US3579494A - C-sulfonated tyrosyl peptides related to cholecystokinin-pan-creozymin (cck-pz) - Google Patents

Abstract

NOVEL PEPTIDFE AMIDES OF THE GENERAL FORMULA

R-MET-GLY-TRP-MET-ASP-PHE-NH2

WHEREIN R REPRESENTS L-ASPARTYL-3''-SULFONYL-L-TYROSYL, L-ASPARTYL-L-ARGINYL-L-ASPARTYL-3''-SULFONYL-L-TYROSYL, OR ISOLEUCYL - L - SERYL-LASPARTYL-L-ARGINYL-L-ASPARTYL-3''SULFONYL-L-TYROSYL, AND INTERMEDIATES IN THE PRODUCTION THEREOF. THE PEPTIDE AMIDES OF THIS INVENTION AS WELL AS THE PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF HAVE BEEN FOUND TO POSSES CHOLECYSTOKININ ACTIVITY.

Description

United States Patent 3,579,494 C-SULFONATED TYROSYL PEPTIDES RE- LATED T0 CHOLECYSTOKININ-PAN- 'CREOZYlVIIN (CCK-PZ) Miguel A. Ondetti, North Brunswick, John T. Sheehan, Middlesex, and Josip Pluscec, East Brunswick, N.J., assignors to E. R. Squibb & Sons, Inc., New York,

No brawing. Filed July 18, 1968, Ser. No. 745,688 Int. Cl. C07c 103/52 U.S. Cl. 260112.5 7 Claims ABSTRACT OF THE DISCLOSURE Novel peptide amides of the general formula R-MetGlyTrp-MetAspPhe-NH wherein R represents L-aspartyl-3'-sulfonyl-L-tyrosyl, L-aspartyl L arginyl L aspartyl-3'-sulfonyl-L-tyrosyl, or isoleucyl L seryl-L-aspartyl-L-arginyl-L-aspartyl-3'- sulfonyl-L-tyrosyl, and intermediates in the production thereof. The peptide amides of this invention as well as the pharmaceutically acceptable salts thereof have been found to possess cholecystokinin activity.

This invention relates to novel peptide amides of the general formula wherein R represents L-aspartyl-3-sulfonyl-L-tyrosyl,

L-aspartyl L arginyl L aspartyl-3'-sulfonyl-L-tyrosyl,

or isoleucyl L seryl-L-aspartyl-L-arginyl-Laspartyl-3'- 3,579,494 Patented May 18, 1971 sulfonyl-L-tyrosyl, and to the intermediates and their salts employed in the preparation thereof.

Peptide salts encompassed by the above include pharmaceutically acceptable acid addition salts, such as hydrochlorides, hydrobromides, acetates, fluoroacetates, such as trifluoroacetate, chloroacetates such as dichloroacetate, and the like, as well as ammonium salts such as dicyclohexylammonium, triethylammonium, morpholinium, pyridinium, and the like, and inorganic salts, such as alkali or alkaline earth metal salts, e.g., sodium, potassium, calcium, barium, and the like.

The final products of this invention are peptide amides containing amino acid residues of L-isoleucine (Ileu), L-serine (Ser), L-arginine (Arg), L-tyrosine (Tyr), L-aspartic acid (Asp), L-methionine (Met), glycine (Gly), L-tryptophane (Trp) and L-phenylalanine (Phe).

These products have been found to possess cholecystokinin activity. That is, they have the ability to stimulate the contraction of the gall bladder. Thus, they find utility as diagnostic aids in X-ray examination of the gall bladder in the same manner as cholecystokinin. For such purposes, they may be administered either intravenously or subcutaneously to an animal species (e.g., cats or dogs) in a single dosage of about 0.008 to 0.012 mg./kg. of body weight.

For this purpose they may be administered parenterally by incorporating the appropriate dosage of the compound with carriers to form injectables according to standard pharmaceutical practice.

The compounds of this invention may be prepared in accordance with the following reaction schema wherein B represents benzyl and X and Y are as hereinafter set forth:

Met-Gly-Trp-Met-Asp-Phe-NH: X-ASID-TYI-NHNHQ (V)| I B I l l N 0 a O B l X- S er l X-Arg-Asp-Tyr-NHNH-Y (I) X-Asp-Tyr-Met-Gly-Ttp-Met-Asp-Phe-NH; (VI) I N 02 0 B l S OaH' X- S er-NHNH-Y (X) l Arg-Asp-Tyr-NHNHY (II) Asp-Tyr-Met- Gly-Trp-Met-Asp-Phe-NHz (VIII) I O B N O a 0 B S or-NHNH-Y (XI) l X-Asp-Arg-Asp-Tyr-NHNH-Y (III) XA A A T NHNH 1v) s rgspyr- X-Ileus er-NHNH-Y (XII) p I X-Asp-Arg-Asp-Tyr-Met- Gly-Trp-Met-Asp-P he-NHz (VII) X-Ileu-Ser-NHNH, (XIII) 0 3H Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NHz X-Ileu-Ser-Asp-Arg-AsoTyr-Met- Gly-Trp-Met-Asp-Phe-NHz (XIV) lieu-Ser-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH (XV) In these formulae Tyr represents 2-amino-3-(3-sulionyl-4-hydroxy)-pheny1propionic acid.

Two of the starting materials, namely, tertiary butyloxycarbonyl-O-benzyl-L-serine and tertiary butyloxycarbonyl-fi-benZyl-L-aspartyl-tyrosine benzyloxycarbonyl-hydrazide are well known in the art. The starting material L-methionyl-glycyl L tryptophyl-L-methionyl-L-aspartyl-L-phenylalaninamide may be prepared by reacting glycyl-L-tryptophyl L methionyl L-aspartyl-L-phenylalaninamide trifluoroacetate with tertiary butyloxycarbony1-L-methionyl-2,4,S-trichlorophenyl ester. The peptide sequences indicated in the above reaction schema may be joined by any known coupling method of peptide synthesis to form the indicated polypeptides as shown. Partial sequences are first formed by joining together the amino acids one at a time and then joining the resulting sequences one with another to obtain the desired polypeptide product.

Alternatively, the peptides of this invention may be prepared by the sequential addition of the appropriate amino acids one at a time to the L-methionyl-glycyl-L-tryptophyl-L-rnethionyl-L-aspar-tyl-L-phenylalanine amide.

Such additions are accomplished, for example, by activating the carboxylic acid group in the amino acid to be added after protecting the amino group in such amino acid, for instance, by converting it to its tertiary-butyloxycarbonyl derivative, converting this derivative into, for example, a nitrophenyl ester and then reacting this active ester with another amino acid or peptide as desired.

Among the suitable activating groups to be employed in the above process may be mentioned any group which causes the acid function to become more reactive, such as mixed anhydrides (which normally involves the acylation of an amine with the mixed anhydrides of, for instance, an acyl amino acid and isovaleric acid), azides, acid chlorides, reaction products with carbodiimides, reactive N-acyl compounds, O-acyl hydroxylamine derivatives, and active esters, such as alkyl esters with electron attracting (negative) substituents, vinyl esters, enol esters, phenyl esters, halophenyl esters, thiophenyl esters, nitrophenyl esters, 2,4-dinitrophenyl esters, and nitrophenylthiol esters.

In forming the peptide sequences in the above manner, the hydroxyl protecting group employed may be benzyl, tertiary butyl, tetrahydropyranyl, and the like, the carboxyl protecting groups may be methyl, ethyl, tertiary butyl, benzyl, and the like, the guanidino protecting groups may be nitro, tosyl, p-nitrobenzyloxycarbonyl, protonation, and the like, and the amino protecting groups (X and Y in the above formulae) may be benzyloxycarbonyl, t-butyloxycarbonyl, trifiuoroacetyl or o-nitrophenylsulfenyl, except that t-butyloxycarbonyl may not be employed in combination with o-nitrophenylsulfenyl.

It should be noted at this point that since protecting group X in the above formulae must be selectively re moved in order to add the next amino acid in the sequence, it is required that X and Y be different.

Various methods of removing protecting groups X and Y from the peptide claim are known in the art. However, since selective removal of X is required, the method chosen will depend upon the protecting group employed. For instance, Where X reperesents t-butyloxycarbonyl, it may be removed by acid treatment, as by trifiuoroacetic acid, or the like. Where X represents benezyloxycarbonyl, the removal may be by hydrogenolysis, for instance, in the presence of a palladium on charcoal catalyst. In those instances where X represents trifiuoroacetyl, it may be removed by treatment with a nucleophile (e.g., hydrazine in methanol, sodium hydroxide in methanol or an alkoxide such as sodium methoxide or sodium ethoxide, and the like), If X is o-nitrophenylsulfenyl, it may be removed either by treatment with mild acid [e.g., a hydrohaloacid (such as hydrobromic or hydrochloric acids) in a solvent such as ethyl acetate, ether, or other alkyl ester or alkyl ether solvents], or with a sulfur nucleophile, such as sodium thiophenoxide, nitrothiophenoxide, and the like. By proceeding in this manner, selective removal of the X protecting group is achieved. Selective removal of Y to form the protected peptide hydrazide intermediate may be accomplished in the same manner as set forth above, the appropriate method of removal being selected, dependent upon the protecting group employed.

The hydroxyl, carboxyl and guanidino protecting groups may be removed by known reactions, such as hydrogenolysis, treatment with acids such as hydrochloric acid, hydrobromic acid, trifiuoroacetic acid, and the like, treatment with alkali, such as sodium or potassium hydroxide, and the like, or by treatment with sodium in liquid ammonia.

The sulfonation of the peptides of this invention containing tyrosine may be achieved by the reaction of the peptide with concentrated sulfuric acid in the cold. The reaction is conducted for a period of time of about 24 to 72 hours at temperatures below 20 C., preferably below 0 C.

The invention will be further illustrated in the following examples. All temperatures are in degrees centigrade unless otherwise stated:

EXAMPLE 1 L-methionylglycyl-L-tryptophyl-L-methionyl-L-aspartyl- L-phenylalanine amide (A) Tert. butyloxycarbonyl L methionylglycyl-L tryptophyl-L-methionyl L aspartyl L phenylalanine amide. Glycyl-L-tryptophyl-L-methionyl L aspartyl-L- phenylalanine amide trifiuoroacetate (3.8 g.) is dissolved in a mixture of dimethylformamide (45 ml.) and triethylamine (1.4 ml.) and tert.-butyloxycarbonyl-L-methionine 2,4,5-trichlorophenyl ester (2.5 g.) added. After stirring for three hours at room temperature, the reaction mixture is diluted with ethyl acetate and the precipitate is filtered, washed with ethyl acetate and ether and dried. Yield is 3.4 g.; M.P. ISO-182.

(B) The protected hexapeptide of (A) (3.1 g.) is dissolved in cold trifiuoroacetic acid (20 ml.) and the solution kept under nitrogen at room temperature for 25 minutes. Addition of ether (250 ml.) precipitates the grilirgloroacetate which is filtered, washed with ether and EXAMPLE 2 Tert.-butyloxycarbonylnitro-L-arginyl 13 benzyl- L-aspartyl-L-tyrosine benzyloxycarbonylhydrazide (I) p-Benzyl-L-aspartyl L-tyrosine benzyloxycarbonylhydrazide trifluoroacetate (1.25 g.) was dissolved in dimethylformamide ml.) and the solution was cooled in an ice-bath. Triethylamine (0.28 ml.) and tert.-butyloxycarbonylnitro L arginine N-hydroxysuccinimide ester (915 mg.) were added. The reaction mixture was stored at room temperature and three more portions (83 mg. each) of active ester were added after 2, 3 and 6 hours. After a total reaction time of 24 hours, ethyl acetate (200 ml.) was added and the solution was washed with 20% aqueous citric acid and water. After drying (MgSO the solvent was removed in vacuo and the residue crystallized from ethyl acetate ether. Yield 1.31 g.

EXAMPLE 3 Nitro-L-arginyl-p-benZyl-L-aspartyl-L-tyrosine benzyloxycarbonylhydrazide trifiuoroacetate (II) I (1.11 g.) was dissolved in trifiuoroacetic acid (7 ml.) and the solution was kept at room temperature for minutes. The solvent was removed in vacuo and the residue was triturated with ether. Yield 1.04 g.

EXAMPLE 4 Tert.-butyloxycarbonyl ,8 benzyl L aspartylnitro-L- arginyl ,8 benzyl L aspartyl-L-tyrosine benzyloxycarbonylhydrazine (III) Triethylamine (0.17 ml.) was added to an ice-cold solution of II (1.04 g.) in dimethylformamide (5 ml.), followed by tert.-butyloxycarbonyl-B-benzyl L aspartic acid p-nitrophenyl ester (650 mg). After 3 hours standing at room temperature, the mixture was diluted with ethyl acetate (100 ml.) and the solution was Washed with aqueous citric acid and water. After drying (MgS the solvent was removed in vacuo and the residue triturated with a mixture of ethyl acetate-hexane. Yield 1.08 g.

EXAMPLE 5 Tert.-butyloxycarbonyl L aspartyl-L-arginyl-L-aspartyl- L-tyrosine hydrazide (IV) A solution of III (900 mg.) in a mixture of methanol, acetic acid, water (2: 1: 1) was hydrogenated for 24 hours over 10% palladium on charcoal (150 mg). The catalyst was filtered and the filtrate was concentrated to dryness. The residue was distintegrated with ethanol and filtered. Yield 402 mg.

EXAMPLE 6 Tert.-butyloxycarbonyl-L-aspartyl-L-tyrosine hydrazide (V) A solution of tert.-butyloxycarbonyl-fi-benzyl-Laspartyl-L-tyrosine benzyloxycarbonylhydrazide (5 g.) in a mixture of methanol, acetic acid and water (211:1) was hydrogenated for 4.5 hours over 10% palladium on charcoal (800 mg.). The catalyst was filtered and the filtrate was concentrated to dryness in vacuo. The residue was triturated with ethyl acetate. Yield 3.1 g.

EXAMPLE 7 Tert.-butyloxycarbonyl L aspartyl-L-tyrosyl-L-methionylglycyl-L-tryptophyl L methionyl-L-aspartyl-L- phenylalanine amide (VI) Concentrated hydrochloric acid (0.12 ml.) was added to a solution of V (100 mg.) in dimethylformamide (4 ml.), cooled in a Dry Ice-acetone bath at 20. The temperature of the bath was allowed to rise to 15 and an aqueous 14% sodium nitrite solution (0.125 ml.) was added. After 5 minutes the temperature of the bath was lowered to -25 and N-ethylpiperidine (0.26 ml.) was added, followed by 180 mg. of L-methionylglycyl- L-tryptophyl-L-methionyl L aspartyl-L-phenylalanine amide trifiuoroacetate dissolved in dimethylformamide (1 ml.). The reaction mixture was stored at 5 and after 24 hours a second portion of tert.-butyloxycarbonyl-L- aspartyl-L-tyrosine azide (from 40 mg. of V) was added.

After another 24-hour period at 5, the reaction mixture was poured into 30 ml. of water containing 1% acetic acid. The precipitate was dried. Yield 200 mg.

EXAMPLE s Tert.-butyloxycarbonyl L aspartyl-L-arginyl-L-aspartyl- L tyrosyl-L-methionylglycyl L tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide (VII) Concentrated hydrochloric acid (0.12 ml.) was added to a solution of IV mg.) in dimethylformamide (1.2 ml.) and cooled in a Dry Ice-acetone bath at -20. The temperature of the bath was allowed to rise to -15 and an aqueous 14% sodium nitrite solution (0.125 ml.) was added. After 5 minutes the temperature of the bath was lowered to 25 and N-ethylpiperidine (0.25 ml.) was added, followed by mg. of L-methionylglycyl-L- tryptophyl-L-methionyl L aspartyl L phenylalanine amide dissolved in dimethylformamide (1 ml.). The reaction mixture was stored at 5 for 24 hours and a second portion of the tetrapeptide azide (from 68 mg. of IV) was added. After 24 hours the reaction was poured into 30 ml. of water. The precipitate was filtered and dried. Yield 250 mg.

EXAMPLE 9 (A) 3'-sulfonyl-L-tyrosyl L methionylglycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide L-tyrosyl L methionylglycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide (100 mg.) was dissolved in sulfuric acid (6 ml.) precooled to -20 in a Dry Ice-acetone bath. The solution was stored at 20 for 72 hours and poured into cold ether (250 ml.). The precipitate was filtered, washed with cold ether and dried. Yield 92 mg. This material can be purified by ion exchange chromatography on DEAE sephadex [(NH CO buffer] or by countercurrent distribution (n-butanol, pyridine, acetic acid, water system).

(B) L-aspartyl-3'-sulfonyl-L-tyrosyl L methionylglycyl- L-tryptophyl-L-methionyl L aspartyl-L-phenylalanine amide (VIII) Following the procedure of Example 9(A), but substituting L-aspartyl-L-tyrosyl-L-methionylglycyl-L-tryptophyl-L-methionyl-L-aspartyl-Irphenylalanine amide for the L-tyrosyl L methionylglycyl-tL-tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide, there is obtained the desired product.

EXAMPLE 10 L aspartyl L arginyl L aspartyl 3' sulfonyl- L tyrosyl L methionylglycyl L tryptophyl L- methionyl L aspartyl L phenylalanine amide (IX) Following the procedure of Example 9(A), but substituting L aspartyl L arginyl L aspartyl L tyrosyl- L-methionylglycyl L tryptophyl L methionyl L aspartyl L phenylalanine amide for the L tyrosyl-L- methionylglycyl L tryptophyl L methionyl L aspartyl L phenylalanine amide, there is obtained the desired product.

EXAMPLE 11 Tert.-butyloxycarbonyl-O-benzyl-L-serine benzyloxycarbonylhydrazide (X) A solution of tert. butyloxycarbonyl O benzyl-L- serine (3 g.) and benzyloxycarbonylhydrazide (1.7 g.) in dichloromethane (20 ml.) was cooled in an ice-water bath. Dicyclohexylcarbodiimide (2.06 g.) Was added and the mixture was stirred 2 hours in the cold bath and 4 hours at room temperature. The precipitate formed was filtered and the filtrate was washed with 20% citric acid, water, saturated sodium bicarbonate and water. After drying (MgSO the solvent was removed in vacuo to yield an oily residue: 4.3 g.

7 EXAMPLE 12 O-benzyl-L-serine-benxyloxycarbonylhydrazide trifluoroacetate (XI) A solution of X (4.3 g.) in trifiuoroacetic acid (20 ml.) was kept at room temperature for 15 minutes. The trifluoroacetic acid was removed in vacuo and the residue was crystallized from ether-hexane. Yield 3.9 g.

EXAMPLE 13 Tert.-butyloXycarbonyl-L-isoleucyl-benzyl-L-serinebenzyloxycarbonylhydrazide (XII) Triethylamine (1.4 ml.) was added to an ice-cold solution of XI (4.5 g.) in dimethylformamide (20 ml.), followed by tert.butyloxycarbonyl-L-isoleucine N-hydroxysuccinimide ester (4.18 g.). The reaction mixture was kept for 6 hours at room temperature and diluted with ethyl acetate (200 ml.). This solution was washed with 20% citric acid, water, saturated sodium bicarbonate and water. After drying (MgSO the solvent was removed in vacuo and the residue was recrystallized from ether-hexane. Yield 5.2 g.

EXAMPLE l4 Tert.-butyloxyc arbonyl-L-isoleu cyl-L-serine hydrazide (XIII) A solution of XII (2.8 g.) in a mixture of methanol, acetic acid and water (221:1) was hydrogenated for 6 hours over palladium on charcoal (280 mg.). The catalyst was filtered, the filtrate was concentrated to dryness and the residue was crystallized from ether. Yield 1.5 g.

EXAMPLE 15 L isoleucyl L seryl L aspartyl L arginyl L- aspartyl 3' sulfonyl L tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl- L-phenylalanine amide (XV) Concentrated hydrochloric acid (0.48 ml.) was added to a solution of tert. butyloxycarbonyl L isoleucyl-L- serine hydrazide (340 mg.) in dimethylformamide (2 ml.) cooled in a Dry Ice-acetone bath at 20". The temperature of the bath was allowed to rise to 15 and an aqueous 14% solution of sodium nitrite (0.50 ml.) was added. After minutes the temperature was lowered to 25 and N-ethylpiperidine (0.6 ml.) was added, followed by a solution of IX (1 g.) in dimethylformamide (4 ml.). After 24 hours standing at 5, the reaction mixture was concentrated to dryness and the resulting tert.- butyloxycarbonyl dodecapeptide (XIV) was dissolved in cold trifluoroacetic acid (6 ml.). The solution was kept under nitrogen for 15 minutes. Ether was added and the precipitate filtered, washed with ether and dried. The desired dodecapeptide sulfonate was isolated by ion-exchange chromatography on DEAE sephadex. Yield 50 mg. The title compound may also be obtained from benzyloxycarbonyl L isoleucyl L seryl L aspartyl L- arginyl L aspartyl 3 sulfonyl L tyrosyl L- methionylglycyl L tryptophyl L methionyl L aspartyl-L- phenylalanine amide.

EXAMPLE 16 Tert.-butyloxycarbonyl-L-arginyl-L-aspartyl-L-tyrosine hydrazide (XVI) A solution of I (300 mg.) in a mixture of methanolacetic acid-Water (2:1:1) was hydrogenated for 24 hours over palladium on charcoal (50 mg). The catalyst was filtered and the filtrate was concentrated to dryness. The residue was disintegrated with ethyl acetate and filtered. Yield 150 mg.

EXAMPLE 17 Tert. butyloxycarbonyl L arginyl L aspartyl L- tyrosyl L methionylglycyl L tryptophyl L- methionyl L aspartyl L phenylalanine amide (XVII) Concentrated hydrochloric acid (0.12 ml.) was added to a solution of XVI (138 mg.) in dimethylformamide (1.2 m1.) and cooled in a Dry Ice-acetone bath at 20". The temperature of the bath was allowed to rise to 15 and an aqueous 14% sodium nitrite solution (0.125 ml.) was added. After 5 minutes the temperature of the bath was lowered to 25 and N-ethylpiperidine (0.26 ml.) was added, followed by 180 mg. of L methionylglycyl-L- tryptophyl L methionyl L aspartyl L phenylalanine amine dissolved in dimethylformamide (1 ml.). The reaction mixture was stored at 5 for 24 hours and a second portion of the tetrapeptide azide (from 51 mg. of XV) was added. After 24 hours the reaction was poured into 30 ml. of water. The precipitate was filtered and dried. Yield 235 mg.

EXAMPLE 1 8 L arginyl L aspartyl 3' sulfonyl L tyrosyl-L- methionylglycyl L tryptophyl L methionyl L- aspartyl-L-phenylalanine amide (XVIII) Following the procedure of Example 9(A), but substituting 235 mg. of L arginyl L aspartyl L tyrosyl- L methionylglycyl L tryptophyl L methionyl-L- aspartyl-L-phenylalanine amide for L tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl- L-phenylalanine amide, there is obtained the above. Yield 140 mg.

EXAMPLE l9 Tert. butyloxycarbonyl L seryl L aspartyl L- arginyl L aspartyl 3 sulfonyl L tyrosyl L- methionylglycyl L tryptophyl L methionyl L- aspartyl-L-phenylalanine amide (XIX) Concentrated hydrochloric acid (0.24 ml.) was added to a solution of tert.butyloxycarbonyl-L-serine hydrazide (115 mg.) in dimethylformamide (1 ml.) cooled in a Dry Ice-acetone bath at 20". The temperature of the bath was allowed to rise to 15 and an aqueous 14% solution of sodium nitrite (0.25 ml.) was added. After 5 minutes the temperature was lowered to 25 and N- ethylpiperidine (0.6 ml.) was added, followed by a solution of IX (500 mg.) in dimethylformamide (2 ml.). After 24 hours standing at 5 the reaction mixture was concentrated to dryness and the residue was triturated with water and then filtered and washed with ethanol. Yield 290 mg.

EXAMPLE 20 L seryl L aspartyl L arginyl L aspartyl-3'- sulfonyl L tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl L phenylalanine amide XIX (290 mg.) was dissolved in trifiuoroacetic acid (3 ml.) and the solution was kept at room temperature under nitrogen for 15 minutes. Ether was added ml.)

and the precipitate was filtered, washed with ether and dried. Chromatography on DEAE sephadex yielded the desired hendecapeptide sulfate ester. Yield 200 mg.

The invention may be variously otherwise embodied within the scope of the appended claims.

What is claimed is:

1. A compound of the formula R-L-methionylglycylL-trypt0phylL- methionylL-aspartyl-L-phenylalanine-amide and pharmaceutically acceptable acid addition salts, ammonium salts, alkali metal salts and alkaline earth metal salts wherein R is selected from the group consisting of:

3 '-sulfonyl-L-tyrosyl, L-aspartyl-3-sulfonyl-L-tyrosyl, L-arginyl-L-aspartyl-3 'sulfonyl-L-tyro syl, L-aspartyl-L-arginyl-L-aspartyl-3'-sulfonyl-L-tyrosyl, L-seryl-L-aspartyl-L-arginyl-L-aspartyl-3 '-sulfonyl- L-tyrosyl, X-L-seryl-L-aspartyl-L-arginyl-L-aspartyl-3'-sulfonyl- L-tyrosyl,

L-isoleucyl-L-seryl-L-aspartyl-L-arginyl-L-asparty1-3'- sulfonyl-L-tyrosyl, and

X-L-isoleucyl-L-seryl-L-aspartyl-L-arginyl-L-aspartyl- 3-sulfony1-L-tyrosyl,

wherein X is benzyloxycarbonyl, t-butyloxycarbonyl, trifiuoroacetyl or o-nitrophenylsulfenyl.

2. A compound in accordance with claim 1 having the name 3 sulfonyl L tyrosyl L methionylglycyl-L- tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide.

3 A compound in accordance with claim 1 having the name L aspartyl 3' sulfonyl L tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl- L-phenylalanine amide.

4. A compound in accordance with claim 1 having the name L aspartyl L arginyl L aspartyl 3' sulfonyl L tyrosyl L methionylglycyl L tryptophy1- L-methionyl-L- aspartyl-L-phenylalanine amide.

5. A compound in accordance with claim 1 having the name benzyloxycarbonyl L isoleucyl L seryl L- aspartyl L arginyl L aspartyl 3' sulfonyl-L- tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl L phenylalanine amide.

6. A compound in accordance With claim 1 having the name L isoleucyl L seryl L aspartyl L arginyl- L aspartyl 3' sulfonyl L tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl L- phenylalanine amide.

7. A compound in accordance with claim 1 having the name t-butyloxycarbonyl L isoleucyl L seryl L- aspartyl L arginyl L aspartyl 3' sulfonyl L- 10 tyrosyl L methionylglycyl L tryptophyl L methionyl L aspartyl L phenylalanine amide.

References Cited UNITED STATES PATENTS 3,472,832 10/1969 Bernardi et a1. 260-1125 3,488,726 1/1970 Ondetti et a1. 260112.5

OTHER REFERENCES Vagne et al., Am. J. Physiol. 215, 881-884 (1968).

Anastasi et al., Experientia 23,699-700 (1967).

Anastasi et al., Experientia 24, 771-773 1968).

Bernardi et al., Experientia 23, 700-702 (1967).

Erspamer et al., Experientia 23, 702-703 1967).

Morley, Peptides, Proceedings of the Eighth European Peptide Symposium, 1966, John Wiley and Sons, Inc., New York (1967).

Mutt et al., Biochem. Biophys. Res. Commun. 26, 392- 397 (1967).

Tracy et al., Nature 204, 935-938 (1964).

Ondetti et al., Peptides-Chemistry and Biochemistry, Proceedings of the First American Peptide Symposium, Marcel Dekker, Inc., New York (1970), pp. 181-190.

LEWIS GOTTS, Primary Examiner M. M. KASSENOFF, Assistant Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE 56 CERTIFICATE OF CORRECTION Patent No. 3,579,494 Dated May 18, 1971 Inventofls) Miguel A. Ondetti, John T. Sheehan and Josip Pluscec It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

r- Column 3, line 18, "phenylalaninamide" should read phenylalanine amide and on line 20, "alaninamide" should read alanine amide and on line 75, "benezyloxy should read benzyloxy Column 5, line 24, "carbonylhydrazine" should read carbonylhydrazide and on line 32, "MgS should read MgSO Column 6, line 16, "0.25" should read 0.26 Column 8, line 9, "amine should read amide Signed and sealed this 21 at day of September 1 971 (SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Acting Commissioner of: Patents