CA1120030A - Somatostatin analogs and intermediates thereto - Google Patents

Abstract

Abstract of the Disclosure The tetradecapeptide D-Val-Gly-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
is described along with its corresponding non-toxic pharmaceutically-acceptable acid addition salts as well as intermediates useful in the synthesis of this tetradecapeptide. This tetradecapeptide is prepared by reacting the corresponding straight-chain tetradecapeptide with an oxidizing agent to con-vert the two sulfhydryl groups to a disulfide bridge. This tetradecapeptide as well as its pharmaceutically acceptable acid addition salts exhibit various activities including inhibition of the release of growth hormone and reduction of gut motility.

Description

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This in~ention relates to the tetxadecapeptide D-Val-Gly-L-C~s-L~Lys-L-Asn~L-P~e-L-Phe-D~T~p~L~Lys~L~Thr~L -Phe-L-Thr-L-Ser~L-Cys-OH, formula I-, as well as to its pharma-ceutically acceptable acid addition salts and to intermediates produced during the synthes~s of this tetradecapeptide.
Somatostatin (also known as somatotropin release in-hibiting factor) ~s a tetradecapeptide of the formula L-Ala-Gly-L-Cys-L~Lys-L-Asn-L-Phe-L-Phe-L-Trp-L-Lys-L -Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH. 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).
In addition, the compound conveniently designated as D-Trp -somatostatin was previously reported by Brown et al., Endocri _logy, 98, No. 2, 336-343 (1976).
. -- ' The biologically active tetradecapeptides of formula I
have the formula defined above and include the non-toxic acid addition salts thereof. Their structures differ from that of somatostatin by the presence of a D-tryptophan resi~ue in posi-tion 8 in place of an L-tryptophan residue and a D-valine -- residue in position 1 in place of an L-alanine residue. For convenience sake, the tetradecapeptides of formula I can be referred to as D-Val , D-Trp -somatostatin.
Thus, this invention relates to a compound selected from those of the formula H-D-Val-Gly 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 their ; pharmaceutically-acceptable non-toxic acid addition salts, and, as intermediates, ~ Gly-L-Cys(Rl)-L-Lys(R2)-L-Ans-L Phe-L-Phe-D Trp(R5)-L-LysrR2)-L-Thr(R3)-L-phe-L-Thr(R3)-L-ser(R4)-L
Cys(Rl)-X, formula II, in which

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R is hydrogen or an a-amino protecting group;
Rl is hydrogen or a thio protecting group;
R2 is hydrogen or an ~-amino protecting group;
R3 and R4 each are hydrogen or a hydroxy protecting group;
R5 is hydrogen or formyl; and X is hydroxy or 0= ~ ~esin -0-~ --o~

in which the resin is polystyrene; with the proviso that, when X
is hydroxy, each of R, Rl, R2, R3, R4, and R5 is hydrogen, and, when X is o = ~ ~ esin ~o~

each of R, Rl, R2~ R3, and R~ is other than hydrogen.
The no~el tetradecapeptide of formula I above is pre-pared by xeacting the corresponding straight-chain tetradecapep-2Q tide of ormula III, D~Val-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe- ~ -D-Val-Gly-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, with an oxidizing agent. 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, ci~ric, maleic phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-tolue-nesulfonic, 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 Also contemplated as ~ein~ within the scope of this invention are intermediates of the formula II, D-Val~Gly-L-Cys (Rl)~L-Lys(R2)~L-Asn-L-Phe-L~Phe-D~Trp(R5)-L~Lys(R2)-L-Thr(R3)-L-Phe-L-Thr(R3~-I,-Ser(R4)-L~Cys(Rl)-X wherein the various symbols are defined as before.
H-D-Val-Gly-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;
N-(BOC)-D-Val-Gly-L-(PMB)Cys-L-(CBzOC)-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-(CBzOC)-Lys-~-(Bzl)Thr-L~Phe-L(Bzl)Thr-L-(Bzl) ~==~esin Ser-L-(PMB)Cys-O-CH2 ~ ~o ;and The a~o~e formulas defining the intermediates include protect~ng 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 pro- -tecting group is such as can be readily removed with restoration 2Q 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 ~een directed specifically to a description and discussion of methods for using such groups. One such volume is the treatise Protective Groups in Organ~c he-mis-try, J. F. W. McOmie, Editor, Plenum Press, ~ New York, 1973.
; In the abo~e formulas defining the intermediates, R
represents either an a-amino hydrogen or an a-amino protecting group. The ~-amino protecting groups contemplated for R are well recognized by those o~ ordinary skill in the peptide art. Many ''', ~-: . :

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of these are detailed in McOmie~ supra, Chapter 2, authored b~
J. W. Barton. Illustrati~e of such protecting groups are benzyl-loxycarbonyl, _~chlorobenzyloxycarbonyl, ~-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, o bromobenzyloxycarbonyl, p-methoxy-benzyloxycarbonyl, ~nitrobenzyloxycarbonyl, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, 2~ biphenylyl)isopropyloxycarbonyl (BpOC), adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyl-oxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl, tri-phenylmethyl (trityl), and p-toluenesulfonyl. Preferably, the ~-amino protecting group defined by R is t-butyloxycarbonyl.
Rl represents either the hydrogen of the sulfhydryl 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 _-methoxybenzyl, benzyl, p-tolyl, benzyhydryl, acetamido-methyl, trityly _-nitrobenzyl, t-butyl, isobutyloxymethyl, as well as any of a number of trityl deri~atives. For additional groups, see, for example, Houben-Weyl, Methodes der Organischen Chemie, "Synthese von Peptiden", Vols. 15/1 and 15/2, (1974), Stuttgart, Germany. Preferably, the sulfhydryl protecting group defined by Rl is p-methoxybenzyl.
R2 represents either hydrogen on the -amino function of the lysine residu or a suitable ~-amino protecting group.
Illustrative of such groups are the bulk of those mentioned hereinabove as being suitable for use as an ~-amino protecting group. Included as typical such groups are benzyloxycarbonyl, t-butyloxycarbonyl, t-amyloxycarbonyl, cyclopentyloxycarbonyl, : 3a adamantyloxycarbonyl, ~-methoxybenzyloxycarbonyl, p-chlorobenzyl-oxycarbonl, p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, ~ z~1~30 o-bromobenzylQxyc~xbonyl) ~nitxo~enzylox~caxhonyl, isopxop~loxy-carbon~l, cyclohexyloxycarbonyl, cycloheptyloxycarbon~l, and ~-toluenesul~onyl.
As will become apparent hereinafter, the process for the preparation of the tetradecapeptides of formula I involves periodic cleavage of the ~amino protecting group from the ter-minal amino acid present on the peptide chain. Thus, the only limitation with respect to the identity of the -amino protecting group on the lysine residue is that it be such that it will not be cleaved under the conditions employed to selectively cleave the ~-amino protecting group. Appropriate selection of the ~-amino and the - amino protecting groups is a matter well within the knowledge of a peptide chemist of ordinary skill in the art and depends upon the relative ease with which a particular pro-tecting group can be cleaved. Thus, groups such as 2-(_~iphenylyl) -~isopropyloxycarbonyl (BpOC) and tityl 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, adamantyl-oxycar~onyl, and p-methoxybenzyloxycarbonyl. Even stronger acid conditions are re~uired to effect cleavage of other protecting ~-~roups such as benzyloxycarbonyl, haloben~yloxycarbonyl, ~-nitrob~nzyloxycarbonyl, cycloalkyloxycarbonyl, and isopropyloxy-carbonyl. 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 3Q ~roups thus will include the use of a group at the a-amino function which is-more labile than that employed as the -amino protectin~ group coupled with cleavaye conditions designed to 6 ~

, 1~2E)030 selectively re~o~e only the ~Amino function In this context~
R2 preferably is o~chlorobenz~loxycarbonyl or cyclopentyloxy-carbonyl, and, in con~unction therew-i~h, the ~amino protecting group of choice for use in each of the amino acids which is added to the peptide chain prefera~ly is t~utylo~ycarbonyl.
The yroups R3 and R4 represent the hydro~yl hydrogen or 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, Cl-C4 alkyl, such as methyl, ethyl, and t-butyl; benzyl; substituted benzyl, such as ~-methoxybenzyl, ~nitrobenzyl, p-chlorobenzyl, and o-chloroben-zyl; Cl-C3 alkanoyl, such as formyl, acetyl, and propionyl; tri-phenylmethyl (trityl~. Preferably, when R3 and R4 are protecting groups, the protecting group of choice in both instances in benzyl.
The group R5 represents either hydrogen or formyl and defines th~ moiety ~ NR5 of the tryptophan residue. The formyl serves as a protecting group. The use of such a protecting ~roup is optional and, therefore, ~5 properly can be hydrogen (N-unpro-2Q tected) or formyl (N-protected~.
The group X relates to the carboxyl terminal of the tetradecapeptide chain; it can be hydroxyl, in which case a free carboxyl group is defined. In addition, X represents the solid resin support to which the carboxyl terminal moiety of the pep-tide is linked during its synthesis. Irhis solid resin is repre-sented by the formula ~ esin -O--CH2--~
o--~--; 3Q In any of the above, when X represents hydroxyl, each of R, Rl, R2, R3, R4, and R5 is hydrogen. When X represents the solid resin support, each of R, Rl, R2, R3, and R4 is a protec~
ting group.
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The following ~bhreyiations~ most o$ which are ~ell known and commonl~ used in the art, are employed herein:
Ala - Alanine Asn - Asparagine Cys - Cysteine Gly ~ Glycine Lys - Lysine Phe - Phenylalanine Ser - Serine Thr ~ Threonine Trp ~ Tryptophan ~al ~ ~aline DCC ~ N,N'-Dicyclohexylcarbodiimide DMF - N,N-Dimethylformamide BOC - t-Butyloxycarbonyl PMB ~ ~-Methoxybenzyl CBzOC ~ o-Chlorobenzyloxycarbonyl CPOC - Cyclopentyloxycarbonyl Bzl - Benzyl For ~ Formyl - BpOC ~ 2~ biphenylyl~isopropyloxycarbonyl Although the selection of the particular protecting groups to be employed in preparing the compounds of ~ormual I
remains a matter well within the ordinary skill of a synthetic peptide chemistr it is well to recognize that the sequence of reactions which must be carried out gives rise to a selection of particular protecting groups. In other words, 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 3a reaction sequence. For example, as alread~ discussed to some degree hereinabove~ the particular protecting group which is em-plo~ed must be one which remains intact under the conditions - 8 ~

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which are emplo~ed ~or cle~y~n~ the ~min~ pxotecting ~roup o~
the terminal amino acid residue of the peptide fragment in prep-aration for the couplin~ of the next succeeding amino acid frag-ment to the peptide ch~in. It is also important to select, as a protectin~ 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 pro-duct. 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 tetr~de-capeptides 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 hydroxyme~hyl resin. Preparation of a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind.
(London)~ 38 1597~98 (1966). The chloromethylated resin is com-mercially available from Lab Sy~tems, Inc., San Mateo, California.
`, 20 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 accor-dance with the method described by B. F. Gisin, Helv. Chim. Acta, 56, 1476 tl973). 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 ~he pre-` sence of a carboxyl ~roup activating compound such as N,N'-di--~ cyclohexylcarbodiimide (DCC).
Once the free carboxyl cysteine has been appropriately linked to the resin support, the remainder of ~he peptide buil-ding sequence involves the step-wise addition of each amino acid ~' `

to the N~terminal p~rtion of the peptide chain~ Necessaxily~
therefore, the particular sequence which is Lnvolved comprises a cleavage of the ~-amino protecting group from the amino acid ~hich represents the N~terminal portion of the peptide fragment ~ollo~ed by couplin~ of the next succeedin~ amino acid residue to the now-free and reactive N~terminal amino acid. Cleavage of the ~-amino protectinq group can be effected in the presence of an acid such as hydrobromic acid, hydrochloric acid, trifluoro-acetic acid~ ~-toluenesulfonic acid, benzenesulfonic acid, naptha-lenesulfonic acid, and acetic acid, with formation of the respec-tive acid addition salt product. Another method which is avail-able for accomplishing cleavage of the amino protecting group involves the use of boron trifluoride. For example, boron tri-fluoride 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 protec~ing group without disruption of any other protecting ~roups present on the peptide chain. In this regard, it is preferred that the cleavage of the N-terminal pro-tecting group b~ accomplished using trifluoroacetic acid~
Generally, the cleavage will be carried out at a temperature -from about 0C. to about room temperature.
Once the N-terminal cleavage has been effected, the product which results normally will ~e in the form of the acid addition salt o~ the acid which has been employed to accomplish the cleavage of the protecting groupO 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 tri-ethylamine.

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The peptide chain then is xeady fo~ reaction with the next succeeding amino acid. This can ~e accomplished by employ-ing any of several recoqnized 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 sub-jected to conditions ~hich will render the carboxyl function acti~e to the coupling reaction. One such activation technique which can ~e employed in the synthesis involves the conversion o~ the amino acid to a mixed anhydride. Thereby, the free car-boxyl 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 chloroformate, sec~butyl chloroformate, isobutyl chloro-formate, and pivaloyl chloride.
Another method of activating the carboxy 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 penta-chlorophenyl ester~ a ~-nitrophenyl ester, an ester formed from l-h~droxybenzotriazole, and an ester formed from N-hydroxysuccin-imide. Another method for effecting coupling of the C-terminal amino acid to the peptide fragment involves carxying out the couplin~ reaction in the presence of at least an equLmolar quan-tity of N,N~-dicyclohexylcarbodiimide (DCC). This latter method is pre~erred for preparing the tetradecapeptide of formula II

where x is R~, i n -O--CH ~0~ ~

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~2~ 3~3t 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 ~luoride 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 ~-amino pro-tecting group present at N-terminal amino acid. When hydrogen fluoride is employed to effect the cleavage of the peptide from ;~ 10 the resin as well as to remove the protecting groups, it is pre-j,:
ferred 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, it facilitates conversion of the blocked cysteines to their thiol forms. Also, when R5 is formyl, the presence of ethyl mercaptan facili~ates 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 tetrade-capeptide under conditions which will effect its oxidation by con~erting the two sulfhydryl groups present in the molecule, one at each cysteinyl moiety, to a disulfide bridge. This can be accomplished by treating a dilute solution of the linear tetra-decapeptid~ ~-ith any of a variety of oxidizing agents including, for example, iodine, and potassium ferricyanide. Air also can be employed as oxidizing agent, the pH o~ the mixture ~enerally being from about 2.5 to about 9.0, and preferably from about 6.2 ~ 12 -., ,~; , , .- ~ . .
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; to about 7.2. When aiX is used ~as oxidizin~ a~ent~ the concen-tration of the peptide solution ~enerally is not ~reatex than about 0.4 mg. of the peptide per milliliter of solution, and usually is about 50 ~./ml.
The compounds of formula I may be administered to warm-blooded mammals, including humans, and are particularly useful for relaxing smooth muscle. Specifically, the gastrointestinal tract can be relaxed by parenteral administration of small am-ounts o~ these compounds, and preferably, of D-Vall, D-Trp8-somatostatin. This action, resulting in reduction of gut motil-ity, is particularly desirable in hypotonic gastrointestinal radiography. These compounds, furthermore, are useful in treat-ment of spastic colon, pylorospasm, and other spastic conditions of the gastrointestinal tract, as well as for ureteral and bi-liary colic.
Normally, in order to efect xelaxation of smooth muscle, these compounds are administered at a dose of about 0.1 ~g. to about 3 ~g. per kilogram body weight of the recipient and preferably from about 0.3 ~g. to about 1.5 ~g. per kilogram body weight. Administration is parenteral and it can be intra-:
; muscular, subcutaneous, or intravenous; preferably, the compounds are administered intravenously or intramuscularly.
For parenteral administration~ fluid unit dosage forms ~ generally are prepared using the compound in association with a -~ pharmaceutical carrier, such as, for example, isotonic saline, isotonic glycine, lactose, mannitol, dilute acetic acid, bac-teriostatic water, fox example, water containing about 1~ benzyl alcohol, and phosphate buffer solutions, as well as appropriate combinations of any standard carriers. The carrier, relative to the active compound, yenerally is present in a weight ratio of ~rom about 25:1 to about 1000:1.
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, The compound~ depending upon the carrier and the con-centration used~ can either be suspended or dissol~ed in a suitable sterile vehicle, water being preferred. In preparing solutions, the compound and carrier can be dissolved in the selected vehicle, the solution filtered and added to a suitable vial or ampoule, and the vial or ampoule sealed. Advantageously, adjuvants, such as a local anesthetic preservative or a buffering agent, can be dissolved in the vehicle. To enhance stability, the ; compound in association with the carrier can be dissolved in water, and the a~ueous solution placed into a vial and then lyophilized.
The dry lyophilized solid then is sealed in the vial and an ac-! companying vial of the vehicle supplied to reconsitute the com-position prior to use. Parenteral suspensions can be prepared in substantiall~ the same manner except that the compound is suspended in the carrier instead of being dissolved.
;~ The compounds of formula I also are active, although ` not necessarily to an equivalent degree, in inhibiting the re-lease of growth hormone. This inhibitory effect is beneficial in those instances in which the host being treated re~uires a therapeutic treatment for excess secretion of somatotxopin, such secretion being associated with adverse conditions such as juvenile diabetes and acromegaly. These compounds also exhibit other physiological effects~ including the inhibition o~ gastric ~ acid secretion, useful in treatment of ulcer conditions; the in-- hibition of exocrine pancreas secretion, potentially useful in treatment of pancreatitis; and the inhibition of secretion of insulin and glucagon. The compounds may be administered by any of several methods~ including oral, sublingual, subcutaneous, i intramuscular~ and intravenous. Preferably~ the dose range for sublinsual or oral administration is about 1 mg. to about 100 mg.
!~ /kg. of ~ody ~eight. Generally, the intra~enous, subcutaneous, or intrasmuscular dose range for these latter indications is ~.~.ZC~30 from about 1 ~g. to about 1 mg./kg. of body weight, and, pre-ferably, is from about 50 ~g. to about 100 ~g./kg. of body weight.
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.

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~ o~ )o~ s ()1- ~or~ ! c~ cl~lmirli~
oral1y or sublin~l~lall~ ass()ci~ltil-n with a ~-harmace-l~icc carrier, for example, in the forrn o~ tal-lets or capsuLes.
Lnert ciiluents or car~iers, for example, Inaqrlesium (~arbonate or lactose, can be used together with conventional dis-integrating agents, for example, maize starch and alginic acid, and lubricating agents, for example, magnesium stearate.
Typically, the amount of carrier or di]uent will range from about 5 to about 95 percent of the final composition, and preferably from about 50 to about 85 percent of the final composltion. Suitable flavoring agents also can be employed in the final preparation rendering the composition more palatable for administration.
When the compounds of ~ormula :r are to be adminis-tered parenterally, suitable carriers may be employed, such as, for example, any of those described above with reference to the use of these compounds for relaxing smoo-th muscle.
The following examples are illustrative of the preparation of compounds of formula I and intermediates thereto.
Lxam~Le 1 N-t-L~UTYI,OXYC~R~ONYI.-L-CYSTEINYI.(S-~-METIIOXYi3ENZYI) METIIYLATIFID POIYSTYRE;NE RESIN
To 1000 rnl. of N,N-dimethylformamide (DME) con-taining the cesium salt of N-t-butyloxycarbonyl-(S-~-methoxybenzyl)cysteine (prepared from 17.5 g. of the free acid) were added 100 g. of chloromethylated polystyrene resin (Lab Systems, Inc~., 0.75 mmoles Cl/gram). The mixture was stirred at room temperature for five days. The resin .. ~ , . . .. ~.. ,, ;, - ~12~3C~

then was filtered ancl was waslle(l alternately Lhre~ times each with a mi~ture o~ 85 percent DMF and 15 percent water and with DMF, and then twice witll DMF. 1'o the resin sus-pended in 1000 ml. of DMF were added a solution of 16 yrams (83.4 mmoles) of cesium acetate. The mlxture was stirred for nine days at room -tempera-ture. The resin then was filtered and was washed alternately -three times each with a mixture of 85 percent DMF and 15 percent water and with DMF.
The resin was washed with C~IC13 and then was suspended four times in CIIC13 in a separatory funnel, drawing off the liquicl each ti~e to remove fines. The resin was Eiltered, washed with 95~ ethanol and then alternately three times each with benzene and 95 percent ethanol. The resin then was dried in vacuo at 30C. to obtain 115.3 g. of the title product. An amino acid analysis showed 0.254 mmoles of Cys per gram resin. The cysteine was determined as cysteic acid from an acid hydrolysis carried out using a 1:1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethyl sulfoxide was added.
~xample_2 N-t-B~TYLOXYCARBONYL-D-VALYL-GLYCYL-L-(S-~-METHOXY-BENZYL)CYSTEINYL-L-(N~-o-CHLOROBENZYLOXYCARBONYL)-LYSYL-L-A',PARAGINYL-L-PHENYLALANYL-L-PiIENYLAIIANYL-D-TRypTopHrL~L-(NE-o-cllLoRoBENzyLoxycARBoNyL)LysyL-L-(O-BENZYL)TIIREONYL-L-PHENYLALANYL-L-(O-BENZYL)THREONYL-L (O-BENZYL)SERYL-L-(S-~-METHOXYBENZYL)CYSTEINYL
METHYLATED POLYSTYRENE RESIN
The product from Example 1 (5.0 grams) was placed in the reaction vessel of a Beckman 990 automatic peptide synthesizer, ancl twelve of the remaining thirteen amino acids were adclec! employing the automatic synthesizer. The . ., . ~

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resulting protected tridecapeE)tide resin was dividec! into two equal portions, and thc final residue was introduced to one of the portions. The amino acids which were employed as well as the se~[uence o~ their employment is as follows: (1) N-t-butyloxycarbonyl-(O-benzyl)-L-serine; (2) N-t-butyl-oxycarbonyl-(O-benzyl)-L-threonine; (3) N-t-butyloxycarbonyl-L-phenylalanine; (4) N-t-butyloxycarbonyl-(O-benzyl)-L-threonine; (5) Na-t-butyloxycarbonyl-N~-o-chlorobenzyl-oxycarbonyl-L-lysine; (6) N -t-butyloxycarbonyl-D-tryptophan;
(7) N-t-butyloxycarbonyl-L-phenylalanine; (8) N-t-butyl-oxycarbonyl-L-phenylalanine; (9) N-t-butyloxycarbonyl-L-asparagine, _-nitrophenyl ester; (10) N -t-butyloxycarbonyl-N~-_-chlorobenzyloxycarbonyl-L-lysine; (11) N-t-butyloxy-carbonyl-(S-~-methoxybenzyl)-L-cysteine; (12) N t-butyl-oxycarbonyl-glycine; and (13) N-t-butyloxycarbonyl-D-valine. The sequence oE 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 ~roup 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./(3ram resin) of three minutes each with chloroform; (4) one wash (10 ml./gram resin) of three minutes with met-hylene 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) , 1~2~)~3(~

three washes (10 ml./gram resin) of three minutes each withmethylene chloride; (7) neutralization by three treatments of three minutes each wi,th 10 m]./gram resin oE 3 percent triethylamine in methylene chloride; (8) three washes (I0 ml./gram resin) of three minutes each wi-th methylene chloride;
(9) three washes (10 ml./yram 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) oE
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./gram resin) of three minutes each wlth methylene chloride; (15) neutralization by -three treatments of three minutes each with 10 ml./gram resin of 3 perc~nt triethylamine in methylene chloride; (16) three washes (1() ml./gram resin) of three minutes each with methylene chlor~de; (17) three washes (10 ml./gram resin) of three minutes each with a rnixture 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 ~,~2~

a l:l 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 (lO
ml./gram resin) o~ three Ininutes each with methylene chloride;
(23) three washes (lO ml.~granl resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and lO percent t-amyl alcohol; (24) -three washes (10 ml./gram resin) of three minutes each with methylene chloride; (25) neutraliza- -tion by three treatments of three minutes each with 10 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 glycine and asparagine residues. The glycine addition was carried out using only steps 1-18. The asparagine residue was incorporated via its ~-nitrophenyl active ester.
In doing so, Step (]1) above was modified to the following 3-step sequence: (a) three washes (lO 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 lO 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 rnodified to the use of the ~-nitrophenyl ester of N-t-butyloxycarbonyl-L-asparagine in a 3:1 mixture of DMF and methylene chloride followed by mixing Eor 720 minutes.
The linished peptide-resin was dried ln vacuo.
The product was hydrolyzed ~y reEluxincJ for 72 hours in a l:l mixture of concentrated hydrochloric acid and dioxane.
Amino acid ana]ysis of the resulting product gave the following resu~ts, lysine being employed as standard: Asn, l.00; 2Thr, 2~18; Ser, 0.95; Gly, l.00; Val, 0.99; 3Phe, 3.45; 2Lys, 2.00.
Example 3 D-VALYL-GLYCYL-L-CYSTEINYL-L-LYSYL-L-ASPA~AGINYL-L-P~ENYLALANYL-L-PHENYL-ALANYL-D-TRYPTOPHYL-L-LYSYL-L-THREONYL-L-PHENYLAlJANYI.-L-THREONYL-L-SERYL-r.-CYSTEINE
To a mixture of 7.2 ml. of anisole and 7.2 ml. of e-thyl mercaptan were added 3.9l4 grams (at substitution level of 0.155 mmoles/qram) of the protected tetradeca-peptide-resin oE Example 2. The mixture was cooled in liquid nitrogen, and 80 ml. of liquid hydrogen fluoride were added by distilLation. The resulting mixture was allowed to warm to 0C. and was stirred for 2 hours. The hydrogen fluoride then was removed by distillation. E-ther was added to the remainin~ mixture, and it was cooled to 0~C. The resulting solid was collected by filtration and washed with ether. The product was dried, and the deprotected tetra- --decapeptide was extracted from the resin mixture using l~ -acetic acid and 50~ acetic acid. The acetic acid solution then was immediately lyophili~ed to dryness in the dark.

.

ZC~3~

The resulting slightly yellow solid was suspended in a mixture of 10 ml. of degassed 0.2M acetic acid and 4 ml. of glacial acetic acid. The resulting suspension was heated slightly with 6 ml. of 50~ acetic acid until a clean, yellow solution resulted, and the solution was applied to al~ephadex~*
G-25 F column. The chromatographic conditions were:
solvent, degassed 0.2~ acetic acid; column size, 7.5 x 150 cm.; temperature, 26C.; flow rate, 1670 ml./hour; fraction volume, 25.05 ~1.

Absorbance at 280 m~l of each fraction plotted versus fraction number indicated one large broad peak with a following shoulder. UV spectroscopy revealed that the main part of the peak was the product. The fractions which were combined and their effluent volumes are as follows: ~ -Fractions 207-233 (5160-5837 ml., peak = 5515 ml.) This ollection of fractions did not include the back side shoul~er. UV spectroscopy indicated that 470 mg.
of the product were present. (yield = 46.4~). An Ellman titration of an aliquot indicated a free sulfhydryl content Of 95% of theor~tical.
Example 4 ~XIDATIOI~ TO D-Val , D-Trp -SOMATOSTATIN
The sl)lution of the reduced D-Val , D-Trp -somatostatin (6'7 ml.) from Example 3 was diluted with distilled water to achieve a concentration of 50 ~Ig./ml.
Concentrated amllonium hydroxide was added to adjust the p~
of the mixture to 6.7. The solution was stirred at 4C. in the dark for 64 hours after which an Ellman titration indicated that c,xidation was complete.

* Trademark for a hydrophilic, insoluble molecular sievechromatographic medium, made by cross-linking dextran.

~ ~ r 3~

The mixture was concentrated ln vacuo to a volume of 45 ml., and 45 ml. of glacial acetic acid were added.
The mlxture then was desalted on a Sephadex C,-25 F column.
The chromatographic conditions were as ~ollows: solvent, degassed 50% acetic acid; column size, 5.0 x 215 cm.;
temperature, 26C.; flow rate, 148 ml./hour; fraction volume, 17.3 m~.
Absorbance at 280 m~ for each Eraction 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 was collected [fractions 116-155 (2000-2685 ml.)]. UV spectroscopy indicated that 279 mg. of product were present in the sample (yield =
59.4%). The solution was lyophilized to dryness in the dark.
The resulting white solid was rechromatographed in two approximately equal portions. The first portion was dissolved in 25 ml. of degassed 50% acetic acid and was absorbed on a Sephadex G-25 F column. Chromatographic conditions were: solvent, degassed 50% acetic acid; column size, 5.0 x 215 cm.; temperature, 26C.; flow rate, 148 ml./hour; fraction volume, 17.3 ml.
Absorbance at 280 m~ for each fraction plotted versus fraction number showed two large peaks. A conserva- -tive cut of the second peak was made. Fractions 119-125 (effluent volumes 2128-2256 ml.) were combined. UV spec-troscopy indicated that 65.3 mg. of product were present in this sample. The solution was lyophilized to dryness in the dark to obtain the desired product.

,, . . .

The second portion was rechromatographed in the same manner as the flrst with similar results. The two good products were combined totalling 126 mg. by UV spectroscopy (45.2~ recovery of purifiecl product). The combined product was dlssolved in 15 ml. of degassed 0.2~1 acetic acid and was "
applied to a Sephadex G-25 F column. Chromatographic conditions were: solvent, degassed 0.2M acetic acid; column size, 5.0 x 150 cm.; temperature, 26C.; flow rate, 466 ml./hr.;
fraction volume, 16.3 ml.
Absorbance at 280 mll of each fraction plotted versus fraction number indicated one large peak. UV spec-troscopy indicated that the major portion of the peak was excellent product. Fractions 160-180 (2592-2934 ml., peak =
2685 ml.) were combined and were lyophilized to dryness in the dark. UV spectroscopy indicated the presence of 90.
mg. of product (71.7~ recovery).
Op-tLcal rotation [a]26 = -56.1 (1 percent acetic acid).
Amino acid analysis: Val, 1.0; Gly, 0.97; 2Cys, 1.62; 2Lys, 2.Q0; ~sn, 1.01; 3Phe, 2.87; Trp, 1.02; 2Thr, 1.83; Ser, 0.81~
The above results are expressed as ratios of Lys/2 = 1Ø All values are averages from two 21 hour hydrolyses without scavengers. Tryptophan was determined from UV
spectroscopy (as a ratio to Lys/2); serine was not corrected for losses durinc~ hydrolysis.
The above product contains minor guantities of impurities. If desired, the product can be further purified 3~

by subjecting it to preparative high pressure liquid chroma-tography (HPLC).
An alternative method for oxidizing the reduced D-Val , D-Trp8-somatostatin to D-Vall, D-Trp -somatostatin is by treatmenl with potassium ferricyanide. The oxidation is accomplished in an aqueous solution brought to p~l 6.7 as described earlier in this example. An aqueous solution of potassium ferricyanide is added to the mixture to produce a final concentration representinq approximately 3.3 times that of the reduced D-Vall, D-Trp8-somatostatin. The solution is stirred in the dark at room temperature for -about two hours. Completion of the oxidation is verified by an Ellman titration.
D-Vall, D-Trp8-somatostatin was tested in dogs for its in vivo inhibition of gastric acid secretion. In six dogs with chronic fistula and Heidenhain pouch, gastric HCl secretion was induced by infusion of the C-terminal tetra-peptide of gastrin at 0.5 ~g./kg.-hr. Each dog served as its own control. After one hour of steady state secretion of HCl, D-Vall, D-Trp8-somatostatin was infused at 0.15 ~g./kg.-hr. for one hour. 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-Vall, D-Trp8-somatostatin was extrapolated against the dose-response curve of somatostatin, and the relative potency of the analog to that of somatostatin is express~d as percent activity. D-Val , D-Trp -somatostatin inhibited steady state acid secretion induced by the C-terminal ~etrapeptide of gastrin by 48.22 - 6.~5% (standard error of ~2~3(:~
mean). This effect is equivalent to that of 0.175 ~ug./kg.-hr.
of somatostatin. Its activity relative to that of somato-statin thus is 116%. A more highly purified sample of D-Vall, D-Trp~-somatostatin administered at doses of 0.200, 0.166, and 0.138 ~g./kg.-hr. inhibited steady state acid secretion induced by the C-terminal tetrapeptide of gastrin by 77.63, 71.57, and 67.8~, respectively. This activity relative to that of somatostatin is 302-325~.
D-Vall, D-Trp8~somatostatin also was tested for its action on gut motility in conscious dogs. Three dogs having intralumenal catheters placed in the antrum, duodenum, and pylorus were used~ Pressure changes in the gut lumen were recorded on a Visicorder using strain gauges and miniature light beam galvanometers. After a steady state control was established, test compound was infused intravenously over a ten minute period. The test compound initially increased the intralumenal pressure in the pylorus and then decre~sed it whereas the pressure in the duodenum and the antrum remained depressed throughout the test. The minimum effective dose required to increase the p~loric pressure and to decrease the duodenum and antrum pressures is about 0.05 ~y./l-g.-10 minutes for D-Vall, D-Trp - -somatostatin. ~ compares to a value for somatostatin itself of 0.125-0.25 ~g./kg.-10 minutes.
D-Vall, D-Trp8-somatostatin also was tested for its activity wibh respect to the release of growth hormone. The procedure which was employed is carried out using normal male Sprague-Dawley rats weighing 100-120 grams (Laboratory Supply Company, Indianapolis, Indi~na). The test is a modification of the method of P. Brazeau, W. Vale, - 29 ~

~9LZ~ 3~

and R. Guilleman, Endocrinology, ~4 184 (1974). In this assay, a total of five groups of eight rats each weré
employed for the testing of each compound. Sodium pentobarbital was administered intraperitoneally to all o the rats to stimulate ~rowth hormone secretion.
One group s~rved as the control and received onl~ saline.
Two of the groups xeceived somatostatin, one at 2 ~g./rat, subcataneously, and the other at 50 ~g./rat, subcut-aneously. The other two groups received test compound, one at 10 ~g./rat, subcutaneously, and the other at 0.4 ~g./rat, subcutaneously. The serum concentration of growth hormone was measured 20 minutes after simult-aneous a~ministration of so~ium 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 test compound and of somatostatin itself were compared.
At dose levels of 0.4 ~g./rat and 10 ~g./rat, D-Vall, D-Trp8-s~toStatin inhibited the increase in growth hormone secretion by 14% and by 42% over control, respectively. Somatostatir, at a dose level of 2,ug./rat had no effect on the increase in growth hormone secretion whereas at 50 ~g./rat it inhibited the increase in growth hormone secretion ~y 56% over control.
D-Vall, D-Trp3-somatostatin was tested for its in vivo activity in inhibiting glucagon and insulin secretion upon stimulation with L-alanine. Normal mongrel dogs of either sex were ~asted overnight. Control blood samples were obtained, and then an intravenous infusior of saline, somatostatin, or test compound was started.
After 30 minutes~ L-alanine additionally was administered ~3 intravenously for a period of 15 minutes. The infusion of saline, somatostatin, or test compound 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 dose of D-Vall, D-Trp8-somatostatin for the inhibition of glucagon secretion is 0.04 to 0.11 ~g./kg./min. and for the inhibition of insulin secretion is less than 0.004 ,ug./kg./min.

.j

Claims (2)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of the formula formula I, and the pharmaceutically acceptable non-toxic acid addition salts thereof which comprises reacting the corresponding straight-chain tetradecapeptide of formula III, D-Val-Gly-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, with an oxidizing agent.

2, A compound of the general formula , formula I, and the pharmaceutically acceptable non-toxic acid addition salts thereof, whenever prepared by the process of Claim 1 or an obvious equivalent thereof.