GB1577414A - Somatostatin analogues - Google Patents

Description

(54) SOMATOSTATIN ANALOGUES (71) We, ELI LILLY AND COMPANY, a Corporation of the State of Indiana, United States of America, having a principal place of business at 307 East McCarty Street, City of Indianapolis, State of Indiana, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to polypeptide derivatives.

According to the invention there is provided a tetradecapeptide

[formula (I)], in which Y is D-Ala, D-ser, or D-Val, and Z is L-Asn or L-Ala and the pharmaceutically acceptable acid addition salts thereof.

Somatostatin (also known as somatotropin release inhibiting factor) is a tetradecapeptide of the formula

This tetradecapeptide Was isolated from ovine hypothalamic extracts and was found to be active in inhibiting the secretion of growth hormone (GH), also known as somatotropin. In this regard, see P. Brazeau, W. Vale, R. Burgus, N. Ling, M.

Butcher, J. Rivier, and R. Guillemin, Science, 179, 77 (1973).

D-Trp8-somatostatin was reported in J. Rivier, M. Brown, and W. Vale, Biochemical and Biophysical Research Communications, 65, No. 2, 749751 (1975).

The biologically active tetradecapeptides of formula (I) include the non-toxic acid addition salts thereof. Their structures differ from that of somatostatin by the presence of a D-tryptophan residue in position 8 in place of an L-tryptophan residue, and, additionally, insofar as the tetradecapeptides of formula (I) are concerned, by the presence of a D-alanine, D-serine, or D-valine residue in position 2 in place of a glycine residue and/or by the presence of an L-alanine residue in position 5 in place of an L-asparagine residue. For convenience sake, the tetradecapeptides of formula (I) can be referred to, for example, as D-Ala2, D Trp8-somatostatin; D-Ala2, L-Ala5, D-Trp8-somatostatin; D-Ser2, D-Trp8somatostatin; and D-Val2, D-Trp8-somatostatin.

Thus, this invention includes a compound selected from those of the formula

[formula (I)1, and the pharmaceutically acceptable acid addition salts thereof, and, as derivatives, R - L - Ala - Y' - L - Cys(Rt) - L - Lys(R2) - Z - L - Phe - L Phe - D - Trp(R5) - L - Lys(R2) - L - Thr(R3) - L - Phe - L - Thr(R3) - L Ser(R4) - L - Cys(R1) - X, [formula (II)]; in which Y' is D-Ala, D-Ser(R4), or D-Val; Z is L-Asn or L-Ala; R is hydrogen or an a-amino protecting group; Rl is hydrogen or a thio protecting group; R2 is hydrogen or an E-amino protecting group; R3 and R4 each are hydrogen or a hydroxy protecting group; Rs is hydrogen or formyl; and X is hydroxy or

.0 Resin - > CH ~ in which the Resin is polystyrene; with the proviso that, when X is hydroxy, each of R, R1, R2, R3, R4 and R5 is hydrogen, and, when X is

=f Resin - > CH K each of R, R1, R2, R3 and R4 is other than hydrogen.

The novel tetradecapeptides of formula (I) above may be prepared by oxidising the corresponding straight-chain tetradecapeptide of formula (III), H L - Ala -Y -L -Cys -L -Lys -Z -L -Phe - L - Phe - D - Trp - L - Lys - L - Thr - L - Phe - L - Thr - L - Ser - L - Cys - OH, wherein Y and Z are defined as before.

This reaction converts the two sulfhydryl groups to a disulfide bridge.

Pharmaceutically acceptable non-toxic acid addition salts include the organic and inorganic acid addition salts, for example, those prepared from acids such as hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, and propionic. Preferably, the acid addition salts are those prepared from acetic acid. Any of the above salts are prepared by conventional methods.

Examples of intermediates within the scope of this invention are the following compounds of formula (II) R - L - Ala - D - Ala - L - Cys(R1) - L - Lys(R2) L - Asn - L - Phe - L - Phe - D - Trp(Rs) - L - Lys(R2) - L - Thr(R3) - L Phe - L - Thr(R3) - L - Ser(R4) - L - Cys(R1) - X; R - L - Ala - D - Ala - L - Cys(R1) - L - Lys(R2) - L - Ala - L - Phe - L Phe - D - Trp(Rs) - L - Lys(R2) - L - Thr(R3) - L - Phe - L - Thr(R3) - L Ser(R4) - L - Cys(R1) - X; R -L -Ala -D -Ser(R4) -L7Cys(R) -L Lys(R2)ML - Asn -L -Phe L - Phe - D - Trp(Rs) - L - Lys(R2) - L - Thr(R3) - L - Phe - L - Thr(R3) - L Ser(R4) - L - Cys(R1) - X; and R - L - Ala - D - Val - L - Cys(R1) - L - Lys(R2) - L - Asn - L - Phe - L Phe - D - Trp(Rs) - L - Lys(R2) - L - Thr(R3) - L - Phe - L - Thr(R3) - L Ser(R4) - L - Cys(R1) - X.

Preferred intermediates include the following: H - L - Ala - D - Ala - L - Cys - L - Lys - L - Asn - L - Phe - L - Phe D - Trp - L - Lys - L - Thr - L - Phe - L - Thr - L - Ser - L - Cys - OH; H - L - Ala - D - Ala - L - Cys - L - Lys - L - Ala - L - Phe - L - Phe D - Trp - L - Lys - L - Thr - L - Phe - L - Thr - L - Ser - L - Cys - OH; N - (BOC) - L - Ala - D - Ala - L - (PMB)Cys - L - (CPOC)Lys - L Asn - L - Phe - L - Phe - D - Trp - L - (CPOC)Lys - L - (Bzl)Thr - L - Phe L - (Bzl)Thr - L - (Bzl)Ser - L - (PMB)Cys - O -

/ =aS,Resin CH2-Resin N - (BOC) - L -Ala - D - Ala - L - (PMB)Cys - L - (CPOC)Lys - L - Ala L - Phe - L - Phe - D - Trp - L - (CPOC)Lys - L - (Bzl)Thr - L - Phe L - (Bzl)Thr - L - (Bzl)Ser - L - (PMB)Cys - O -

/ Resin CH 2 // H - L - Ala - D - Ser - L - Cys - L - Lys - t - Asn - L - Phe - L - Phe D - Trp - L - Lys - L - Thr - L - Phe - L - Thr - L - Ser - L - Cys - OH; N - (BOC) - L -Ala - D - (Bzl)Ser - L - (PMB)Cys - L - (CBzOC)Lys -L - Asn - L - Phe - L - Phe - D - Trp - L - (CBzOC)Lys - L - (Bzl)Thr - L - Phe L - (Bzl)Thr - L - (Bzl)Ser - L - (PMB)Cys - O -

.=. Resin Cys-O-CH, H - L - Ala - D - Val - L - Cys - L - Lys - L - Asn - L - Phe - L - Phe D - Trp - L - Lys - L - Thr - L - Phe - L - Thr - L - Ser - L - Cys - OH; and N - (BOC) - L - Ala - D - Val - L - (PMB)Cys - L - (CBzOC)Lys - L Asn - L - Phe - L - Phe - D - (For)Trp - L - (CBzOC)Lys - L - (Bzl)Thr - L Phe - L - (Bzl)Thr - L - (Bzl)Ser - L - (PMB)Cys - O -

/e=o Res i n CH 2 S v v The above formulas defining the intermediates include protecting groups for amino, hydroxy, and thio(sulfhydryl) functions. The properties of a protecting group as defined herein are two-fold. First, the protecting group prevents a reactive moiety present on a particular molecule from undergoing reaction during subjection of the molecule to conditions which could cause disruption of the otherwise active moiety. Secondly, the protecting group is such as can be readily removed with restoration of the original active moiety and under conditions which would not undesirably affect other portions of the molecule. Groups which are useful for these purposes, that is, for protecting amino, hydroxy, and thio groups, are well recognized by those skilled in the art. Indeed, entire volumes have been directed specifically to a description and discussion of methods for using such groups. One such volume is the treatise Protective Groups in Organic Chemistry, M.

F. W. McOmie, Editor, Plenum Press, New York, 1973.

In the above formulas defining the intermediates R is either hydrogen or an aamino protecting group. The a-amino protecting groups contemplated for R are well recognized by those of ordinary skill in the peptide art. Many of these are detailed in McOmie, supra, Chapter 2, authored by J. W. Barton. Illustrative of such protecting groups are benzyloxycarbonyl p - chlorobenzyloxycarbonyl, p bromobenzyloxycarbonyl, o- chlorobenzyloxycarbonyl, 2,6 dichlorobenzyloxycarbonyl, 2,4 - dichlorobenzyloxycarbonyl, o- bromobenzyloxycarbonyl, p - methoxybenzyloxycarbonyl, p nitrobenzyloxycarbonyl, t - butyloxycarbonyl (BOC), t - amyloxyearbonyl,2 -(p - biphenylyl)isopropyloxycarbonyl (BpOC), adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, eyeloheptyloxyearbonyl, triphenylmethyl (trityl), and p - toluenesulfonyl.

Preferably, the a-amino protecting group defined by R is t - butyloxycarbonyl.

R, represents either the hydrogen of the sulfyhydryl group of the cysteine or a protecting group for the sulfhydryl substituent. Many such protecting groups are described in McOmie, supra, Chapter 7, authored by R. G. Hickey, V. R. Rao, and W. G. Rhodes. Illustrative suitable such protecting groups are p-methoxybenzyl, benzyl, p-tolyl, benzhydryl, acetamidomethyl, trityl, p-nitrobenzyl, t-butyl, isobutyloxymethyl, as well as any of a number of trityl derivatives. For additional groups, see, for example, Houben-Weyl, Methods der Organischen Chemie, "Synthese von Peptiden", Vols. 15/1 and 15/2, (1974), Stuttgart, Germany.

Preferably, the sulfhydryl protecting group defined by R, is p-methoxybenzyl.

R2 represents either hydrogen of the E-amino function of the lysine residue or a suitable E-amino protecting group. Illustrative of such groups are the bulk of those mentioned hereinabove as being suitable for use as an a-amino protecting group.

Included as typical such groups are benzyloxycarbonyl, t - butyloxycarbonyl, t amyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, pmethoxybenzyloxycarbonyl, p - chlorobenzyloxycarbonyl, p bromobenzyloxycarbonyl, o- chlorobenzyloxycarbonyl, 2,6 dichlorobenzyloxycarbonyl, 2,4 - dichlorobenzyloxycarbonyl, o- bromobenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, isopropyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl and p - toluenesulfonyl.

As will become apparent hereinafter, the above process of preparing the tetradecapeptide of formula (I) involves periodic cleavage of the a-amino protecting group from the terminal amino acid present on the peptide chain. Thus, it will be appreciated that the identity of the E-amino protecting group on the lysine resldue Is preferably such that it will not be cleaved under the conditions employed in selectively cleaving the a-amino protecting group. Appropriate selection of the a-amino and the E-amino protecting groups is a matter well within the knowledge of a pentide chemist of ordinary skill in the art and depends upon the relative ease with which a particular protecting group can be cleaved. Thus, groups such as 2 (p - biphenylyl)isopropyloxycarbonyl (BpOC) and trityl are very labile and can be cleaved even in the presence of mild acid. A moderately strong acid, such as hydrochloric acid, trifluoroacetic acid, or boron trifluoride in acetic acid, is required to cleave other groups such as t - butyloxycarbonyl, t - amyloxycarbonyl, adamantyloxycarbonyl, and p - methoxybenzyloxycarbonyl. Even stronger acid conditions are required to effect cleavage of other protecting groups such as benzyloxycarbonyl, halobenzyloxycarbonyl, p - nitrobenzyloxycarbonyl, cycloalkoxycarbonyl, and isopropyloxycarbonyl. Cleavage of these latter groups requires drastic acid conditions such as the use of hydrogen bromide, hydrogen fluoride, or boron trifluoroacetate in trifluoroacetic acid. Of course, any of the more labile groups will also be cleaved under the stronger acid conditions.

Appropriate selection of the amino protecting groups thus will include the use of a group at the a-amino function which is more labile than that employed as the Eamino protecting group coupled with cleavage conditions designed to selectively remove only the a-amino function. In this context, R2 preferably is o- chlorobenzyloxycarbonyl or cyclopentyloxycarbonyl, and, in conjunction therewith, the a-amino protecting group of choice for use in each of the amino acids which is added to the peptide chain preferably is t - butyloxvcarbonyl.

The groups R3 and R4 both represent hydrogen or, separately, a protecting group for the alcoholic hydroxyl of threonine and serine, respectively. Many such protecting groups are described in McOmie, supra, Chapter 3, authored by C. B.

Reese. Typical such protecting groups are, for example, C1-C4 alkyl, such as methyl, ethyl, and t-butyl; benzyl; substituted benzyl, such as p-methoxybenzyl, pnitrobenzyl, p-chlorobenzyl, and o-chlorobenzyl; C1C3 alkanoyl, such as formyl, acetyl, and propionyl; triphenylmethyl (trityl). Preferably, when R3 and R4 are protecting groups, the protecting group of choice in both instances is benzyl.

The group R5 represents either hydrogen or formyl, the latter being a protecting group for the

of the tryptophan residue. The use of such a protecting group is optional and therefore R,can be hydrogen (N-unprotected) or formyl (N-protected).

The group X represents the carboxyl terminal of the tetradecapeptide chain and can be hydroxyl in which case a free carboxyl group thereby is defined. In addition, X represents the solid resin support to which the carboxyl terminal moiety of the peptide is linked during its synthesis. This solid resin is represented by the formula

Resin .7 wherein Resin is polystyrene.

In any of the above, when X represents hydroxyl, each of R, R1, R2, R R and R5 is hydrogen. When X represents the solid resin support, each of R, R1, R2, R3 and R4 is a protecting group.

The following abbreviations, most of which are well known and commonly used in the art, are employed herein:

Ala - Alanine Asn -- Asparagine Cys -- Cysteine Gly - Glycine Lys - Lysine Phe - Phenylalanine k as radicals Ser - Serine Thr -- Threonine Trp -- Tryptophan Val - Valine DCC N,N'-Dicyclohexylcarbodiimide DMF -- N,N-Dimethylformamide BOC -- t-Butyloxycarbonyl PMB -- p-Methoxybenzyl CBzOC -- o-Chlorobenzyloxycarbonyl CPOC -- Cyclopentyloxycarbonyl Bzl - Benzyl For - Formyl BpOC -- 2-(p-biphenylyl)isopropyloxycarbonyl Although the selection of the particular protecting groups to be employed in preparing the compounds of formula (I) remains a matter well within the ordinary skill of a synthetic peptide chemist, it is well to recognize that the proper selection of the protecting groups is dependent upon the particular succeeding reactions which must be carried out. Thus, the protecting group of choice must be one which is stable both to the reagents and under the conditions employed in the succeeding steps of the reaction sequence. For example, as already discussed to some degree hereinabove, the particular protecting group which is employed must be one which remains intact under the conditions which are employed for cleaving the a-amino protecting group of the terminal amino acid residue of the peptide fragment in preparation for the coupling of the next succeeding amino acid fragment to the peptide chain. It is also important to select, as a protecting group, one which will remain intact during the building of the peptide chain and which will be readily removable upon completion of the synthesis of the desired tetradecapeptide product. All of these matters are well within the knowledge and understanding of a peptide chemist of ordinary skill in the art.

As is evident from the above discussion, the tetradecapeptide of formula (I) can be prepared by solid phase synthesis. This synthesis involves a sequential building of the peptide chain beginning at the C-terminal end of the peptide.

Specifically, cysteine first is linked at its carboxyl function to the resin by reaction of an amino-protected, S-protected cysteine with a chloromethylated resin or a hydroxymethyl resin. Preparation of a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London), 38 1597-98 (1966). The chloromethylated resin is commercially available from Lab Systems, Inc., San Mateo, California.

In accomplishing linkage of the C-terminal cysteine to the resin, the protected cysteine first is converted to its cesium salt. This salt then is reacted with the resin in accordance with the method described by V. F. Gisin, Helv. Chim. Acta, 56, 1476 (1973). Alternatively, the cysteine can be linked to the resin by activation of the carboxyl function of the cysteine molecule by application of readily recognized techniques. For example, the cysteine can be reacted with the resin in the presence of a carboxyl group activating compound such as N,N' - dicyclohexylcarbodiimide (DCC).

Once the free carboxyl cysteine has been appropriately linked to the resin support, the remainder of the peptide building sequence involves the step-wise addition of each amino acid to the N-terminal portion of the peptide chain.

Necessarily, therefore, the particular sequence which is involved comprises a cleavage of the a-amino protecting group from the amino acid which represents the N-terminal portion of the peptide fragment followed by coupling of the next succeeding amino acid residue to the now free and reactive N-terminal amino acid.

Cleavage of the a-amino protecting group can be effected in the presence of an acid such as hydrobromic acid, hydrochloric acid, trifluoroacetic acid, ptoluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and acetic acid, with formation of the respective acid addition salt product. Another method which is available for accomplishing cleavage of the amino protecting group involves the use of boron trifluoride. For example, boron trifluoride diethyl etherate in glacial acetic acid will convert the amino-protected peptide fragment to a BF3 complex which then can be converted to the deblocked peptide fragment by treatment with a base such as aqueous potassium bicarbonate. Any of these methods can be employed as long as it is recognized that the method of choice must be one which accomplishes cleavage of the N-terminal a-amino protecting group without disruption of any other protecting groups present on the peptide chain. In this regard, it is preferred that the cleavage of the N-terminal protecting group be accomplished using trifluoroacetic acid. Generally, the cleavage will be carried out at a temperature from OOC. to room temperature.

Once the N-terminal cleavage has been effected, the product which results normally will be in the form of the acid addition salt of the acid which has been employed to accomplish the cleavage of the protecting group. The product then can be converted to the free terminal amino compound by treatment with a mild base, typically a tertiary amine such as pyridine, or triethylamine.

The peptide chain then is ready for reaction with the next succeeding amino cid. This can be accomplished by employing any of several recognized techniques.

In order to achieve coupling of the next-succeeding amino acid to the N-terminal peptide chain, an amino acid which has a free carboxyl but which is suitably protected at the a-amino function as well as at any other active moiety is employed.

The amino acid then is subjected to conditions which wi;l render the carboxyl function active to the coupling reaction. One such activation technique which can be employed in the synthesis involves the conversion of the amino acid to a mixed anhydride. Thereby, the free carboxyl function of the amino acid is activated by reaction with another acid, typically a carbonic acid in the form of its acid chloride.

Examples of such acid chlorides which can be used to form the appropriate mixed anhydrides are ethyl chloroformate, phenyl chloroform ate, sec-butyl chloroformate, isobutyl chloroformate, and pivaloyl chloride.

Another method of activating the carboxyl function of the amino acid to achieve coupling is by conversion of the amino acid to its active ester derivative.

Examples of such active esters are, for example, a 2,4,5 - trichlorophenyl ester, a pentachlorophenyl ester, a p-nitrophenyl ester, an ester formed from 1 - hydroxybenzotriazole, and an ester formed from N - hydroxysuccinimide. Another method for effecting coupling of the C-terminal amino acid to the peptide fragment involves carrying out the coupling reaction in the presence of at least an equimolar quantity of N,N' - dicyclohexylcarbodiimide (DCC). This latter method is preferred for preparing the tetradecapeptide of formula (II) wherein X is

Resin .7 -CH K Once the desired amino acid sequence has been prepared, the resulting peptide can be removed from the resin support. This is accomplished by treatment of the protected resin-supported tetradecapeptide with hydrogen fluoride.

Treatment with hydrogen fluoride cleaves the peptide from the resin; in addition, however, it cleaves all remaining protecting groups present on the reactive moieties located on the peptide chain as well as the a-amino protecting group present at Nterminal amino acid. When hydrogen fluoride is employed to effect the cleavage of the peptide from the resin as well as removal of the protecting groups, it is preferred that the reaction be carried out in the presence of anisole. The presence of anisole has been found to inhibit the potential alkylation of certain amino acid residues present in the peptide chain. In addition, it is preferred that the cleavage be carried out in the presence of ethyl mercaptan. The ethyl mercaptan serves to protect the indole ring of the tryptophan residue and, furthermore, facilitates conversion of the blocked cysteines to their thiol forms. Also, when R5 is formyl, the presence of ethyl mercaptan facilitates hydrogen fluoride cleavage of the formyl group.

Once the cleavage reaction has been accomplished, the product which is obtained is a straight-chain peptide containing 14 amino acid residues. In order to obtain the final product of formula (I), it is necessary to treat the straight-chain tetradecapeptide under conditions which will effect its oxidation by converting the two sulfhydryl groups present in the molecule, one at each systeinyl moiety, to a disulfide bridge. This can be accomplished by treating a dilute solution of the linear tetradecapeptide with any of a variety of oxidizing agents including, for example, iodine, and potassium ferricyanide. Air also can be employed as oxidizing agent, the pH of the mixture preferably being from 2.5 to 9.0, and more preferably from 7.0 to 7.6. When air is used as oxidizing agent, the concentration of the peptide solution preferably is not greater than 0.4 mg. of the peptide per milliliter of solution, and more preferably is about 50 g./ml.

The compounds of formula (I) may be administered to warm-blooded mammals, including humans, by any of several methods, including orally, sublingually, subcutaneously, intramuscularly and intravenously. Administration of these compounds will inhibit the release of growth hormone. This inhibitory effect is beneficial in those instances in which the host being treated requires a therapeutic treatment for excess secretion of somatotropin, such secretion being associated with adverse conditions such as juvenile diabetes and acromegaly. These compounds also exhibit other physiological effects, including the inhibition of gastric acid secretion, useful in treatment of ulcer conditions; the inhibition of exocrine pancreas secretion, potentially useful in treatment of pancreatitis; the inhibition of secretion of insulin and glucagon; and the reduction of gut motility, useful in gastrointestinal radiology. Preferably, the dose range for sublingual or oral administration is 1 mg. to 100 mg./kg. of body weight per day. Preferably, the dose range for intravenous, subcutaneous, or intramuscular administration is from 10 ,ug.

to 1 mg./kg. of body weight per day, and, more preferably, is from 50 ,ug. to 100 Mg./kg. of body weight per day. It is evident that the dose range will vary widely depending upon the particular condition which is being treated as well as the severity of the condition.

It is also possible to administer the compounds of formula (I) in the form of tablets containing other inocuous ingredients. Inert diluents or carriers, for example, magnesium carbonate or lactose, can be used together with conventional disintegrating agents, for example, maize starch and alginic acid, and lubricating agents, for example, magnesium stearate. Preferably, the amount of carrier or diluent will range from 5 to 95 wt. percent of the final composition, and preferably from 50 to 85 wt. percent of the final composition. Suitable flavoring agents also can be employed in the final preparation rendering the composition more palatable for administration.

When the compounds of formula (I) are to be administered intravenously, suitable carriers may be employed, such as, for example, isotonic saline, and phosphate buffer solutions.

The following examples are illustrative of the preparation of compounds of this invention. In these examples, percentage compositions and ratios are expressed on a volume basis unless otherwise indicated.

Example 1 N - t - Butyloxycarbonyl - L - cysteinyl (S - p - methoxybenzyl) Methylated Polystyrene Resin To 20.0 g. of chloromethylated polystyrene resin (Lab. Systems, Inc., 0.75 mmoles/gram) suspended in 150 ml. of N,N - dimethylformamide (DMF) were added 3.7 grams (7.8 mmoles) of the cesium salt of N - t - butyloxycarbonyl - (S p - methoxybenzyl) - L - cysteine. The mixture was stirred at room temperature for three days. The resin then was filtered and washed successively with DMF, a mixture of 90 percent DMF and 10 percent water, and DMF. To the resin suspended in DMF was added a solution of 5.5 grams of cesium acetate in hot DMF. The mixture was stirred overnight at room temperature, for eight hours at 50 C., overnight at room temperature, for eight hours at 50 C., and for three days at room temperature. The resin then was filtered and was washed successively with DMF, a mixture of 90 percent DMF and 10 percent water, DMF, a mixture of 90 percent DMF and 10 percent water, DMF, and 95 percent ethanol. The resin then was dried in vacuo at 500 C. to obtain the title product containing 0.45 percent nitrogen (0.32 mmole/gram) and 0.80 percent sulfur (0.25 mmole/gram).

Example 2 t - Butyloxycarbonyl - L - alanyl - D - alanyl - L - S - p methoxybenzyl)cysteinyl - L - (cyclopentyloxycarbonyl)lysyl L - asparaginyl - L - phenylalanyl - L - phenylalanyl - D - tryptophyl L - (N - cyclopentyloxycarbonyl)lysyl - L - (O - benzyl)threonyl L - phenylalanyl - L - (O - benzyl)threonyl - L - (0 - benzyl)seryl L - (S - p - methoxybenzyl)cysteinyl Methylated Polystyrene Resin To a reaction vessel were added 16.26 g. of the product from Example 1.

Sequences of deprotection, neutralization, coupling, and a recoupling were carried out for the addition of each amino acid to the peptide. The alcohol and 10 percent t-amyl alcohol; and (28) three washes (10.0 ml./gram resin) of three minutes each with methylene chloride.

The above treatment sequence was employed for addition of each of the amino acids with the exception of the L-asparagine residue. The L-asparagine residue was incorporated via its p-nitrophenyl active ester. In doing so, Step (11) above was modified to the following 3-step sequence: (a) three washes (7.5-15 ml./gram resin) of three minutes each with DMF; (b) addition of 1.0 mmole/gram resin of the p-nitrophenyl ester of N - t - butyloxycarbonyl - L - asparagine in 7.5-15 ml./gram resin of a 1:1 mixture of DMF and methylene chloride followed by mixing for 720 minutes; and (c) three washes (7.5-15 ml./gram resin) of three minutes each with DMF. Also, Step (20) was altered to duplicate the above Step (b) with the exception that a 3:1 mixture of DMF and methylene chloride was employed.

The amino acid analysis of the resulting product gave the following results, lysine being employed as standard: Asn, 1.01; 2Thr, 2.02; Ser, 0.96; 2Ala, 2.24; 3Phe, 2.85; 2Lys, 2.00; Trp, 0.57. The presence of cysteine was not determined since it is destroyed by the method of analysis.

Example 3 L - alanyl - D - alanyl - L - cysteinyl - L - lysyl - L - asparaginyl L - phenylalanyl - L - phenylalanyl - D - tryptophyl L - lysyl - L - threonyl - L - phenylalanyl - L - threonyl L - seryl - L - eysteine To a mixture of 10 ml. of anisole and 10 ml. of ethyl mercaptan were added 2.25 g. of the protected tetradecapeptide-resin of Example 2. The mixture was cooled in liquid nitrogen, and 40 ml. of liquid hydrogen fluoride were added by distillation. The resulting mixture was allowed to warm to OOC. and was stirred for 1.5 hours. The hydrogen fluoride then was distilled off, and ether was added to the remaining mixture. The resulting solid material was collected by filtration and washed with ether. The product was dried, and the deprotected tetradecapeptide was extracted from the resin mixture using 1 M acetic acid. The acetic acid solution then was immediately lyophilized to dryness in the dark. The resulting solid was suspended in a mixture of 15 ml. of 1 M acetic acid and 5 ml. of glacial acetic acid.

Purification of the product was accomplished by chromatography on a Sephadex (Registered Trade Mark) G-25 F column. The chromatographic conditions were: solvent, degassed 0.2 M acetic acid; column size, 7.5x150 cm.; temperature, 260C.; flow rate, 700 ml./hour; fraction volume, 24.5 ml.

Absorbance at 280 m,u of each fraction plotted versus fraction number indicated one very large peak with several small peaks. A collection of four sets of fractions was made. The fractions which were combined and their effluent volumes are as follows: Fractions 127-189 (3087-4630 ml.) Fractions 19199 (4631-4875 ml.) Fractions 200212 (4876-5194 ml.) Fractions 213-310 (5195-7595 ml.) The four samples were lyophilized to dryness in the dark and collected. UV spectroscopy indicated that the third sample (140.4 mg.) was the best product.

Example 4 Oxidation to D-Ala2, D-Trp8-somatostatin A portion of the reduced D-Ala2, D-Trp8-somatostatin from Example 3 (18 mg.) was dissolved in 70 ml. of 1 M acetic acid. A UV spectrum of the resulting solution indicated a concentration of 200 yg./ml. The solution was diluted with 210 ml. of distilled water to achieve a 50 Mg./ml. concentration. Concentrated ammonium hydroxide was added to adjust the pH of the mixture to 8.1. The solution was stirred at room temperature in the dark for 24 hours after which an Ellman titration (Arch. Biochem. Biophys. 82, 70 (1959)) indicated that oxidation was complete. The mixture was acidified with 3 ml. of glacial acetic acid and lyophilized to dryness.

The resulting solid was dissolved in 5 ml. of deoxygenated 0.2 M acetic acid.

The product was absorbed on a Sephadex G-25 F column. The chromatographic conditions were as follows: solvent, degassed 50 /E acetic acid; column size 5.0x90 cm.; temperature, 260C.; flow rate, 343 ml./hour; fraction volume, 20 ml.

Absorbance at 280 m for each fraction plotted versus fraction number indicated two large peaks. The first peak represented aggregated forms of the product, and the second peak represented good monomeric product. The product represented by the second peak was collected, diluted with distilled water, and lyophilized to dryness. The resulting solid was dissolved in 12 ml. of degassed 0.2 M acetic acid, and the solution was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 0.2 M acetic acid; column size, 5.0xl50 cm.; temperature, 260C.; flow rate, 516 ml./hour; fraction volume, 17.2 ml.

Absorbance at 280 m of each fraction plotted versus fraction number indicated one large peak with some low shoulders. UV spectroscopy showed the large peak to be good product. Fractions 150173 (effluent volumes of 2563- 2977 ml.) were combined and lyophilized to dryness in the dark to obtain 44.56 mg.

of the desired product of formula (I), D-Ala2, D-Trp8-somatostatin. The product was combined with a sample of the same product obtained from a parallel oxidation using the same lot of reduced D-Ala2, D-Trp-8-somatostatin.

Optical rotation [al6=-39.90 (1 percent acetic acid).

Amino acid analysis: Ala+D-Ala,9ocyso 89lJys,.oAsno 87phees6pheo ss D-Trp0.96Lys1.0Thr0.89Phe0.98Thr0.89Ser0.79Cys0.89.

Example 5 N - t - Butyloxyearbonyl - L - alanyl - D - (0 - benzyl)seryl - L (S - p - methoxybenzyl)cysteinyl - L - (NE O chlorobenzyloxycarbonyl)lysyl - L - asparaginyl - L phenylalanyl - L - phenylalanyl - D - (formyl)tryptophyl - L (N' - o - chlorobenzyloxycarbonyl)lysyl - L - (O - benzyl)threonyl L - phenylalanyl - L -(O - benzyl)threonyl - L - (0 - benzyl)seryl L - (S - p - methoxybenzyl)cysteinyl Methylated Polystyrene Resin The product from Example 1(10.0 grams) was placed in the reaction vessel of a Beckman 990 automatic peptide synthesizer, and eleven of the remaining thirteen amino acids were added employing the automatic synthesizer. The resulting protected dodecapeptide resin was divided into three equal portions, and the final two residues were introduced to one of the portions. The amino acids which were employed as well as the sequence of their employment is as follows: (1) N - t butyloxycarbonyl - (0 - benzyl) - L - serine; (2) N - t - butyloxycarbonyl - (O benzyl) - L - threonine; (3) N - t - butyloxycarbonyl - L - phenylalanine; (4) N t - butyloxycarbonyl - (O - benzyl) - L- threonine; (5) N - t butyloxycarbonyl - NE - o - chlorobenzyloxycarbonyl - L - lysine; (6) Na - t - butyloxycarbonyl - (N - formyl) - D - tryptophan; (7) N - t - butyloxycarbonyl L - phenylalanine; (8) N - t - butyloxycarbonyl - L - phenylalanine; (9) N - t butyloxycarbonyl- L- asparagine, p-nitrophenyl ester; (10) N- t butyloxycarbonyl - NE - o - chlorobenzyloxycarbonyl - L - lysine; (II) N - t butyloxycarbonyl - (S - p - methoxybenzyl) - L - cysteine; (12) N - t - butyloxycarbonyl - (0 - benzyl) - D - serine; and (13) N - t - butyloxycarbonyl L- alanine. The sequence of deprotection, neutralization, coupling, and recoupling for the introduction of each amino acid into the peptide is as follows: (1) three washes (10 ml./gram resin) of three minutes each with chloroform; (2) removal of BOC group by treatment twice for twenty minutes each with 10 ml./gram resin of a mixture of 29 percent trifluoroacetic acid, 48 percent chloroform, 6 percent triethylsilane, and 17 percent methylene chloride; (3) two washes (10 ml./gram resin) of three minutes each with chloroform; (4) one wash (10 ml./gram resin) of three minutes with methylene chloride; (5) three washes (10 ml.1gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (6) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (7) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (8) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (9) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (10) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (11) addition of 1.0 mmole/gram resin of the protected amino acid and 1.0 mmole/gram resin of N,N' dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of methylene chloride followed by mixing for 120 minutes; (12) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (13) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent tamyl alcohol; (14) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (15) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (16) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (17) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (18) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (19) three washes (10 ml./gram resin) of three minutes each with DMF; (20) addition of 1.0 mmole/gram resin of the protected amino acid and 1.0 mmole/gram resin of N,N' dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of a 1:1 mixture of DMF and methylene chloride followed by mixing for 120 minutes; (21) three washes (10 ml./gram resin) of three minutes each with DMF; (22) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (23) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (24) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (25) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (26) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (27) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; and (28) three washes (10 ml./gram resin) of three minutes each with methylene chloride.

The above treatment sequence was employed for addition of each of the amino acids with the exception of the L-asparagine residue. This residue was incorporated via its p-nitrophenyl active ester. In doing so, Step (11) above was modified to the following 3-step sequence: (a) three washes (10 ml./gram resin) of three minutes each with DMF; (b) addition of 1.0 mmole/gram resin of the pnitrophenyl ester of N - t - butyloxycarbonyl - L - asparagine in 10 ml./gram resin of a 1:3 mixture of DMF and methylene chloride followed by mixing for 720 minutes; and (c) three washes (10 ml./gram resin) of three minutes each with DMF.

Also, Step (20) above was modified to the use of the p-nitrophenyl ester of N - t butyloxycarbonyl - L - asparagine in a 3:1 mixture of DMF and methylene chloride followed by mixing for 720 minutes.

The finished peptide-resin was dried in vacuo. A portion of the product was hydrolyzed by refluxing it for 21 hours in a 1:1 mixture of concentrated hydrochloric acid and dioxane. Amino acid analysis of the resulting product gave the following results, lysine being employed as standard: Asn, 1.17; 2 Thr, 2.28; 2 Ser, 2.12; Ala, 1.22; 3 Phe, 2.94; 2 Lys, 2.00, Trp, 0.74.

Dimethyl sulfoxide was added to the tryptophan hydrolysis mixture. Cysteine was not determined since it is destroyed by the method of analysis.

Example 6 L - alanyl - D - seryl - L - cysteinyl - L - lysyl - L - asparaginyl L - phenylalanyl - L - phenylalanyl - D tryptophyl - L - lysyl - L - threonyl - L - phenylalanyl - L threonyl - L - seryl - L - cysteine To a mixture of 5 ml. of anisole and 5 ml. of ethyl mercaptan were added 2.805 grams (at substitution level of 0.149 mmoles/gram) of the protected tetradecapeptide-resin of Example 5. The mixture was cooled in liquid nitrogen, and 56 ml. of liquid hydrogen fluoride were added by distillation. The resulting mixture was allowed to warm to OOC. and was stirred for 2 hours. The hydrogen fluoride then was removed by distillation, and ether was added to the remaining mixture. The mixture was cooled to OOC., and the resulting solid was collected by filtration and washed with ether. The product was dried, and the deprotected tetradecapeptide was extracted from the resin mixture using 1 M acetic acid and a small amount of glacial acetic acid. The acetic acid solution was then lyophilized to dryness in the dark. The resulting white solid was suspended in a mixture of 10 ml.

of degassed 0.2 M acetic acid and 4 ml. of glacial acetic acid. The resulting suspension was heated; however, the solid did not completely dissolve. The insoluble portion was filtered off, and the clear, slightly yellow filtrate was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 0.2 M acetic acid; column size 7.5x150 cm.; temperature, 260C.; flow rate, 629 ml./hour; fraction volume, 22.0 ml.

Absorbance at 280 my of each fraction plotted versus fraction number indicated one large peak with a following shoulder. UV spectroscopy revealed that the main peak was good product. The fractions which were combined and their effluent volumes are as follows: Fractions 235-260 (5148-5720 ml., peak=5496 ml.) This collection of fractions did not include the following shoulder. UV spectroscopy indicated that 237 mg. of the product were present, (yield=34.00/).

An Ellman titration of an aliquot indicated a free sulfhydryl content of 82.40/ of theoretical.

Example 7 Oxidation to D-Ser2, D-Trp8-somatostatin The solution of the reduced D-Ser2, D-Trp8-somatostatin (572 ml., theoretically 237 mg. of product) from Example 6 was diluted with 120 ml. of 0.2 M acetic acid and 4040 ml. of distilled water to achieve a concentration of 50 g./ml.

Concentrated ammonium hydroxide was added to adjust the pH of the mixture to 6.8. The solution was stirred at room temperature in the dark for 41 hours after which an Ellman titration indicated that oxidation was complete.

The mixture was concentrated in vacuo to a volume of about 10 ml. The concentrate was diluted with 10 ml. of glacial acetic acid and then was desalted on a Sephadex G-25 F column. The chromatographic conditions were as follows: solvent, degassed 50% acetic acid; column size, 5.0x90 cm.; temperature, 260 C.; flow rate, 252 ml./hour; fraction volume, 16.8 ml.

Absorbance at 280 m,u for each fraction plotted versus fraction number indicated two large peaks. The first peak represented the aggregated forms of the product, and the second peak represented monomeric product. The material represented by the second peak [Fractions 5468 (8901142 ml.)] was collected and lyophilized to dryness in the dark. The resulting solid was dissolved in 15 ml. of degassed 0.2 M acetic acid and was applied to a Sephadex G-25 F column.

Chromatographic conditions were: solvent, degassed 0.2 M acetic acid; column size. 5.0x 150 cm.; temperature, 260C.; flow rate, 477 ml./hour; fraction volume, 16.7 ml.

Absorbance at 280 my for each fraction plotted versus fraction number showed one large peak having a front side shoulder. UV spectroscopy indicated that the main peak was good product. Fractions 155-170 (effluent volumes of 2572-2839 ml., peak=2664 ml.) were combined and lyophilized to dryness in the dark. UV spectroscopy indicated 70.2 mg. of the desired product (yield from reduced form=29.6 /n).

Optical rotation [a]026=-43.40 (1 percent acetic acid).

Amino acid analysis: Ala, 0.99; 2 Ser, 1.60; 2 Cys, 1.88; 2 Lys, 2.0; Asn, 1.02; 3 Phe, 2.79; Trp, 0.79; 2 Thr, 1.88.

The above results are expressed as ratio to (Ala+Lys)/3=1.0. The following three 21 hour hydrolyses were run: (1) In presence of dimethyl sulfoxide to oxidize cysteine to cysteic acid.

(2) Thioglycolic acid scavenged.

(3) Without scavenger or oxidizer.

All of the above values are averages of the three hydrolyses except the following: Cys and Ser; (1) and (3) only; D-Trp; (2) only; Phe; (2) and (3) only.

Example 8 N - t - butyloxycarbonyl - L - alanyl - D - valyl - L - (S - p - methoxy benzyl)cysteinyl - L (NE - O - chlorobenzyloxycarbonyl)lysyl L - asparaginyl - L - phenylalanyl - L - phenylalanyl - D (formyl)tryptophyl - L - (NE - O - chlorobenzyloxycarbonyl)lysyl L - (O - benzyl)threonyl - L - phenylalanyl - L - O - benzyl)threonyl - L - (O - benzyl)seryl - L - (S - p - methoxybenzyl)cysteinyl Methylated Polystyrene Resin The product from Example 1(10.0 grams) was placed in the reaction vessel of a Beckman 990 automatic peptide synthesizer, and eleven of the remaining thirteen amino acids were added employing the automatic synthesizer. The resulting protected dodecapeptide resin was divided into three equal portions, and the final two residues were introduced to one of the portions. The amino acids which were employed as well as the sequence of their employment is as follows: (1) N - t butyloxycarbonyl - (0 - benzyl) - L - serine; (2) N - t - butyloxycarbonyl - (O benzyl) - L - threonine; (3) N - t - butyloxycarbonyl - L - phenylalanine: (4) N t - butyloxycarbonyl - (0 - benzyl) - L- threonine; (5) N - t butyloxycarbonyl - NE o - chlorobenzyloxycarbonyl - L - lysine; (6) N - t - butyloxycarbonyl - (N - formyl) - D - tryptophan; (7) N - t - butyloxycarbonyl L - phenylalanine; (8) N - t - butyloxycarbonyl - L - phenylalanine; (9) N - t butyloxycarbonyl - L- asparagine, p-nitrophenyl ester; (10) N - t butyloxycarbonyl - NE o - chlorobenzyloxycarbonyl - L - lysine; (II) N - t butyloxycarbonyl - (S - p - methoxybenzyl) - L - cysteine; (12) N - t - butyloxycarbonyl - D - valine; and (13) N - t - butyloxycarbonyl - L - alanine.

The sequence of deprotection, neutralization, coupling, and recoupling for the introduction of each amino acid into the peptide is as follows: (1) three washes (10 ml./gram resin) of three minutes each with chloroform; (2) removal of BOC group by treatment twice for twenty minutes each with 10 ml./gram resin of a mixture of 29 percent trifluoroacetic acid, 48 percent chloroform, 6 percent triethylsilane, and 17 percent methylene chloride; (3) two washes (10 ml./gram resin) of three minutes each with chloroform; (4) one wash (10 ml./gram resin) of three minutes with methylene chloride; (5) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (6) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (7) neutralization by three elements of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (8) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (9) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent tamyl alcohol; (10) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (11) addition of 1.0 mmole/gram resin of the protected amino acid and 1.0 mmole/gram resin of N,N' - dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of methylene chloride followed by mixing for 120 minutes; (12) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (13) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol, (14) three washes (10 ml./granti; resin) of three minutes each with methylene chloride; (15) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (16) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (17) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (18) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (19) three washes (10 ml./gram resin) of three minutes each with DMF; (20) addition of 1.0 mmole/gram resin of the protected amino acid and 1.0 mmole/gram resin of N,N' - dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of a 1:1 mixture of DMF and methylene chloride followed by mixing for 120 minutes; (21) three washes (10 ml./gram resin) of three minutes each with DMF; (22) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (23) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent t-amyl alcohol; (24) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (25) neutralization by three treatments of three minutes each with 10 ml./gram resin of 3 percent triethylamine in methylene chloride; (26) three washes (10 ml./gram resin) of three minutes each with methylene chloride; (27) three washes (10 ml./gram resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent tamyl alcohol; and (28) three washes (10 ml./gram resin) of three minutes each with methylene chloride.

The above treatment sequence was employed for addition of each of the amino acids with the exception of the L-asparagine residue. This residue was incorporated via its p-nitrophenyl active ester. In doing so, Step (11) above was modified to the following 3-step sequence: (a) three washes (10 ml./gram resin) of three minutes each with DMF; (b) addition of 1.0 mmole/gram resin of the pnitrophenyl ester of N - t - butyloxycarbonyl - L - asparagine in 10 ml./gram resin of a 1:3 mixture of DMF and methylene chloride followed by mixing for 720 minutes; and (c) three washes (10 ml./gram resin) of three minutes each with DMF.

Also, Step (20) above was modified to the use of the p-nitrophenyl ester of N - t butyloxycarbonyl - L - asparagine in a 3:1 mixture of DMF and methylene chloride followed by mixing for 720 minutes.

The finished peptide-resin was dried in vacuo. A portion of the product was hydrolyzed by refluxing for 72 hours in a 1:1 mixture of concentrated hydrochloric acid and dioxane. Amino acid analysis of the resulting product gave the following results, lysine being employed as standard: Asn, 1.24; 2 Thr, 2.64; Ser, 1.16; Ala, 1.30; Val, 1.14; 3 Phe, 3.66; 2 Lys 2.00; Trp, 0.77.

Dimethyl sulfoxide was added to the tryptophan hydrolysis mixture. Cysteine was not determined since it is destroyed by the method of analysis.

Example 9 L - alanyl - D - valyl - L - cysteinyl - L - lysyl - L - asparaginyl L - phenylalanyl - L - phenylalanyl - D tryptophyl - L - lysyl - L - threonyl - L - phenylalanyl L - threonyl - L - seryl - L - cystein To a mixture of 5 ml. of anisole and 5 ml. of ethyl mercaptan were added 2.70 grams (at substitution level of 0.151 mmoles/gram) of the protected tetradecapeptide-resin of Example 8. The mixture was cooled in liquid nitrogen, and 54 ml. of liquid hydrogen fluoride were added by distillation. The resulting mixture was allowed to warm to OOC. and was stirred for 2 hours. The hydrogen fluoride then was removed by distillation, and ether was added to the remaining mixture. The mixture was cooled to 0 C., and resulting solid was collected by filtration and washed with ether. The product was dried, and the deprotected tetradecapeptide was extracted from the resin mixture using 1 M acetic acid. The acetic acid solution then was immediately lyophilized to dryness in the dark. The resulting white solid was suspended in a mixture of 10 ml. of degassed 0.2 M acetic acid and 4 ml. of glacial acetic acid. The resulting suspension was heated; however, the solid did not completely dissolve. The insoluble portion was filtered off, and the clear, slightly yellow filtrate was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 0.2 M acetic acid; column size, 7.5x 150 cm.; temperature, 260C.; flow rate, 628 ml./hour; fraction volume, 22.0 ml.

Absorbance at 280 my of each fraction plotted versus fraction number indicated one large, broad peak with a following shoulder. UV spectroscopy revealed that the main peak was good product. The fractions which were combined and their effluent volumes are as follows: Fractions 233-262 (5104--5764 ml., peak=5499 ml.).

This collection of fractions did not include the following shoulder. UV spectroscopy indicated that 176 mg. of the product were present (yield=25.70/,). An Ellman titration of an aliquot indicated a free sulfhydryl content of 76.70/c of theoretical.

Example 10 Oxidation to D-Val2, D-Trp8-somatostatin The solution of the reduced D-Val2, D-Trp8-somatostatin (660 ml., theoretically 176 mg.) from Example 9 was diluted with distilled water to achieve a concentration of 50 g./ml. Concentrated ammonium hydroxide was added to adjust the pH of the mixture to 6.7. The solution was stirred at room temperature in the dark for 64 hours after which an Ellman titration indicated that oxidation was complete.

The mixture was concentrated in vacuo to a volume of about 10 ml. The concentrate was diluted with 10 ml. of glacial acetic acid and then was desalted on a Sephadex G-25 F column. The chromatographic conditions were as follows: solvent, degassed 50( acetic acid; column size, 5.0x90 cm.; temperature, 260C.; flow rate, 225 ml./hour; fraction volume, 17.0 ml.

Absorbance at 280 mA4 for each fraction plotted versus fraction number indicated two large peaks. The first peak represented the aggregated forms of the product, and the second peak represented monomeric product. The material represented by the second peak [Fractions 48-65 (799-1105 ml.)] was collected and lyophilized to dryness in the dark. The resulting solid was dissolved i dark. UV spectroscopy indicated 55.6 mg. of the desired product (yield from reduced form=31.6 /).

The resulting solid was dissolved in 6 ml. of 50 /n acetic acid and was rechromatographed on a Sephadex G-25 F column. The chromatographic conditions were: solvent, degassed 50 /n acetic acid; column size, 2.5x 180 cm.; temperature, 260C.; flow rate, 53.9 ml./hour; fraction volume, 8.98 ml.

Absorbance at 280 m,u of each fraction plotted versus fraction number indicated one large peak. A central cut of this peak [Fractions 55-61(485-548 ml., peak=531 ml.) was combined and lyophilized to dryness in the dark. UV spectroscopy indicated the presence of 49.2 mg. of product in the sample (88.5 /n recovery).

Optical rotation [al26=S2.20 (1 percent acetic acid).

Amino acid analysis: Ala, 0.98; Val, 0.80; 2 Cys, 1.76; 2 Lys, 2.02; Asn, 1.0; 3 Phe, 2.85; Trp, 1.03; 2 Thr, 1.86; Ser, 0.78.

The above results are expressed as ratios to (Ala+Lys)/3=l.0. The following two 21 hour hydrolyses were run: (1) In presence of dimethyl sulfoxide to oxidize cysteine to cysteic acids.

(2) Without scavenger or oxidizer.

The Trp value is determined from UV spectroscopy. All other of the above values are averages of the two hydrolyses except Phe, which is determined from the second hydrolysis only.

D-Ala2, D-Trp8-somatostatin was tested for its activity in inhibiting gastric acid secretion. Large 5-6 inch bullfrogs were pitched. The gastric mucosa was freed from the muscle layers and was bisected longitudinally. The two halves were mounted in separate acrylic plastic chambers. The secretory area which was exposed was 2.85 square centimeters, and the volume of each half of the chamber was 5 ml. The solutions which were used to bathe the mucosa were the same as those used by Durbin et al., Biochemica et Biophysics, Acta, 321, 553-560 (1973), with the exception that the serosal fluid contained sodium dihydrogen phosphate at a 1 millimolar concentration. Both sides of the chamber were aerated with a mixture of 95% oxygen and 5% carbon dioxide by volume. The acid secretory rate was followed by maintaining the secretory solution at a pH of 4.5.

A concentration of 1x10-5 moles per liter of pentagastrin was used on the serosal side of the tissue to stimulate the acid secretory response. The serosal fluid was renewed every 40 minutes to prevent lowering of pentagastrin concentration by enzymatic hydrolysis of the peptide bonds. Addition of the compound to be tested was done by placing it in the serosal fluid each time the bathing solution was changed.

Spontaneous acid outputs for pentagastrin-stimulated secretion producing no less than 8 microequivalents/hour of acid served as controls. The effect of inhibition on gastric acid secretion was expressed as percent of inhibition from the control periods preceding the introduction of the test compound into the serosal buffer. Only one of the halves of the gastric mucosa was treated with the test compound, the other half serving as control to ensure continued viability of the tissue. After establishing steady state secretion, the test compound was added to the nutrient solution in an amount sufficient to attain an inhibitor concentration of lx10-5 moles/liter. The acid was continually titrated to pH 4.5, and the volume of 12.5 mM sodium hydroxide utilized each 20 minutes was used to determine the acid secretory rate. The results were expressed as micro equivalents of acid secreted per hour.

Using this method of evaluation, somatostatic itself produced a percent inhibition of gastric acid secretion of 54.64 plus or minus 6.05 whereas D-Ala2, D Trp8-somatostatin produced a percent inhibition of gastric acid secretion of 78.10 plus or minus 6.21.

D-Ser2, D-Trp5-somatostatin and D-Val2, D-Trp5-somatostatin were independently tested in dogs for its in vivo inhibition of gastric acid secretion. In two groups of six dogs in each group with chronic fistula and Heidenhain pouch, gastric HCI secretion was induced by infusion of the C-terminal tetrapeptide of gastrin at 0.5,ug/kg-hr. Each dog served as its own control, receiving on a separate day only the tetrapeptide. On another day, the dogs received the tetrapeptide, and, after one hour of steady state secretion of Hcl, D-Ser2, D-Trp8-somatostatin was infused at 0.20 ,ug/kg-hr. for one hour in 1 group of dogs and D-Val2, D-Trp8 somatostatin was infused at 0.25 Mg/kg-hr. for one hour in the other group of dogs.

Collection of gastric acid samples was continued for an additional 1.5 hours at 15 minute intervals. The samples were titrated to pH 7 with an automatic titrator. The maximal inhibitory effect of the D-Ser2, D-Trp6-somatostatin was extrapolated against the dose-response curve of somatostatin, and the relative potency of the analog to that of somatostatin is expressed as percent activity. D-Ser2, D-Trp8- somatostatin inhibited steady state acid secretion induced by the C-terminal tetrapeptide of gastrin by 73.5+4.8% standard error of mean. This effect is equivalent to that of 0.545 ,ug/kg-hr. of somatostatin. Its activity relative to that of somatostatin thus is 273%. In a similar manner, the maximal inhibitor effect of the D-Val2, D-Trp5-somatostatin was extrapolated against the dose-response curve of somatostatin, and the relative potency of the analog to that of somatostatin is expressed as percent activity. D-Val2, D-Trp6-somatostatin inhibited steady state acid secretion induced by the C-terminal tetrapeptide of gastrin by 61.9+3.4 / standard error of mean. This effect is equivalent to that of 0.324 ,4g/kg-hr. of somatostatin. Its activity relative to that of somatostatin thus is 130%.

D-Ser2, D-Trp-somatostatin and D-Val2, D-Trp8-somatostatin also were tested for their action on gut motility in conscious dogs. Two groups of three dogs each having intralumenal catheters placed in the antrum, duodenum, and pylorus were used. Pressure changes in the gut lumen were recorded on a Visicorder (Registered Trade Mark) using strain gauges and miniature light beam galvanometers. After a steady state control was established, D-Ser2, D-Trp8-somatostatin was infused intravenously over a ten minute period in 1 group of dogs and D-Val2, D-Trp8somatostatin was infused intravenously over a ten minute period in the other group of dogs. Both compounds initially increased the intralumenal pressure in the pylorus and then decreased it whereas the pressure in the duodenum and the antrum remained depressed throughout the test. The minimum effective dose required to increase the pyloric pressure and to decrease the duodenum and antrum pressures was > 0.05 to < 0.125 Mg/kg.-10 minutes for D-Ser2, D-Trp8somatostatin and > 0.125 to < 0.25 ssg./kg.-10 minutes for D-Val2, D-Trp8 somatostatin. This compared to a minimum effective dose of 0.125--0.25 pg./kg.-l0 minutes for somatostatin itself.

D-Ser2, D-Trp8-somatostatin and D-Val2, D-Trp8-somatostatin also were shown to inhibit pancreatic secretion. In two groups of three dogs each having both pancreatic and total gastric fistula, secretion from the pancreas was induced by infusion of secretin at 2 units/kg-hr. and cholecystokinin at 0.45 unit/kg-hr., and secretion of gastric Hcl by infusion of tetragastrin at 0.5 ssg/kg-hr. After a steady response was established, each dog in 1 group received D-Ser2, D-Trp somatostatin for one hour at 0.2 yg/kg-hr. and each dog in the other group received D-Val2, D-Tep8-somatostatin for one hour at 0.25 ssg/kg.-hr. The peak inhibitor effect expressed as percent change over control for total protein was -76% for D Ser2, D-Trp8-somatostatin and 72 /n for D-Val2, D-Trp8-somatostatin. This corresponds to the inhibitory effect obtained by administration of about 0.5-0.6 ,ug/kg-hr. of somatostatin.

D-Ser2, D-Trp8-somatostatin and D-Val2, D-Trp8-somatostatin also were tested for their activity with respect to the release of growth hormone. The procedure which was employed is carried out using male Spraque-Dawley rats weighing 100120 grams (Laboratory Supply Company, Indianapolis, Indiana).

The test is a modification of the method of P. Brazeau, W. Vale, and R. Guilleman, Endocrinology, 94 184 (1974). In this assay, five groups of eight rats each were employed for each compound. Sodium pentobarbital was administered intraperitoneally to all of the rats to stimulate growth hormone secretion. One group for each compound served as the control and received only saline. Two of the groups for each compound received somatostatin, one at 2 ,ug./rat, subcutaneously, and the other at 50 g./rat, subcutaneously. The other two groups for each compound received D-Ser2, D-Trp8-somatostatin or D-Val2, D-Trp8somatostatin one group at 2 ,ag./rat, subcutaneously, and the other group at 50 g./rat, subcutaneously. The serum concentration of growth hormone was measured 20 minutes after simultaneous administration of sodium pentobarbital and test compound. The degree of inhibition of serum growth hormone concentration then was determined with respect to the control group, and the relative activities of D-Ser2, D-Trp8-somatostatin, D-Val2, D-Trp8-somatostatin, and somatostatin itself was compared.

At a dose level of 2 Mg./rat, D-Ser2, D-Trp8-somatostatin had the same effect on growth hormone secretion as somatostatin which ranged from no effect to 45% inhibition of growth hormone secretion over control. At a dose level of 50 Mg./rat, D-Ser2, D-Trp8-somatostatin inhibited growth hormone secretion by 66 percent over control. At a dose level of 2 ssg./rat, D-Val2, D-Trp8-somatostatin inhibited the increase in growth hormone secretion by 21 /" over control whereas somatostatin had no effect on the increase in growth hormone secretion. At a dose level of 50 Mg./rat, D-Val2, D-Trp8-somatostatin inhibited the increase in growth hormone secretion by 88% over control. Somatostatin itself produced a 64--800/, inhibition.

D-Ser2, D-Trp8-somatostatin and D-Val2, D-Trp8-somatostatin were tested for their in vivo activity in inhibiting glucagon and insulin secretion upon stimulation with L-alanine. Normal mongrel dogs of either sex were fasted overnight. Control blood samples were obtained, and then an intravenous infusion of saline, somatostatin, D-Ser2, D-Trp8-somatostatin or D-Val2, D-Trp8-somatostatin was started. After 30 minutes, L-alanine additionally was administered intravenously for a period of 15 minutes. The infusion of saline, somatostatin, D-Ser2, D-Trp8- somatostatin or D-Val2, D-Trp8-somatostatin was continued for 15 minutes after completion of the L-alanine infusion. The infusion of L-alanine caused an abrupt increase in serum concentration of glucagon and insulin which returned to control concentration upon termination of the L-alanine infusion. From the above it was determined that the minimal effective concentration of D-Ser2, D-Trp8somatostatin is about 25 nanograms/kg./min. and the minimal effective concentration of D-Val2, D-Trp8-somatostatin is about 194 nanograms/kg./min., whereas the minimal effective concentration of somatostatin is 100200 nanograms/kg./min.

D-Val2, D-Trp8-somatostatin also was evaluated for in vivo activity in inhibiting glucagon secretion upon stimulation with insulin. Normal mongrel dogs of either sex were fasted overnight. After control blood samples had been obtained arz intravenous infusion of saline, somatostatin, or D-Val2, D: Trp8-somatostatin was commenced. After 15 minutes, insulin, 0.3 units/kg., was injected intravenously. The infusion of saline, somatostatin, or the test compound was continued for two hours, and blood samples were obtained at various intervals throughout the test. The total dose of the somatostatin or test compound ranged from 120--260 g./dog (0.07-0.13 Mg/kg/min.). Administration of insulin produced a reduction in the blood glucose concentration and an increase in serum glucagon concentration. Infusion of somatostatin blocked in the increase in serum 4teagon concentration but had no affect on the reduction of the blood glucose concentration.

In comparison, when, instead of somatostatin, D-Val2, D-Trp8-somatostatin was infused at a rate of 0.109 Mg/kg/min., it was found that a partial inhibition of the rise in serum glucagon concentration produced by insulin hypoglycemia resulted.

It will be appreciated that the invention also comprises a pharmaceutical formulation comprising a compound of formula (I) as hereinbefore defined or a pharmaceutically-acceptable acid-addition salt thereof in association with a pharmaceutically-acceptable carrier therefor.

The invention also includes a method of inhibiting release of growth hormone, and/or gastric acid secretion, and/or exocrine pancreas secretion, and/or secretion of insulin and glucagon, and/or of reducing gut motility, in a non-human animal, which method comprises administering to the animal a compound of formula (I) as hereinbefore defined, or a pharmaceutically-acceptable acid-addition salt thereof.

WHAT WE CLAIM IS: 1. A compound of the formula (I)

in which Y is D-Ala, D-Ser, or D-Val, and Z is L-Asn or L-Ala or a pharmaceutically acceptable acid addition salt thereof.

2. A compound of Claim 1, wherein Y is D-Ala.

3. A compound of Claim I, wherein Y is D-Ser.

4. A compound of Claim 1, wherein Y is D-Val.

5. A compound of any one of Claims 1--4, wherein Z is L-Asn.

6. A compound of any one of Claims 1--4, wherein Z is L-Ala.

7. A process for preparing the compounds of formula (I) as defined in Claim 1, which comprises oxidising the corresponding straight-chain tetradecapeptide of formula (III), H - L - Ala - Y - L - Cys - L - Lys - Z - L - Phe - L - Phe - D

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (23)

**WARNING** start of CLMS field may overlap end of DESC **. increase in growth hormone secretion by 21 /" over control whereas somatostatin had no effect on the increase in growth hormone secretion. At a dose level of 50 Mg./rat, D-Val2, D-Trp8-somatostatin inhibited the increase in growth hormone secretion by 88% over control. Somatostatin itself produced a 64--800/, inhibition. D-Ser2, D-Trp8-somatostatin and D-Val2, D-Trp8-somatostatin were tested for their in vivo activity in inhibiting glucagon and insulin secretion upon stimulation with L-alanine. Normal mongrel dogs of either sex were fasted overnight. Control blood samples were obtained, and then an intravenous infusion of saline, somatostatin, D-Ser2, D-Trp8-somatostatin or D-Val2, D-Trp8-somatostatin was started. After 30 minutes, L-alanine additionally was administered intravenously for a period of 15 minutes. The infusion of saline, somatostatin, D-Ser2, D-Trp8- somatostatin or D-Val2, D-Trp8-somatostatin was continued for 15 minutes after completion of the L-alanine infusion. The infusion of L-alanine caused an abrupt increase in serum concentration of glucagon and insulin which returned to control concentration upon termination of the L-alanine infusion. From the above it was determined that the minimal effective concentration of D-Ser2, D-Trp8somatostatin is about 25 nanograms/kg./min. and the minimal effective concentration of D-Val2, D-Trp8-somatostatin is about 194 nanograms/kg./min., whereas the minimal effective concentration of somatostatin is 100200 nanograms/kg./min. D-Val2, D-Trp8-somatostatin also was evaluated for in vivo activity in inhibiting glucagon secretion upon stimulation with insulin. Normal mongrel dogs of either sex were fasted overnight. After control blood samples had been obtained arz intravenous infusion of saline, somatostatin, or D-Val2, D: Trp8-somatostatin was commenced. After 15 minutes, insulin, 0.3 units/kg., was injected intravenously. The infusion of saline, somatostatin, or the test compound was continued for two hours, and blood samples were obtained at various intervals throughout the test. The total dose of the somatostatin or test compound ranged from 120--260 g./dog (0.07-0.13 Mg/kg/min.). Administration of insulin produced a reduction in the blood glucose concentration and an increase in serum glucagon concentration. Infusion of somatostatin blocked in the increase in serum 4teagon concentration but had no affect on the reduction of the blood glucose concentration. In comparison, when, instead of somatostatin, D-Val2, D-Trp8-somatostatin was infused at a rate of 0.109 Mg/kg/min., it was found that a partial inhibition of the rise in serum glucagon concentration produced by insulin hypoglycemia resulted. It will be appreciated that the invention also comprises a pharmaceutical formulation comprising a compound of formula (I) as hereinbefore defined or a pharmaceutically-acceptable acid-addition salt thereof in association with a pharmaceutically-acceptable carrier therefor. The invention also includes a method of inhibiting release of growth hormone, and/or gastric acid secretion, and/or exocrine pancreas secretion, and/or secretion of insulin and glucagon, and/or of reducing gut motility, in a non-human animal, which method comprises administering to the animal a compound of formula (I) as hereinbefore defined, or a pharmaceutically-acceptable acid-addition salt thereof. WHAT WE CLAIM IS:

1. A compound of the formula (I)

in which Y is D-Ala, D-Ser, or D-Val, and Z is L-Asn or L-Ala or a pharmaceutically acceptable acid addition salt thereof.

2. A compound of Claim 1, wherein Y is D-Ala.

3. A compound of Claim I, wherein Y is D-Ser.

4. A compound of Claim 1, wherein Y is D-Val.

5. A compound of any one of Claims 1--4, wherein Z is L-Asn.

6. A compound of any one of Claims 1--4, wherein Z is L-Ala.

7. A process for preparing the compounds of formula (I) as defined in Claim 1, which comprises oxidising the corresponding straight-chain tetradecapeptide of formula (III), H - L - Ala - Y - L - Cys - L - Lys - Z - L - Phe - L - Phe - D

Trp - L - Lys - L - Thr - L - Phe - L - Thr - L - Ser - L - Cys - OH, wherein Y and Z are defined as in Claim 1.

8. A compound of the formula (II), R - L - Ala - Y' - L - Cys(Rl) - L Lys(R2) - Z - L - Phe - L - Phe - D - Trp(R5) - L - Lys(R2) - L - Thr(R3) - L Phe - L - Thr(R3) - L - Ser(R4) - L - Cys(R1) - X; in which Y' is D-Ala, D-Ser(R4), or D-Val; Z is L-Asn or L-Ala; R is hydrogen or an a-amino protecting group; R1 is hydrogen or a thio protecting group; R2 is hydrogen or an E-amino protecting group; R3 and R4 each are hydrogen or a hydroxy protecting group; R5 is hydrogen or formyl; and X is hydroxy or

Res Resin - > CH .

K in which Resin is polystyrene; with the proviso that, when X is hydrogen, each of R, R1, R2, R3, R4 and R5 is hydrogen, and, when X is

.0 Resin - > CH > each of R, R1, R2, R3 and R4 is other than hydrogen.

Y

9. A compound of Claim 8, in which X is hydroxy.

10. A compound of Claim 8, in which R is t-butyloxycarbonyl.

11. A compound of Claim 8 or 10, in which R1 is p-methoxybenzyl.

12. A compound of any of Claims 8, 10 or 11, in which R2 is cyclopentyloxycarbonyl.

13. A compound of any of Claims 8, 10 or 11, in which R2 is o- chlorobenzyloxycarbonyl.

14. A compound of any of Claims 8, or 10 to 13, in which R3 and R4 are benzyl.

15. A compound of any of Claims 8 to 14, in which Y' is D-Ala and Z is L-Asn.

16. A compound of any of Claims 8 to 14, in which Y' is D-Ser and Z is L-Asn.

17. A compound of any of Claims 8 to 14, in which Y' is D-Val and Z is L-Asn.

18. A compound according to Claim 1 substantially as hereinbefore described in any of Examples 4, 7 and 10.

19. A compound according to Claim 8 substantially as hereinbefore described in any of Examples 2, 3, 5, 6, 8 and 9.

20. A process according to Claim 7 substantially as hereinbefore described in any one of Examples 4, 7 and 10.

21. A compound of formula (I) as defined in Claim 1 prepared by a process according to Claim 7 or Claim 20.

22. A pharmaceutical formulation comprising a compound according to any one of Claims 1 to 6, 18 and 21, or a pharmaceutically-acceptable acid addition salt thereof in association with a pharmaceutically-acceptable carrier therefor.

23. A method of inhibiting release of growth hormone, and/or gastric acid secretion, and/or exocrine pancreas secretion, and/or secretion of insulin and glucagon, and/or of reducing gut motility, in a non-human animal, which method comprises administering the animal a compound according to any one of Claims 1 to 6, 18 and 21.