US3761461A - D-ser1-nle4-lys17-lys18-val-nh2 25-alpha-1-25 acth and derivatives thereof - Google Patents

Abstract

THE PRESENT INVENTION PROVIDES THE PENTACOSAPERPTIDES OF GENERAL FORMULA D-SERYL-L-TYROSYL-L-SERYL-L-NORLENCYLX-L-HISTIDYL-L-PHENYLALANYL-L-ARGINYL-L-TRYPTOPHANYL-GLYCYL-L-LYSYL-L-PROTYL-L-VALYL-GLYCYLL-LYSYL-L-LLYSYL-L-LYSYL-L-LYSYL-L-PROLYL-LVALYL-L-LYSYL-L-VALYL-L-TYROSYL-L-PROLYL-L-VALINAMIDE, IN WHICH X SIGNIFIES AN L-GLUTAMYL OR L-GLUTAMINYL RADICAL, THEIR THERAPEUTICALLY ACTIVE ACID ADDITION SALTS AND HEAVY METAL COMPLEXES. THE PENTACOSAPEPTIDES OF THE ABOVE FORMULA, THEIR SALTS AND HEAVY METAL COMPLEXES EXHIBIT A HIGH ADRENOCORTICOTROPIC EFFECT. THE PRODUCTION OF THESE PEPTIDES IS FURTHERMORE DESCRIBED.

Description

United States Tatent O 3,761,461 1D-S'er -Nle -l[.ys -Lys -Val-NI-l t- ACTH AND DERIVATIVES THEREOF Janos Pless, Basel, Stephan Guttmann, Allscllwil, and

Roger Boissonnas, Bottmingen, Switzerland, assignors to Saudoz Ltd. (also known as Sandoz A.G.), Basel, Switzerland No Drawing. Filed Jan. 10, 1967, Ser. No. 608,448 Claims priority, application /Svgitzerland, Ian. 12, 1966,

Int. Cl. C07c 103/52 US. Cl. 260112.5 3 Claims ABSTRACT OF THE DISCLUSURE The present invention provides the pentacosapeptides of general formula D-seryl-L-tyrosyl-L-seryl-L-norleucyl- X L histidyl L phenylalanyl L arginyl L-tryptophanyl glycyl L lysyl L prolyl L valyl-glycyl- L lysyl L lysyl L lysyl L lysyl L prolyl-L- valyl L lysyl L valyl L tyrosyl-L-prolyl-L-valinamide, in which X signifies an L-glutamyl or L-glutaminyl radical, their therapeutically active acid addition salts and heavy metal complexes. The pentacosapeptides of the above formula, their salts and heavy metal complexes exhibit a high adrenocorticotropic effect. The production of these peptides is furthermore described.

The present invention relates to new polypeptides and a process for their production.

The present invention provides the pentacosapeptides of general formula D-seryl-L-tyrosyl-L-seryl-L-norleucy1- X L histidyl L phenylalanyl L arginyl-L-tryptophanyl glycyl L lysyl L prolyl L valyl-glycyl-L- lysyl L lysyl L lysyl L lysyl L prolyl-L-valyl- L-lysyl L valyl L tyrosyl L prolyl-L-valinamide, in which X signifies an L-glutamyl or L-glutaminyl radical, their therapeutically active acid addition salts and heavy metal complexes. The pentacosapeptides of the above formula, their salts and heavy metal complexes exhibit a high adrenocorticotropic effect. The pentacosapeptides of the above formula are hereinafter named as follows: D Ser Nle Lys Lys Val-NH pentacosapeptide and D Ser -Nle -Gln -Lys -Lys -Val- NH -pentacosapeptide.

The pentacosapeptide of formula L-seryl-L-tyrosyl-L- seryl L norleucyl L glutamyl L histidyl L phenylalanyl L arginyl L tryptophanyl-glycyl-L- lysyl L prolyl L valyl glycyl L lysyl L lysyl- L lysyl L lysyl L prolyl L valyl L lysyl L- valyl- L tyrosyl L prolyl L valinamide, having a corticotropic effect and hereinafter named Nle -Lys Lys -Val-NH -pentac0sapeptide, is known.

An advantage of Nle -Lys -Lys -Val-NH -pentacosapeptide over natural ACTH is that the former has no antigenic effects. A further advantage is that it is devoid of a methionine radical in the 4-position, which methionine radical is easily oxidized whereby the hormone becomes inactive; Nle Lys -Lys -Val-NH -pentacosapeptide contains a norleucine radical in place of the methionine radical present in ACTH, which norleucine radical has the same steric properties as the methionine radical, but is stable to oxidation. Furthermore, Nle -Lys -Lys Val-NH -pentacosapeptide has two lysine radicals in the 17- and 18-positions in place of the arginine radicals present in these positions in natural ACTH. This simplifies the synthetic production of Nle -Lys -Lys Val-NH -pentacosapeptide and, surprisingly, no lowering of the biological activity has been ascertained in the new compound.

Furthermore, Nle Lys" Lys Val-NH -pentacosapeptide has a valinamide radical in the 25-position,

ice

which is not present in this position in natural ACTH.

This valinamide radical protects the peptide chain from enzymatic degradation. Despite these advantages of Nle Lys Lys Val-NH -pentacosapeptide over natural ACTH, this compound has the disadvantage that it is easily affected by aminopeptidase degradation.

In accordance with the present invention, compounds having all the advantages of Nle -Lys -Lys Val-NH but which are not affected by aminopeptidases have been produced. This has been achieved by replacing the terminal L-serine radical of Nle Lys Lys -Val-NH pentacosapeptide by a D-serine radical yielding D-ser Nle Lys Lys Val-NH -pentacosapeptide. As D-amino acid radicals are not found in the natural, biologically active peptide hormones, it was not to be expected that the replacement of a natural amino acid radical by an antipode not occurring in nature would result in a compound having biological and therapeutical properties not only qualitatively equal but also quantitatively superior to those of natural ACTH. The other compound of the invention has been produced by replacing the glutaminic acid radical in the 5-position of D-ser -Nle Lys Lys Val NH pentacosapeptide by a glutamine radical. Surprisingly this may be done without a loss of the biological activity so that D-ser -Nle -Gln Lys Lys Val NH -pentacosapeptide has all the advantages of D ser Nle 4 Lys" Lys -Val-N'H cosapeptide.

The pentacosapeptides of the invention may be produced by methods for the synthesis of compounds of this type in actual use of described in the literature on the subject, it being possible to join together the amino acids in the order indicated in the above formula one at a time or by first forming constituent peptide units and joining these together.

One method of producing the pentacosapeptides of the invention consists in that L-valyl-glycyl-e-N-R-L-lysyl-e- N-R-L-lysyl-e-N-R-L-lysyl-e-N-R-L-lysyl prolyl-L-valyle-N-R-L-lysyl-L-valyl-L-tyrosyl-L-prolyl-L-valinamide, in which R signifies a carbo-tert-butoxy or carbo-tert-amyloxy radical, is condensed with N-triphenylmethyl-L-glutaminyl (or 'y-O-tert-butyl-L-glutamyl) Im triphenylmethyl-L-histidyl-L-phenylalanyl-L-arginyl L tryptophanyl-glycyl-e-N-R-L-lysyl L proline-2,4,5-trichlorophenyl-ester, in which R has the above significance, the resulting N-triphenylmethyl-L-glutaminyl (or 'y-O-tertbutyl-L-glutamyl)-Im-triphenylmethyl L histidyl I.- phenylalanyl-L-arginyl L triptophanylglycyl-e-N-R-L- lysyl-L-prolyl L valyl glYCYl-e-N-R-L-IYSYI-e-NR-L- lysyl-e-N-R-L-lysyl-e-N-R-L-lysyl L prolyl-L-valyl-e- N-R-L-lysyl-L-valyl-L-tyrosyl-L-prolyl-L valinamide, in which R has the above significance, is condensed with N-R'-D-seryl-L-tyrosyl-L-seryl-L-norleucylazide, in which R signifies a triphenylmethyl, a carbo-tert-butoxy or a carbo-tert-amyloxy radical, after removal of the N-triphenylmethyl radical of the glutaminyl (or 'y-O-tertbutyl-L-glutamyl) radical, and all the protective radicals of the resulting new, protected pentacosapeptides N-R'- D-seryl-L-tyrosyl-L-seryl-L-norleucyl L glutaminyl (0r 'y-O-tert-butyl-L-glutamyl)-Imtriphenylmethyl L histidyl-L-phenylalanyl-L-arginyl L tryptophanyl-glycyl-e- N-R-L-lysyl-L-prolyl-L-valyl-glycyl e N R-L-lysyl-e- N-R-L-lysyl-e-N-R-L-lysyl-e-N-R-L-lysyl L prolyl I- valyl-e-N-R-L-lysyl-L-valyl-L-tyrosyl-L-prolyl L valinamide, in which R and R have the above significance, are removed in one or more stages in an acid medium.

The starting materials for producing the pentacosapeptides of the invention, insofar as they were hitherto unknown, may be obtained by methods for the synthesis of peptides in actual use or described in the literature, it being possible to join together the amino acids one at a time or by first forming constituent peptide units and joining these together.

It should be noted that the pentacosapeptides of the invention may likewise be obtained or used in the form of their salts. Examples of acids for acid addition salt formation are: acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, maleic, fumaric, tartarc, citric, benzoic, cinnamic, salicylic, 2-phenoxyor 2-acetoxy-benzoic, mandelic, methanesulphonic, ethauesulphonic, hydroxyethanesulphonic, benzeneor toluenesulphonic, naphthalenesulphonic and sulphanilic acid, polymeric acids, e.g. tannic, alginic or polygalacturonic acid, polyphloretinic phosphate or carboxymethyl cellulose, and halogen hydracids, e.g. hydrochloric or hydrobromic acid, nitric, thiocyanic, sulphuric and phosphoric acid. Zinc may, for example, be used for the heavy metal complex.

A major advantage of the synthetic pentacosapeptides over the natural hormone extracted from animal material is that the former have no antigenic effects. Absence of antigenic effect in a substance indicates no objection to its use even in the face of an earlier allergic reaction to natural ACIH.

According to the usual and accepted standard tests the pentacosapeptides of the invention have the following biological activity:

5701-140 corticotropin IU for every mg. of D-Ser -Nle Lys -Lys -Val-NH -pentacosapeptide 550:140 corticotropin IU for every mg. of D-Ser -Nle Gln -Lys -Lys -Val-NH -pentacosapeptide.

The pentacosapeptides of the invention were tested in ac cordance with the third International Standard for corticotropin which is available in the form of an International Standard for Corticotropin and permits the standardization of ACTH preparations in International Units.

It has been found that, upon intravenous administration, the new pentacosapeptides have a longer duration of action than the hitherto known naturally occurring and synthetic ACTH compounds.

The surprisingly high activity of the new pentacosapeptides, which was ascertained upon standardization, has been confirmed upon therapeutic application, so that on a weight basis the new pentacosapeptides are more active than all the hitherto known naturally occurring and synthetic ACTH preparations.

The dose of the pentacosapeptides of the invention ranges between about 40 and 60 IU daily, in exceptional cases between and 100 IU daily.

The pentacosapeptides of the invention may be used as medicaments, for example in the form of pharmaceutical preparations. These may contain the said compounds in mixture with an organic or inorganic carrier material which is suitable for parenteral administration. Appropriate carrier materials are substances which do not react with the new compounds, e.g. gelatin, lactose, starch, magnesium stearate, talcum, vegetable oils, benzyl alcohols, gum arabic, polyalkylene glycols, Vaseline, cholesterol and other known pharmaceutical carrier materials. The pharmaceutical preparations may, for example, be used in the liquid form as solutions, suspensions or emulsions. They may be sterilized and/or they may contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents. However, they may contain other therapeutically valuable substances. The new compounds may also be administered in the form of depot preparations as is the case with natural ACTH.

Examples of radicals for blocking the amino radical of the serine radical during the synthesis of the new pentacosapeptides are the triphenylmethyl, the carbo-tertbutoxy and the carbo-tert-amyloxy radical, but other suitable protective radicals, e.g. the carbobenzoxy, the trifiuoroacetyl, the acetyl, the chloroacetyl and the formyl radical, may likewise be used.

Suitable radicals for blocking the e-amino radical of the lysine radical are the carbo-tert-butoxy and the carbotert-amyloxy radical, but other suitable protective radicals, e.g. the carbobenzoxy, toluenesulphonyl, phthalyl, formyl and trifiuoroacetyl radical, may likewise be used.

A suitable radical for blocking the 'y-carboxyl radical of the glutaminic acid radical is the tert-butyloxy radical, but other suitable protective radicals, e.g. the methoxy, the ethoxy, the tert-amyloxy, the amide or the benzyloxy radical, may likewise be used.

A suitable radical for the blocking of the imidazole radical of the histidine radical is the triphenylmethyl radical, but other suitable protective radicals, e.g. the carbo-tert-butoxy, carbo-tert-amyloxy, carbobenzoxy or benzyl radical, may likewise be used.

The following abbreviations are used in the text and in the drawings:

CBO=carbobenzoxy Gly=glycyl Trit:trityl=triphenylmethyl His 'L-histidyl OEt=ethoxy Arg=L-arginyl Glu =L-glutamyl Gln=L-glutaminyl Try=L-tryptophanyl Tyr=L-tyrosy1 Val=L-valyl Irn=imidazolyl In the following non-limitative examples all temperatures are indicated in degrees centigrade.

RRRR R III! RRR R I l R-:-T)-Scr-Tyr-Scr-Nle-i-Na H-F ilwHis-Phe-Arg-Try- Gly-l lys-P ro-Val- Gly-Ilys-II ys-Il ys- L ys-Pro-Val-Ijys-Val-T yr-Pro-Val -l-NH;

R R R I H- gD -Ser-'Iyr-Ser-Nle-(illu-Hls-Phe-Arg-Try- Gly-Lys-Pro-val-Gly Lys- L ys-Lys-L ys-Pro-Val-Lys-Val-Tyr-Pro-Val-i-NII;

EXAMPLE 1 Carbo tert butoxy D seryl L tyrosyl L seryl- L-norleucine hydrazide. (CTB D Ser Tyr Ser-Nle- NHNH 54 g. of H-Ser-Nle-Ome-HCl and 63 g. of CBO-Tyr- OH are dissolved in 860 ml. of acetonitrile, cooling is effected to 28 ml. of triethylamine are added, cooling is effected to --10 and 41 g. of dicylohexyl carbodiimide are added. The mixture is stirred at 0 for 16 hours and is then filtered. The precipitate is washed with 1400 ml. of pyridine. The combined filtrates are evaporated and the residue crystallized from ethyl acetate. 101 g. of CBO Tyr Ser Nle OMe (melting point 140-142, M1 15" in dimethyl formamide) are obtained. 51 g. of the product obtained above are dissolved in 2 litres of a 1 N solution of hydrochloric acid in methanol and hydrogenation is effected in the presence of 10 g. of palladium/charcoal. After about 2 hours hydrogen is no longer taken up. Filtration and evaporation are effected and the residue is crystallized from a mixture of methanol and ether (3:1). 42 g. of H-Tyr-Ser-Nle-OMe-HCl, having a melting point of 227, [u] 7" in dimethyl formamide, are obtained.

60 g. of D-serine methyl ester hydrochloride are dissolved in 200 ml. of dimethyl formamide and 54 ml. of triethylamine, cooling is effected to 0 and the triethylamine hydrochloride is filtered oif. Dimethyl formamide is evaporated in a high vacuum and the residue dissolved in 150 ml. of pyridine. 100 g. of tert-butyl-oxy-carbonyl azide are added dropwise and the mixture is allowed to stand at 20 for 2 days. The solvent is evaporated and the product is taken up in ethyl acetate. After washing with water, dilute hydrochloric acid and potassium bicarbonate solution, drying is effected over sodium sulphate. After evaporating the ethyl acetate CTB-D-Ser-OMe results as an oil. The ester is dissolved in 500 ml. of methanol and is allowed to stand at 20 with 50 ml. of hydrazine hydrate for 2 days. After evaporating the methanol, the hydrazide crystallizes. After recrystallization from hot ethyl acetate, 53 g. of CTB-D-ser-NHNH having a melting point of 114, [u] -3 in dimethyl formamide, are obtained. 10 g. of CTB-D-serine hydrazide are dissolved at -10 in 136 ml. of 1 N hydrochloric acid containing 15 g. of sodium chloride. 160 ml. of ethyl acetate and subsequently 3.8 g. of sodium nitrite are added in 3 portions at the same temperature. The mixture is allowed to react for a further minutes at whilst stirring continuously. The ethyl acetate phase is separated, washed with a cold 10% potassium bicarbonate solution and dried with sodium sulphate. A solution of 13 g'. of H-Tyr-Ser-Nle-OMe hydrochloride in 60 ml. of dimethyl formamide and 6 ml. of triethylamine is added to the dried solution. The ethyl acetate is subsequently evaporated in a vacuum and the solution allowed to stand at 20 for 16 hours. The remaining solvent is evaporated in a vacuum and the residue dissolved in ethyl acetate. Washing with dilute phosphoric acid and potassium bicarbonate solution and drying over sodium sulphate are effected. After evaporating the solvent and precipitating with ether g. of CTB D-ser-Tyr-ser-Nle-OMe, having a melting point of 135, [a] 6 in methanol, are obtained. 11 g. of CTBD-Ser-Tyr-Ser-Nle-OMe are dissolved in 100 ml. of methanol and 4.5 ml. of hydrazine hydrate are added. The mixture is allowed to stand over night at 20, whereupon the product crystallizes. Filtration and washing with methanol and petroleum ether are effected. 7.7 g. of CTBD-S'er-Tyr-Ser-Nle-hydrazide, having a melting point of 210, [a] +6.4 in dimethyl formamide, are obtained.

6 EXAMPLE 2 D seryl L tyrosyl L seryl-L-norleucyl-L-glutamyl-L- histidyl L phenylalanyl L arginyl-L-tryptophanylglycyl L lysyl L prolyl L valyl-glycyl-L-lysyl-L- lysyl L lysyl L lysyl L prolyl-L-valyl-L-lysyl-L- valyl L tyrosyl-L-prolyl-L-valinamide. (H-D-Ser-Tyr- Ser Nle Glu His Phe Arg Try-Gly-Lys-Pro-Val- Gly Lys Lys Lys Lys Pro-Val-Lys-Val-Tyr-Pro- Val-NH 2.0 g. of CTB-DSer-Tyr-Ser-Nle-NHNH (Example 1) are dissolved in 12 ml. of dimethyl formamide, 4 ml. of water are added, cooling is effected to 10", 2 ml. of 6 N hydrochloric acid and subsequently 280 mg. of sodium nitrite are added, stirring is effected at 5 for 5 minutes, 300 ml. of 0.2 N potassium bicarbonate solu- {tion are added and the mixture is centrifuged. The resulting CTB-D-Ser-Tyr-Ser-Nle-N is dissolved in 50 ml. of dimethyl formamide, 10.5 g. of H-Glu(OTB)-(Trit) His-Phe-Arg-Try-Gly- (CTB)Lys-Pro-Val Gly (CTB) Lys-(CTB)Lys-(CTB)Lys-(CTB)Lys-Pro Val (CTB) Lys-Val-Tyr-Pro-Val-N-H acetate are added, the mixture is allowed to stand at 0 for 12 hours, a further amount of tetrapeptide azide produced from 2.0 g. of CTB-D-Ser- Tyr-Ser-Nle-NHNH is added, the mixture is allowed to stand at 0 for 6 hours, is evaporated, treated with ethyl acetate, washed with hot acetone and ethyl acetate and dried in a vacuum. The resulting product is dissolved in ml. of 90% trifluoroacetic acid, the solution is allowed to stand at 20 in an atmosphere of nitrogen for one hour, is evaporated, treated with ethyl acetate, filtered and dried. The resulting product is dissolved in 500 ml. of Amberlite IRA-410 in the acetate form, is filtered and lyophilized. After drying over sodium hydroxide, 7.3 g. of H D-Ser-Tyr-SerNle-Glu-His-Phe-Arg-Try-Gly-Lys- Pro-Val-Gly-Lys-Lys-Lys-Lys-ProVal-Lys-Val-Tyr Pro- Val-NH- heptaacetate decahydrate, having a homogeneous behaviour in chromatography and electrophoresis, are obtained. (Total hydrolysis gives the following composition of amino acids:

Microanalysis.-Ca1culated (percent): C, 52.6; H, 7.6; N, 14.9; 0, 24.7. Found (percent): C, 52.4; H, 7.5; N, 15.0; 0, 24.8. Melting point 202 with decomposition, [a] =-82 in 1 N acetic acid.

EXAMPLE 3 Trityl-L-glutaminyl-Im-trityl L histidyl L phenylalanyl-L-arginyl L tryptophanyl-glycyl-e-N-carbotert-butoxy L lysylL-proline-2,4,S-trichlorophenyl ester. ('Irit Glu-(Trit)His-Phe-Arg-Try-Gly-(CTB) Lys-Pro-OOP) 457 g. of H-His-Phe-OMeQ HBr and 266 ml. of triethylamine are dissolved in 1000 ml. of dimethyl formamide, stirring is effected at 0 for 10 minutes, filtration is effected, 445 g. of OBO-Gln-OCP are added to the filtrate and the mixture is allowed to stand at 20 for 16 hours. The dimethyl formamide is removed by evaporation, the residue is dissolved in ethyl acetate, washed with dilute acetic acid/water and a 1 N sodium bicarbonate solution. Drying over sodium sulphate and evaporation are effected and the residue is crystallized from ethyl acetate. 328 g. of CBO-Gln-His-Phe-OMe, having a melting point of 195, [a] -=20 in dimethyl formamide, are obtained.

The tripeptide obtained above is dissolved in 3 litres of methanol, hydrogenation is effected in the presence of 50 g. of 10% palladium/charcoal, filtration and evaporation are effected, the residue is dissolved in 1.5 litres of methylene chloride, cooling is effected to 0, ml. of triethylamine and then 270 g. of triphenylchloromethane are added, the mixture is allowed to stand at 20 for 16 hours, washing is effected with dilute acetic acid, water and 1 N sodium bicarbonate solution, drying and evaporation are effected. The residue is dissolved in diethyl ether and precipitation is effected with petroleum ether. 480 g. of Trit-Gln-(Trit)His-Phe-OMe (melting point 90 with decomposition) are obtained and are dissolved in 1000 ml. of methanol. 50 ml. of hydrazine are added, the mixture is allowed to stand at 20 for 24 hours, concentration is effected to 500 ml., 5 litres of diethyl ether are added, washing with 0.1 N common salt solution, drying, concentrating to 500 ml. and precipitating with petroleum ether are effected. 380 g. of Trit-Gln- (Trit)His-Phe-NHNH having a melting point of 100 with decomposition, [a] =12 in dimethyl formamide, are obtained.

41 g. of Trit-Gln-(Trit)His-Phe-NHNH are dissolved in 100 ml. of dimethyl formamide and 100 ml. of isopropanol, cooling is effected to -10 and40 ml. of 4 N hydrochloric acid are added; 9 ml. of 5 N sodium nitrite solution are subsequently added whilst stirring. After 5 minutes 28 ml. of triethylamine and 1000 ml. of ice water are added, filtration is effected with suction, the precipitate is dissolved in ethyl acetate, washing with a saturated common salt solution, drying and evaporating at are effected; the residue is dissolved in 100 ml. of dimethyl formamide, 26 g. of H-Arg-Try-Gly-(CTB)Lys-Pro-OH, 3 AcOH and 4.5 ml. of triethylamine are added, the mixture is allowed to stand at 0 for 16 hours, 1000 ml. of ethyl acetate are added, washing with 0.5 N acetic acid, water and 0.5 N pyridine and evaporation are effected. The residue is dissolved in 100 ml. of ethyl acetate and precipitation is effected with diethyl ether. 46 g. of Trit- Gln- (Trit) His-Phe-Arg-Try-Gly- (CTB Lys-Pro-OH, having a melting point of 190, [a] =12 in dimethyl formamide, are obtained.

The peptide obtained above is dissolved in 200 ml. of pyridine, cooling is effected to '20, 30 ml. of a 1 N solution of hydrochloric acid in dioxane are added, stirring is effected at -20 for minutes, 7.8 ml. of triethylamine are added, the mixture is concentrated to half its volume, 30 g. of Tri-(trichlorophenyl)-phosphite are added, the mixture is allowed to stand at 20 for 16 hours, is evaporated, the residue is treated with diethyl ether and dissolved in ethyl acetate. Precipitation with diethyl ether, filtration and drying are effected. 51 g. of Trit-Gln-(Trit)His-Phe-Arg-Try-Gly (CTB)Lys Pro- OCRHCI, having a melting point of 180 with decomposition, are obtained.

EXAMPLE 4 D-seryl-L-tyrosyl-L-seryl-L-norleucyl L glutaminyl-L- histidyl-L-phenylalanyl L arginyl L tryptophanylglycyl-L-lysyl-L-pro1yl-L-valylglycyl-L-lysyl L lysyl- L-lysyl-L-lysyl-L-prolyl-L-valyl L lysyl L valyl-L- tyrosyl-L-prolyl-L-valinamide. (H-D-Ser-Tyr-Ser Nle- Gln-His-Phe-Arg-Try-Gly-Lys-Pro-Val-Gly Lys Lys- Lys-Lys-Pr0-Val-Lys-Val-Tyr-Pro-Va1-NH 6 g. of Trit-Gln-(Trit)His-Phe-Arg Try Gly(CT.B) Lys-Pro-OCP-HCl and 5.7 g. of H-Val-Gly-(CTB)Lys- (CTB)Lys-(OTB)Lys-(CTB)Lys-Pro Val (CTB)Lys- Val-Tyr-Pro-Val-NH are dissolved in 20 ml. of dimethyl formamide, 0.3 g. of imidazole are added and the mixture is allowed to stand at 20 for 3 days. A further 0.6 g. of the same octapeptide hydrochloride are added, the mixture is allowed to stand at 20 for a further 16 hours, precipitation with ethyl acetate and filtration are effected; the precipitate is dissolvedin ethanol, the procedure is repeated, precipitation with ethyl acetate, filtration and drying are effected. 8.0 g. of Trit-Gln-(Trit)His-Phe-Arg- Try-Gly- (CTB)Lys-Pro-Val-Gly-(CTB)Lys (CTB)Lys- (CTB Lys- (CTB Lys-Pro-Val- (CTB Lys-Val-Tyr Pro- Val-NH (melting point 198 with decomposition, [a] =26 in dimethyl formamide) are obtained and are dissolved in 200 ml. of acetic acid/water (8:2). The

solution is allowed to stand at 30 for 2 hours, is evaporated, washed with ethyl acetate, filtered and dried. 7 g. of H-Gln-(Trit)His-Phe-Arg-Try-Gly (CTB)Lys Pro- Val-Gly-(CTB)Lys-(CTB)Lys (CTB)Lys (CTB)Lys- Pro-Val-(CTB)Lys-Val-Tyr-Pro-Val-NH acetate are obtained. 1.0 g. of CTB-D-Ser-Tyr-Ser-Nle-NH-NH is dissolved in 6 ml. of dimethyl formamide, 2 ml. of water are added, cooling is effected to 10, 1 ml. of 6 N hydrochloric acid and then 140 mg. of sodium nitrite are added, stirring is effected at 5 for 5 minutes, 300 ml. of 0.2 N potassium bicarbonate solution are added and the mixture is centrifuged. The resulting CTB-D-Ser-Tyr- Ser-Nle-N is dissolved in 50 ml. of dimethyl formamide, 5.0 g. of H-Gln-(Trit)His-Phe-Arg-Try-Gly (CTB)Lys- Pro-Val-Gly-(CTB)Lys-(CTB)Lys (CTB)Lys (CTB) Lys-Pro-Val-(CTB)Lys-Val-Tyr-Pro-Val-NH acetate are added, the mixture is allowed to stand at 0 for 12 hours, a further amount of tetrapeptide azide produced from 1.0 g. of CTB-D-Ser-Tyr-Ser-Nle-NHNH is added, the mixture is allowed to stand at 0 for 6 hours, is evaporated, treated with ethyl acetate, washed with hot acetone and ethyl acetate and dried in a vacuum. The resulting product is dissolved in 100 ml. of trifiuoroacetic acid, the solution is allowed to stand at 20 in an atmosphere of nitrogen for one hour, is evaporated, treated with ethyl acetate, filtered and dried. The resulting product is dissolved in 500 ml. of 0.2 N acetic acid, the solution is treated with Amberlite IRA-410 in the acetate form, is filtered and lyophilized. After drying over sodium hydroxide 3.5 g. of H-D-Ser-Tyr-Ser-Nle-Gln-His-Phe-Arg- Try-Gly-Lys-Pro-Val-Gly-Lys-Lys Lys Lys Pro Val- Lys-Val-Tyr-Pro-Val-NH acetate, having a homogeneous behaviour in chromatography and electrophoresis, are obtained. (Total hydrolysis gives the following composition of amino acids:

Microanalysis.Calculated (percent): C, 52.7; H, 7.7; N, 15.3; 0, 24.3. Found (percent): C, 52.4; H, 7.8; N, 15.1; 0, 24.6. Melting point 204 with decomposition, [a] in l N acetic acid.

What is claimed is:

1. A compound selected from the group consisting of a compound of formula: D-seryl-L-tyrosyl-L-seryl-L-norleucyl-X-L-histidyl-L-phenylalanyl-L-arginyl L tryptophanyl-glycyl-L-lysyl-L-prolyl-L-valylglycyl L lysyl-L- lysyl L-lysyl-L-lysyl-L-prolyl-L-valyl-L-lysyl L valyl-L- tyrosyl-L-prolyl-L-valinamide, in which X is L-glutamyl or L-glutaminyl, and the pharmaceutically acceptable acid addition salts and heavy metal complexes thereof.

2. A compound according to claim 1, in which the compound is D-seryl-L-tyrosyl-L-seryl L norleucyl-L- glutamyl-L-histidyl-L-phenylalanyI-Larginyl L tryptophanyl-glycyl-L-lysyl-L-prolyl-L-valyl-glycyl L lysyl- L-lysyl-L-lysyl-L-1ysyl-L-prolyl-L-valyl-L-lysyl L valyl- L-tyrosyl-L-prolyl-L-valinamide.

3. A compound according to claim 1, in which the compound is D-seryl-L-tyrosyl-L-seryl L norleucyl-L- glutaminyl-L-histidyl L-phenylalanyl-L-arginyl-L tryptophanyl-glycyl-L-lysyl-L-prolyl-L-valyl-glycyl L- lysyl-L- lysyl L-lysyl-L-lysyl-L-proly1-?.-valyl-L-lysy1 L valyl-L- tyrosyl-L-prolyl-L-valinamide.

References Cited UNITED STATES PATENTS 3,228,926 1/1966 Kappeler et al. 260--112.5

FOREIGN PATENTS 668250 2/1966 Belgium 260-1125 10 OTHER REFERENCES Doepfner: Experientia 22, 527-528 (1966). Guttmann et a1.: Acta Chim. Acad. Sci. Hung. 44, 141- 142 (1965).

ELBERT -L. ROBERTS, Primary Examiner US. Cl. X.R. 424-179 UNl'lED STATES PATENT @FIFHCE QER'HFMA'E @F @EQHWN Patent No. 31,761,461 Dated September 25, 1973 Inventor(s) Janos PLESS e1: a1,

It is certified that error appears in the aboveidentified patent and that said Letters Patent are hereby corrected as shown below:

Column 2, line 31, delete "of" and insert therefor or line 5 9 delete "valyl glycyl" and insert therefor, valyl glycl -u T Column 3, lines 28 30, delete in entirety.

Column 3, chart, line 2, delete. "R" and insert therefor --R chart, at beginning of'all boxes delete and insert therefor minus signs Signed and sealed this 26th day of March 19714..

(SEAL) Attest:

EDWARD MQF'LETGHERJR 0., MARSHALL DANN Attesting Officer Commissioner of Patents FORM PO-IOSO (10-69) U\ USCOMM-DC 60376-P69 A t U.S GOVERNMENT PRINTING OFFICE I969 0-366-334