 em3 2.5-n. HBr in glacial acetic acid and the mixture kept at 21 for 11/2 hours with exclusion of moisture, the material slowly going into solution.

   Finally, the HBr-glacial acetic acid mixture is completely removed in a high vacuum at a bath temperature of 250 ° C and the remaining resin is washed with acetone, which solidifies to a granular powder. The extremely hygroscopic product is suction filtered and washed with a little acetone. After drying, 50 mg of crude L-asparaginyl-L-lysyl-L-valyl-L-tyrosyl-L-leucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester trihydrobromide with a melting point of about 180 to 2200 ( Decomposition). The crude product is processed further without further purification.



  When testing for blood pressure-increasing effects on rats, the product shows on the kidney-less rat: an immediate, clearly hypertensin-like increase in blood pressure. The strength of the effect is about 1 / 10-1 / 15 compared to norepinephrine.



  c) L-asparaginyl-L-lysyl-L-valyl-L-tyt-osyl-L-leucyl-I-histidyl-L-prolyl-L-phenylalanate.



  34 mg (27;., Mol) L-asparaginyl-L-lysyl-L-valyl-L-tyrosyl - L - leu cyl - L-histidyl-L-proiyl-L-phenylalanine methyl ester trihydrobromide of m.p. 180- 2200 are dissolved in 1 cm3 of methanol and 0.1-n dropwise. NaOH was added until the pH of the solution remained at 10-10.5 for 15 minutes. A total of 1.81 cm3 becomes 0.1-n. Added NaOH (6.7 equivalents).

   The pH is then brought to 8 by blowing in CO, the methanol is removed in vacuo, the pH of the aqueous solution is brought to 8.5-9.0 by adding a little soda solution and the aqueous solution is treated with 3 portions of water-saturated n-butanol ( 30, 20, 20 cm3) extracted. The butanol extracts are washed once with 3 cm 31/2 saturated Na, SO4 solution, dried with Na, SO4 and give 18 mg of crude L-asparaginyl-L-lysyl-L-valyl-L-tyrosyl-L-leucyl-L on evaporation -histidyl-L-prolyl-L-phenyl-alanine as a colorless, fine-grain coating.

   The crude product is taken up three times in 3 cm3 of water, with 4 mg of water-sparingly soluble impurities being separated off. The aqueous solutions combined and evaporated to give 14 mg of residue. This is washed with three times 1 cm3 of dry n-butanol.

 <Desc / Clms Page number 15>

 see, with 1 mg of n-butanol more easily soluble components can be removed.



  The N-carbobenzyloxy-L-asparaginyl- (N-carbobenzyloxy) -L-lysine used as starting material can be prepared as follows: 1. N-carbobenzyloxy-L-asparaginyl- (Nf-carbobenzyloxy) - L -lysine methyl ester.



  To a solution, cooled to 0, of 12.0 g (0.041 mol) of N, -carbobenzyloxy-L-lysine methyl ester and 8.6 cm3 (0.061 mol) of triethylamine in 60 cm3 of absolute toluene is added within 15 minutes with the exclusion of moisture A solution, cooled to 01, of 7.4 g of 90% diethyl chlorophosphite (0.048 mol) in 20 cm of absolute toluene was added dropwise.

   The reaction mixture is then kept at room temperature for 2 hours; Then the precipitated tri- @: thylamine hydrochloride is sucked off, washed with a little absolute toluene and the toluene filtrate evaporated to dryness in vacuo, the diethyl phosphitamide of the N-carbobenzyloxy-L-lysine methyl ester as a tough resin is obtained (weight 17.0 g).

   It is dissolved in 60 cm3 of diethyl phosphite, 10.9 g of dry substance. Finely powdered N-carbobenzyloxy-L-asparagine (0.041 mol) was added and the mixture was heated to 80 "for 1 hour, the N-carbobenzyloxy-L-asparagine going into solution after a few minutes. After 1 hour, the diethyl phosphite was completely distilled off under reduced pressure , the remaining resin was taken up in a lot of ethyl acetate and the ethyl acetate solution was washed with dilute hydrochloric acid, water, dilute soda solution and water.

   After drying with Na, SO4 and evaporation, the ethyl acetate phase leaves behind 13.0 g of crude N-carbo-benzyloxy - L-asparaginyl - (N @ - carbobenzyloxy) - L-lysine methyl ester as a gelatinous mass. After reprecipitating twice from methanol-ether, 8.5 g of the product are obtained as a flaky precipitate; M.p. 152 to 161, yield 38% of theory.



  2. N-carbobenzyloxy-L-asparaginyl- (N ± -carboben- @yloxy) -L-lysine.



  5.00 g (0.0092 mol) of N-carbobenzyloxy-L-asparaginyl- (NF-carbobenzyloxy) -L-lysine methyl ester with a melting point of 152-161 are dissolved in 60 cm3 of dimethylformamide and 0.1 N in portions. Sodium hydroxide solution is added until the pH value of the solution remains at 10-10.5 for 20 minutes (a total of 140 cm3 of 0.1N NaOH added). The pH is then brought to 8 by adding some solid CO 2 and the dimethylformamide-water mixture is concentrated to a small volume first in vacuo, then in high vacuum.

   The residue is mixed with water, the sodium salt of N-carbobenzyloxy-L-asparaginyl- (NE-carbobenzyloxy) -L-lysine precipitating as a thick, gelatinous mass. It is brought into solution by adding a lot of water (about 200 cm3), something undissolved is filtered off and the filtrate is treated with conc. HCl brought dropwise to pH = 1.

   The flaky precipitate is filtered off with suction, washed with ice water, dried and methanol-ether recrystallized twice; 3.2 g of N-carbobenzyloxy-L-asparaginyl-NF-carbobenzyloxy) -L-lysine (fine needles) of melting point 17.5-181 are obtained (yield 740/0 of theory). The product is soluble in very dilute alkalis, with 2-n. Sodium hydroxide or soda solution creates a gelatinous, poorly soluble sodium salt. Example S a) N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-lettcyl-L-t), rosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester.



  To the solution of 802 mg (1 miMol) L-leucyl-tyrosyl-L-isoleu cyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester in 6 ml freshly distilled dimethylformamide are added 360 mg (1.5 mmol) dicyclohexylcarbodiimide and after a short time 710 mg (1.5 mmol) N-carbobenzyloxy-L-asparaginyl-nitro-L-arginine in 6 mg dimethylformamide. Allow to react overnight at room temperature,

      the dicyclohexylurea is filtered off and the solvent is evaporated off at 40 in a high vacuum. The foamy residue is dried for a further hour at 40 and 0.01 mm Hg, then triturated with a lot of ethyl acetate and the powdery product is dried again in a high vacuum. For further purification, the crude product is washed repeatedly with plenty of acetone and ice-cold methanol. 280 mg (22%) of N-carbobenzyloxy-octapeptide methyl ester are obtained; F. = 204 to 206.



  b) L-asparaginyl-L-arginyl-L-leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolv l-L-pheny [alanine methyl ester trihydrochloride.



  210 mg (0.22 mmol) N-carbobenzyloxy-L-asparaginyl-nitro-L-arginyl-L-leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine-meth @ ylesters are in 30 m1 abs. Hydrogenated methanol in the presence of 4 equivalents of hydrochloric acid and 200 mg of Pd-charcoal (10 0/0 Pd) under normal conditions. (The CO2 formed is absorbed in a second hydrogenation vessel with dilute sodium hydroxide solution.) After about 17 hours, slightly more than the calculated amount of hydrogen has been absorbed.

   The catalyst is filtered off, the solution is evaporated to a small volume at 40 and a lot of ether is added. The trihydrochloride of the octapeptide ester separates out as a white granular product in addition to ammonium chloride. Yield 240 mg (270 mg calculated for trihydrochloride and equimolar amount of ammonium chloride).



  The product is processed further without further purification. It is easily soluble in methanol, ethanol and water.



  The carbonamide group of the asparaginyl residue can be converted into the carboxyl group as follows: 100 mg L-asparaginyl-L-arginyl-L-leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are concentrated in 2 ml. pure hydrochloric acid

 <Desc / Clms Page number 16>

 Held at 37 for 85 minutes. The hydrochloric acid is quickly evaporated off in vacuo at 25 and the residue is dried over sodium hydroxide and phosphorus pentoxide.



  For cleaning, the foamy crude product is dissolved in 1.5 ml of ethanol with gentle warming and the octapeptide trihydrochloride is precipitated with 8 ml of acetone. Centrifugation and rewashing with 2 portions of 2 ml acetone yield 90 mg L-asparagus, -yl-L-arginyl-L-leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine trihydrochloride.



  In the kidneyless rat, it shows 20 times the activity of noradrenaline.



  By Craig partition or chromatography on paper powder in suitable solvent mixtures (e.g. butanol-methanol-0.33 m NH4-COOH or sec-butanol-3 0.10 NH; 120:44), L-asparagyl L-arginyl -1, - leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-proiyl-L-phenylalanine from L-asaraainyl-L-ar, -inyl-L- leucyl-L-tyrosyl-L-isoleucyl-L- histidyl-L-prolyl-L-phenylalanine.



  The one used as the raw material. L-Leucyl-L-tyrosyl - L - isoleucyl - L - histidyl-L-prolyl-L-phenylalanine methyl ester can be prepared as follows: 1. N-Cnrbobenzyloxy-L-leucyl-L-tyrosine methyl ester .



  940 mg (3.5 mmol) of N-carbobenzyloxy-L-leucine are dissolved in 10 ml of absolute tetrahydrofuran, 800 mg (10%, 1J excess) of dicyclohexyl-carbodiimide are added and then 690 mg (3.5 mmol) of tyrosine methyl ester, dissolved in 15 m1 abs. Tetrahydrofuran. The mixture is left to react overnight, the dicyclohexylurea which has separated out is filtered off and the solvent is evaporated off in vacuo at 40.

   The evaporation residue is washed in ethyl acetate with 1-n. Hydrochloric acid, water, 2-n. Sodium bicarbonate solution and water neutral; the ethyl acetate solution dries over sodium sulfate and the solvent evaporates in vacuo. 1.4 g (90%) of crude carbobenzoxy dipeptide ester are isolated. Reprecipitating the crude ester twice from ethyl acetate / petroleum ether gives pure,

   but amorphous N-carbobenzyloxy-L-leucyl-Lrtyrosine methyl ester. [apD = -14 -i_- 4 (c = 1.108 in methanol).



  The same carbobenzyloxy-L-leucyl-L-tyrosine methyl ester prepared by the mixed anhydride method shows [a] D = -11 2 (c = 1.631 in ethanol).



  2. N-carbobenzyloxy-L-leucyl-L-tyrosine.



  To 2.37 g (5.3 mmol) of N-carbobenzyloxy-L-leucyl-L-tyrosine methyl ester are added 13.5 ml of 1-n. NaOH, shake until completely dissolved (5 minutes) and saponify for 1 hour at room temperature. The alkaline solution is extracted once with 5 ml of ethyl acetate, the organic phase is washed once more with a little water and the combined aqueous solutions are freed from ethyl acetate in vacuo for 10 minutes. With 13.5 ml 1-n. HCl, the carbobenzoxydipeptide is precipitated as a sticky mass that solidifies in the ice box, but dissolves again at room temperature.

   The mother liquor is decanted off, the gummy residue is washed thoroughly three times with water and dried over KOH and phosphorus pentoxide in a high vacuum. 1.9 g (800/0 of amorphous carbobenzyloxy-L-leucyl-L-tyrosine, which could not crystallize, are obtained.

   The multiplicative distribution (Craig basic process) over 30 stages between 80% methanol as the upper phase and chloroform: carbon tetrachloride 1: 1 as the lower phase (K = 1.54) also results in a non-crystalline product.



  3. N-carbobenzyloxy-L-leucyl-L-tyroYyl-L-isoletrcyl-L-histidyl-L-prolyl-L-phenylalartine-nietlayl ester. The solution of 1.03 g (2.4 mmol) of N-carbobenzyloxy-L-leucyl-L-tyrosine in 10 ml of dry acetonitrile is mixed with 520 mg (5% excess) of dicyclohexylcarbodiimide and the Solution of 1.38 g (2.5 mmol) of L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester (cf. Example 1) in 10 ml of acetonitrile. The dicyclohexylurea is currently eliminated.

   After about 6 hours, the N-carbobenzoxy-hexapeptide ester begins to separate out as a gelatinous mass. The mixture is left to stand in the dark at room temperature for 2 days and the acetonitrile is then evaporated off at 40 ° under reduced pressure. The residue is digested with 5 ml of ice-cold methanol, the dicyclohexylurea is filtered off and washed three more times with 1 ml of ice-cold methanol each time. The methanol is evaporated off in vacuo and the residue is dissolved in chloroform three times with 2 ml of 2-n. Hydrochloric acid, washed five times with 2 ml of ice-cold sodium bicarbonate solution and with water.

   The solution, dried over sodium sulfate, is briefly boiled with a little activated charcoal, filtered and the chloroform is completely evaporated off. Single reprecipitation from chloroform "ether yields 1.7 g (76%) carbobenzoxyhexapeptide methyl ester.



  After three reprecipitations from chloroform / ether, the analysis preparation shows F. = 139-141, [a] D = -51 4o (c = 1.87 in methanol). 4. L-Leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanite methyl ester.



  1.2 g (1.28 mmol) of N-carbobenzyloxy-L-leucyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine methyl ester are dissolved in 20 ml of abs. Methanol containing 2 equivalents of hydrochloric acid is split hydrogenolytically in the presence of 300 mg Pd-charcoal (10% Pd). After 2 hours, a little more than the calculated amount of hydrogen has been absorbed.

   The solution freed from the catalyst is evaporated to dryness in vacuo at 40, the residue is dissolved in 10 ml of ice-cold water and 2.6 mmol of sodium carbonate, dissolved in 10 ml of water, are added to the clear solution. The excreted hexapeptide ester is with 25 ml and

 <Desc / Clms Page number 17>

 extracted twice with 10 ml of n-butanol / chloroform 1: 1, the aqueous phases washed three times with 5 ml of water, dried over sodium sulfate and concentrated in vacuo to a small volume. By adding a lot of ether, the hexapeptide ester precipitates out quantitatively. Single reprecipitation from chloroform / ether gives 840 mg (81 1 / o-) amorphous product, m.p. = 130 to 140.

   The product is sufficiently pure for further processing.

 

Claims (1)

PATENT CLAIM Process for the production of new octapeptides of the formula EMI17.9 wherein R1 is an amino-lower alkyl group and R2 is a lower alkyl group, characterized in that an L-asparaginyl-La- (amino-lower-alkyl) -amino-acetic acid, a La-amino-lower alkyl-acetic acid, L-tyrosine, a La- Amino-lower alkyl-acetic acid, L-histidine, L-proline and L-phenylalanine with intermediate protection of amino resp. Carboxyl groups combined with one another by condensation. SUBClaims 1. The method according to claim, characterized in that an L-asparaginyl-nitro-L-arginine, the α-amino group of which is protected by a group which can be split off by means of hydrogenating agents, in the presence of a carbodiimide with an L-valyl-L-tyrosyl -L-valyl-L-histidyl-L-prolyl-L-phenylalanine ester condensed. 2. Process according to patent claim, characterized in that an L-asparaginyl-nitro-L-arginine, the α-amino group of which is protected by a group which can be split off by means of hydrogenating agents, is reacted with an L-valyl-L-tyrosine ester in the presence of a carbodiimide , the tetrapeptide ester obtained is converted into the free acid using an alkaline agent and the tetrapeptide is condensed in the presence of a carbodiimide with an L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine ester. 3. Process according to patent claim and dependent claims 1 and 2, characterized in that the carbonylamide group of the asparagine residue is saponified to form the carboxyl group in the octapeptide obtained.