Somatostatin analogs and intermediates thereto

i '3 i

J / 1 ί

L

3Ü

MINISTRY OF INDUSTRY

FECfSTRO OF IA INDUSTRIAL PROPERTY

<img file="ES476901A1_D0001.tif" />

SPAIN

Coneedldo ol provide data that f | _β, _cOtl.

or NUMBER © EN X © ©

UMERO xe. w

DATE DS PRESENTATION

16-1-1979

PATENT OF INVENTION

<td>θ PRIORITIES: (^ NUMBER</td><td>θ DATE</td><td>^ 3) COUNTRY</td>

<td> 789.472</td><td> 21-4-1977</td><td>U.S</td>

<td>^ rCCHA ADVERTISING</td><td>© INTERNATIONAL CLASSIFICATION</td><td>© PATENT HEARS WHAT IS SIVISIONARY</td>

<td></td><td>£ é&gt;</td><td> 469.005</td>

© TITLE HEARS THE INVENTION

A PROCEDURE FOR THE PREPARATION OF NEW SOMATOSTATINE DERIVATIVES © APPLICANT (YES

EU LILLY AND COMPANY

OOMICILIO OEL APPLICANT

307 East McCarty Street, INDIANÚPOLIS, Indiana, United States © INVENTOR IES)

James Edwin Shields, of United States nationality, who has assigned his rights to the requesting entity © HEADING HOLDER)

The same applicant © REPRESENTATIVE

Mr. BERNARDO UNGRIA GOIBURU

UNC A-4 MOO. 910 «

BE USED AS THE FIRST MEMORY PAGE

POOR

QUALITY

This invention relates to a method for preparing the D-Vai-Giy ~ L-Cys-L-Lys-L ~ Asn-L-PheL-Ehe-L-Trp-L-Lys-L-Tlir-L tetradecapeptide. -Phe-L-Thr-L-Sér-L-Cys-OH, formula I, as well as its addition salts of. Pharmaceutically acceptable acid and intermediates produced by the synthesis of the tetradecapeptide.

Somatostatin (also known as the factor that inhibits the release of somatotropin) is a tido tetradecape of the formula L-Ala-Gly-L-Cys-Ir-Lys-L-Asn-Ir-Phe-L-Phe! L-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH. This tetradeca peptide was isolated from the hypothalamic extracts of sheep and was found to be active in inhibiting the secretion of developmental hormone (HG), also known as sómatotro pina. In this regard, see P. Brazeau, -W. Ok, E. Eurgus,

H. Ling, H. Butcher, J. Eivier, and E. Guiilemin, Science 179, 77 (1973).

In addition, US Patent 3,904,594 describes natural somatostatin as well as a generic class of other compounds having the sequence of dodecaptide represented by positions 5-14 of the natural hormone.

In addition, the compound conveniently designated as L-Ala ^ -somatostatin was previously disclosed in Perland et al., Molecular and Cellular Endocrinology, 4, 79-88 (1976) · Ea D-Ala ^ -somatostatin, although structurally it is a stereoisomer of the natural L-Ala ^ -somatostatin, is about half as active as natural somatostatin in the in vivo inhibition of gastric acid secretion. The compound of this invention, D-Val ^ -somatostatin, differs from D-Ala ^ -somatostatin by the substitution of

1S ί -Λ · * I ί

and two hydrogens per methyl groups. If something could be expected with respect to the pharmacological activity of D-Val ^ -soma-tostatin, it would be that its activity was similar to that of D-Ala ^ -somatostatin; in this way, the natural hormone would be less active as an in vivo inhibitor of gastric acid sectioning. However, D-Val ^ -sometostatin exhibits an activity that is slightly higher than the natural hormone. This result is what demonstrates the unpredictability of L-Val ^ -somatostatin when compared to structurally similar compounds of the prior art.

The biologically active tetradecapeptide of the formula I defined above includes the non-toxic addition salts thereof. Its structure differs from that!

!

of somatostatin due to the presence of a D-valine na residue in position 1, instead of an L-alanine residue.

For convenience, the tetradecapeptide of formula I may be referred to as D-Val ^ -somatostatin.

Thus, this invention relates to a process for preparing a compound selected from those of the formula HD-Val-Gly-Ir-Cys-L-Lys-L-Asn-L-Phe-L - Phe-LTrp- L-ñys-L-Thr-L-Phe-L-Thr-L-Ser-L-Gys-OH and its pharmaceutically acceptable non-toxic acid addition salts, and as an intermediate, RD-Val-Gly-L-Cys - (R ^) - L-Dys- (R2) -L-AsnIr-Phe-L ~ Phe-L-Trp (R₅) L-] 4ys- (R₂) -L-Thr (E₅) -L-Phe-L-Thr (R₅) -L · Ser (R¿pL-Cys (Rp-X, formula II; where

R is hydrogen or a "-amino protecting group;

R ^ is hydrogen or a thio protection group;

I? 2 ^{it is} hydrogen or a £ -amino protection group;

Rj and R ^ each are hydrogen or a hydroxy protecting group;

Hidrógenoss hydrogen or formyl; and X is hydroxy or

<img file="ES476901A1_D0002.tif" />

Resin where the resin is polystyrene: with the proviso that, when X is hydroxy, each of R, Rp Rg? 7 R5 is hydrogen, and when X is

<img file="ES476901A1_D0003.tif" />

.Resin each; of R, Rp Rg:^ANDj Y ^R4 ^{it is} different from hydrogen.

The new tetradecapeptide of formula I, D-íal-GlyL-Cys-L^;-Lys-L-Asn-LI^&gt;herL-Phe-L-Trp-L-Lys-Ir-Thr-Ir-Phe''Ij-Thr ~

L-Ser-L-Cys-OH, is prepared by reacting the corresponding linear chain tetradecapeptide of formula III, HB-Val-Gly-Ir &lt;! Ys-Ir-Iiys -]&gt; Asn-L-Phe-l &gt; Phe-L-Trp-L-Iys-L-ThrL-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 salts include organic or inorganic acid addition salts, for example those prepared from hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric acid, succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalene sulfonic, and propionic. Preferably, the acid addition salts are those prepared from acetic acid. Any of the above salts are prepared&gt;

by conventional methods.

₅ Also contemplated within the scope of this invention are the intermediaries of formula II, RD-Va 1-Gly-L-Cy s ^) - L-I and s (l?₂ ) -L-Asn-L-Phe-Ir-Phe-L-Trp (R ^) L-Lys- (R₂) -L-Thr (R₅) -L-Phe-L-Tbr (R₃) -L-Ser (R₄) -L-Cys (R₁) -X.

They include preferred intermediaries:

HD-Val-Gly-L-Cys-L-Lys-L-Asn-L-Phe-Í ^ ·

Pbe-L-Trp-L-Lys-L-Thr-L-Phe-L-Thr-L-SerL-Cys-OH, formula III; and the

N- (BOC) -D-Val-Gly-L- (PMB) Gys-L- (CBzOG) í Ly s — L — A sn- L-Ph e - L-Phe - Ir- (For) Tr p - L- (C Bz OC) Ly sL- (Bzl) Thr-L-Phe-L- (Bzl) Thr-L- (Bzl) Resin

<img file="ES476901A1_D0004.tif" />

! The above formulas defining the intermediates include protection groups for the amino, hydroxy and thio (sulfhydryl) functions. The properties of a protection group, as defined herein, are twofold. First, the protection group prevents a reactive portion present in a particular molecule from undergoing a reaction during the subject's submission to conditions that could cause the otherwise active portion to rupture. Secondly, the protection group is such that it can be easily separated by restoring the original active portion and under conditions that would not undesirably affect other portions of the molecule. The groups that are useful for these purposes, that is, to protect the amino, hydroxy and thio groups, are well known to those skilled in the art. Actually, complete volumes have been edited specifically to a description and discussion of the methods for using such groups. One such volume is the treatise, Protective Gropps in Organic Chemistry,

- 6 10

JPW McOmie, Editor, Plenum Press, New York, 1973

In the above formulas that define the intermediates Rios represents either an oc_amino hydrogen or a &lt;&lt; -amino protecting group. The et-araino protecting groups considered for R are well known to those with ordinary experience in the peptide art. -Many of these are detailed in McOmie, previously cited, Chapter 2, written by JW Barton. Illustrative of such protection groups are benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichloro ^ encyloxycarbonyl, o-bromobenzyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyl , p-nitrobenzyloxycarbonyl, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, 2- ~ (p-nifenyl) isopropyloxycarbonyl (BpOC), adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl, triphenylmethyl (trityl), and p-toluenesulfonyl. Preferably, 1 the c &lt;-amino protecting group defined by R is t-butyloxycarbonyl.

R¹ it represents either the hydrogen of the sulfhydryl group of the cistern or a protecting group for the sulfhydryl substituent. Many such protection groups are described by McOmie, previously mentioned, chapter 7, written by RG Hickey, VR Razo, and VG Rhodes. Of such suitable protection groups, p-metc xbenzyl, benzyl, p-totyl, benzhydryl, midomethyl aceta, trityl, p-nitrobenzyl, t-butyl, isobutyloxymethyl, as well as also any of a number of trityl derivatives. For additional groups see, by

T¡

IS | example, Houben-Weyl, Methodes der Organischen Chemie, | Synthese von Peptiden, Volumes 15/1 and 15/2, (19/4), Stuttgart, Germany. Preferably, the sulfhydryl protecting group defined by is p-methoxybenzyl.

Rg represents either hydrogen on the £ -ami function or the lysine residue or a suitable -amine protecting group. Illustrative of such groups are all of those mentioned hereinbefore which are suitable for use as an amino protection group. Included as typical of such groups are benzyloxycarbonyl, t-butyloxycarbonyl, t-amyloxy

J carbonyl, cyclo-pentyloxycarbonyl, adamantyloxycarboxyl, and p-methoxybenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, l p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, e'l 2,6-dichlorobenzyloxycarbonyl, 2,4-dichloroxycarbonyl, 2,4-dichloroxycarbonyl o-bromobenzyloxycarbonyl, p-nitroten cyloxycarbonyl, isopropyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl and p-toluenesulfonile. i

As will be evident later, the process for the preparation of the tetradecapeptides of formula I involves the periodic dissociation of the cC-amino protection group from the terminal amino acid present in the peptide chain. Thus, the only limitation with respect to the identity of the £ -araino protection group in the lysine residue is that it is such that it will not dissociate under the conditions used to selectively dissociate the &lt;deci-amino protection group . The appropriate selection of the &lt;-amino and £ -amino protection groups is a matter · that remains within the knowledge of a chemist related to peptides of ordinary experience in the field and depends on the relative ease with which a group can dissociate of particular protection. In this way, groups such as 2- (p-biphenylyl) isopropyloxycarbonyl (BpOC |) and trityl are very labile and can be dissociated even in the presence of a moderate acid. A moderately strong acid, such as hydrochloride acid, trifluoroacetic acid or boron trifluoride in acetic acid, is required to dissociate other groups such as t-hutoxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl and p-methoxybene ciloxycarbonyl . Even stronger acid conditions are required to dissociate other protection groups such as benzyloxycarbonyl, halogenobenzyl.

I '' ~ i

xic'arbonyl, p-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl and isopropyloxycarbonyl. The dissociation of the latter groups requires drastic acid conditions such as the use of hydrobromic acid, hydrofluoric acid or boron trifluoroacetate in trifluoroacetic acid. Of course, any of the weakest groups will dissociate and also under the strongest acidic conditions. Appropriate selection of the amino protection groups in this manner will include the use of a group in the "&lt;-amino" function that is more labile than that employed as the S-amino protection group with the dissociation conditions designed to selectively separate only the oC-amino function. In this context, it is preferably o-chlorobenzyloxycarbonyl or cyclopentyloxycarbonyl and, together with it, the &lt;&lt; - amino protection group chosen for use in each of the amino acids that are added to the peptide chain, preferably is t- butyloxycarbonyl. t

The Rj 7 groups represent the hydrogen of the hydrc

IS jxilo or a protection group for the alcoholic hydroxyl of threonine and serine, respectively. Many of the protection group :: are described in McOmie, previously cited, Chapter 3, written by CB Reese. They are typical., Of such protecting groups, for example, C-^ -C ^ alkyl, such as methyl, ethyl, and t-butyl; blessing benzyl substitute tuido, such as p-raethoxybenzyl, p-nitrobenzyl, p-chlorobenzyl and o-chlorobenzyl; C ^ -Cj alkanoyl, such as formyl, acetyl and propionyl; triphenylmethyl (trityl). Preferably, when Rj and iR ^ are protection groups, the selection protection group in both cases is benzyl.

i

The group R ^ represents either hydrogen or formyl and defines the portion of the tryptophan residue. The formyl serves as a protection group. The use of such a protection group is optional and, therefore, approximately R ^ can be hydrogen (N-deprotected) or formyl (N-protegi do).

Group X refers to the terminal carboxyl of the tetradecapeptide chain; This may be hydroxyl, in which case a free carboxyl group is defined. In addition, X represents the solid resin support, to which the carboxyl terminal portion of the peptide is linked during its synthesis. This solid resin is represented by the formula

<img file="ES476901A1_D0005.tif" />

Resin

In all of the above, when X represents hydroxyl, each of R, Rp R₂, R ^, R ^ and R ^, is hydrogen. When X represents the solid resin support, each of R, Rp R ^,

Rj and is a protection group.

ΙΟ

SW

The following abbreviations are used herein most of which are well known and commonly used in. The technique:

Ala - Alanina '' Asn - Asparagine *

Cys - Cysteine ​​-Gly - Glycine

Lys - Lysine

Phe - Phenylalanine

Ser - Serina

Thr - Tre onina

Tpp - Tryptophan ¿1 - Valina '

DCC - N, N^Z-dicyclohexylcarbodiimide

I

DMF - Ν, Ν-dimethylformamide

BOC - t-butyloxycarbonyl • PMB - p-methoxybenzyl

CBzOC - £ -chlorobenzyloxycarbonyl • i

CPOC - cyclopentyloxycarbonyl Bzl - Benzyl

For - Formilo

BpOC - 2- (p = biphenylin-isopropyloxycarbonyl)

Although the selection of the particular protecting groups to be used to prepare the compounds of formula I remains a matter that falls within the ordinary experience of a chemical related to synthetic peptides, it is well recognized that the sequence of the reactions that must be carried out result in a selection of a particular protection group. In other words, the selection protection group can be one that is stable © - • t

IS

11 reagent to reagents as to the conditions used in the subsequent stages of the reaction sequence. By pious axis, as discussed to some extent hereinbefore, the particular protection group employed must be one that remains intact under the conditions used to dissociate the amino-amino acid protection group from the amino acid residue. terminal of the peptide fragment in preparation for coupling the next amino acid fragment below, to the peptide chain. It is also important to select, as a protection group, the one that remains intact during the construction of the peptide chain or that is easily separable at the end of the syndhesis of the desired tetradecapeptide product. All these matters / are within the knowledge and understanding of a chemist related to peptides of ordinary experience in the field.

As is evident from the previous discussion, the tetra-; Decapeptides of formula I can be prepared by solid phase synthesis. This synthesis involves a sequence construction in the peptide chain that begins at the C-terminal end of the peptide. Specifically, cysteine ​​is first linked in its carboxyl function to the resin by reacting an S-protected, amino-protected cysteine ​​with a chloroethylated resin or a hydroxymethyl resin. The preparation of a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London), 58 1597-98 (1966). Chloromethylated resin is commercially available from Lab Systems, Inc., San Mateo, California.

To achieve the C-terminal cysteine ​​bond with_?the resin, the protected cysteine ​​first becomes its

<td> .</td><td> -12 -</td>

<td> 1</td><td>cesium salt This salt is then reacted with the re sina according to the method described by BP Gisin, Helv.</td>

<td> 5</td><td>Chim. Minutes, 56? 14-76 (1973) «Alternatively, the cistern can be bound to the resin by activating the carboxyl function of the la-apply medial cistern molecule tion of easily known techniques. For example, the cis-</td>

<td> 10</td><td>Theine can be reacted with the resin in the presence of an activation compound of the carboxyl group such as N, N'- drci &lt;-; lohexylcarbodiiinide (DCC). 1 Once the carboxyl-free cistern has been properly interlaced with the resin support, the rest of the the sequence of construction of the peptide involves the adi-</td>

<td> 15</td><td>step-by-step ratio of each amino acid to the N-terminal portion of the peptide chain. Necessarily, therefore, the sequel The particular company involved involves a dissociation of the &lt;-amino amino acid protection group, which represents the N-terminal portion of the peptide fragment followed by the coupling of the next amino acid residue to the now free and reactive N-terminal amino acid. The dis-</td>

<td> 20</td><td>ciation of the deoC-amino protection group can be carried out in the presence of an acid such as hydrobromic acid, acid hydrochloric, trifluoroacetic acid, p-toluenesulfonic acid benzenesulfonic acid, naphthalenesulfonic acid and acetic acid</td>

<td> 25</td><td>co, with the formation of the acid addition salt product respective. Another method that is available to achieve dissociation of the amino acid protection group involves the use of boron trifluoride. For example, diethyl ether of boron trifluoride in glacial acetic acid will convert</td>

<td> 30</td><td>the amino-protected peptide fragment to a complex of BP ~ which can then be converted to the peptide fragment</td>

* tr f

I unlocked by treatment with a base such as aqueous potassium bicarbonate. Any of these methods can be used as long as it is recognized that the selection method must be the one that achieves the dissociation of the &lt;&lt; - amino H-terminal protection group without breaking any other protection group present in the chain of peptide In this regard, it is preferred that the dissociation of the N-terminal protecting group be achieved using trifluoroacetic acid. In general terms, the dissociation will be carried out at a temperature of approximately OSC. at room temperature. .

Once the N-terminal dissociation has been performed, i

The resulting product will normally be in the form of the acid acid salt of the acid that has been used to cause the dissociation of the protection group. The product can then be converted to the free amino terminal compound by treatment with a moderate base, typically a tertiary amine such as pyridine or triethylamine.

i

The peptide chain is then ready for the area?

I tion with the following amino acid. This can be achieved through the use of any of several recognized techniques. In order to achieve the coupling of the following amino acid to the N-terminal peptide chain, an amino acid is used that has a free carboxyl but is adequately protected in the oC-amino function, as well as in any other active portion. The amino acid is then subjected to conditions that will make the carboxyl function active with respect to the coupling reaction. One such activation technique, which can be used in the synthesis, involves the conversion of the amino acid to a mixed anhydride. Therefore, the

10

The amino acid free earboxyl function is activated by reaction with another acid, typically a carbonic acid in the form of its acid chloride. Examples of such acid chlorides can be used to form the appropriate mixed anhydrides, ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chlorine formate, and pivaloyl chloride.

Another method of activating the earboxyl function of the amino acid ami to achieve coupling is by converting 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-ni trophenyl ester, an ester formed of 1-hydroxybenzotriaxol, and an ester formed, of N-hydroxysuccinimide. Another method for effecting the coupling of the G-terminal amino acid to the peptide fragment involves carrying out the coupling reaction in the presence of at least an equimolar amount in N, N'dicyclohexylcarbodiimide (DCC). This latter method is preferred to prepare the tetradecapeptide of formula II, wherein X is

-0-CH.

<img file="ES476901A1_D0006.tif" />

Resin

Once the desired amino acid sequence has been prepared, the resulting peptide can be separated from the resin support. This is achieved by treatment of the resin-supported tetrade cappeptide protected with hydrochloric acid. The hydrochloric acid treatment separated the resin peptide, in addition, however, dissociates all the groups.

Remaining protection is present in the reactive portions placed in the peptide chain as well as the 'X-aniino protection group present in the N-terminal amino acid. When hydrofluoric acid is used to dissociate the resin peptide as well as to separate the protection groups, it is preferred that the reaction is carried out in the presence of anisole. The presence of anisole s'e 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 dissociation is carried out in the presence of ethyl mercaptan. Ethylmerf captane serves to protect the indole ring from tryptophan residue and also facilitates the conversion of blocked cysteines to their thiol forms. Also, when Rj is formyl, the presence of ethyl mercaptan facilitates the dissociation of hydrofluoric acid from the formyl group.

Once the dissociation reaction has been achieved, the product 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 linear chain tradecapeptide under conditions that will effect its oxidation by converting the two sulphydryl groups into the molecule one in each portion of cysteinyl to a disulfide point. This can be achieved by treating a dilute solution of the linear tetradecapeptide with any of a variety of oxidation agents including, for example, iodine and potassium ferricyanide. Air can also be used as an oxidizing agent, the pH of the mixture being generally from about 2.5 to 9.0, and preferably from about 7.0 to 7.6. When air is used as an oxidation agent, the

'* «*» · * “' -V **. '*; · Π' * '! ??'? ' The concentration of the peptide solution is generally not greater than about O.⁷! mg of the peptide for me. of solution, and is usually about 50 / ug / ml.

The compounds of formula I can be administered to warm-blooded mammals, including humans, by any of several methods, including oral, sublingual, subcutaneous, intramuscular, intravenous and other suitable routes of administration. Each of these compounds is active, although not necessarily to an equivalent degree, to inhibit the release of developmental hormone. This inhibitory effect is beneficial in those cases in which the host being treated requires a therapeutic treatment for excessive secretion.

/ _z . of soma.totropin, such secretion is associated with adverse conditions such as acromegaly and juvenile diabetes. These compounds also inhibit other physiological effects including inhibition of gastric acid secretion, they are useful in the treatment of ulcerative conditions; inhibition of exocrine secretion of the pancreas is potentially useful in the treatment of pancreatitis; the inhibition of insulin and glucagon secretion, and the reduction of nerves useful in gastrointestinal radiology. Preferably, the dose scale for sublingual or oral administration is approximately 1 mg. at 100 ag./'kg. of body weight per day. Generally, a dose scale for intravenous, subcutaneous or intramuscular administration is approximately IO; ug, at 1 mg / kg. of body weight per day and ', preferably is about 50 Jug. at 100 ug / kg. of body weight per day. It is clear that the scale of the dose will vary widely depending on the particular state that is being treated as well as the severity of the condition.

IS

It is also possible to administer the compounds of formula I in association with a pharmaceutical carrier, for example, in the form of tablets or capsules. Inert carriers or diluents can be used, for example, carbonate. of magnesium or lactose, together with conventional disintegrating agents such as corn starch and alginic acid, and lubricating agents, for example magnesium stearate. Typically, the amount of carrier or diluent will vary from about 5 to 95 percent of the final composition, and preferably about 50 to 85% of the final composition will be preferred. Suitable flavoring agents may also be used.

I in the final preparation that make the taste composition more enjoyable in the administration.

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

The following examples are illustrative of the preparation of the compounds of formula I, and their intermediates.

Example 1

PESIMA LE METYL POLYSTYRENE OF Nt-HJTILOXICARBONIIr-LCISTEINIL (Sp-METOXIEEMCILO)

500 mi. of Ν, Ν-dimethylformamide (DME) containing the cesium salt of Nt-butyloxycarbonyl (Sp-methoxybenzyl) cis theine / "prepared from 9.06 g. (26.5 mmol) of libr acid θ_7. 51-0 g were added. of chloromethylated polystyrene resin (Lab Systems, Inc., 0.75 mmol / gram). The mixture was stirred at room temperature for six days. The resin was then filtered and washed successively three times with a mixture of 90 percent dimethylformamide and 10 percent water, three times with 95 percent ethanol, and three times with dimethylformamide. A. the resin suspended in 500 ml. of dimethylformamide, a solution of 10.5 grams of cesium acetate was added. The mixture was stirred for six days at room temperature. The resin was then stirred and washed successively, once with aqueous dimethylformamide, three times with a mixture of 90 percent dimethylformamide and 10 percent water, three times with 95 percent ethanol, three times with chloride. methylene, three times with 95 percent ethanol 'and three times with chloroform. Be known

I · raron the pieces by suspending the resin in chlorine formed four times and each time extracting liquid. The iesin was then dried under vacuum at 40SC. overnight to get 44.8 g. of the product of the title. An amino acid analysis showed 0.25 mmol of Cys per gram of resin. Cysteine ​​was determined as cysteic acid from a hydrolysis s

acid carried out using a 1: 1 mixture of dioxane and concentrated hydrochloric acid, to which a small amount of dimethyl sulfoxide was added.

Example 2

METHODED POLYSTYRENE RESIN EE Nt-BUTILOXICARBONIL-DVALIL-GLICIL-L- (Sp-METO: HBENCIL) CISTEINIL-L- (N- £ -o-CLOROBER CILOXICARBONIL) -LISIL-L-ASPARAGINIL-L-PENILALANL NIL-L- (PORMIL) TRITOPIL-L- (N- Eo-CLOROBENCI LOXICARBONIL) -LISI Go · (0-BENCIL) TREONIL-L-PENILALANIL-L- (O-BENCIL) TREONIL-L- (0BENOIL) SERIL- L (Sp-METOXIBENCIL) CYSTEINYL.

The product of example 1 (? .O grams) was placed in; the reaction vessel of an auto peptide synthesizer

IS

-₁₉Mystic Beckman 990 and twelve of the remaining thirteen amino acids were added, using the automatic sinter. The resulting protected tridecapeptide resin was divided into two equal portions and the final residue was introduced into one of the portions. The amino acids that were used, as well as the sequence of their use, is the following: (1) Nt-butyloxycarbonyl- (O-benzyl) L-serine; (2) Nt-butyloxycarbonyl ~ (0-benzyl) -L-threonine; (3) Nt-butyloxycarbonyl-L-phenylalanine; (4) Nt-butyloxycarbonyl (O-benzyl) -L-threonine; (5) N- -t-Butyloxycarbonyl-N- -o-chlor.) Benzyloxycarbonyl-L-lysine; (6) N- -t-Butyloxycarbonyl- (N-fo.j. Thousand) -L-tryptophan; (7) Nt-butyloxycarbonyl-L-phenylalanine;

l (8) Nt-butyloxycarbonyl-L-phenylalanine; (9) N-t-butoxycarboyl n-L-asparagine, p-nitrophenic ester; (10) li- -t-buuyloxycar bonyl-N- -o-chlorobenzyloxycarbonyl-L-lysine; (11) Nt-butyl xicarbonyl- (Sp-methoxybenzyl) -L-cysteine; (12) Nt-butxloxycarbonylglycine; and (15) Nt-butyloxycarbonii-D-valine. The sequence of deprotection, neutralization, coupling and rea. Coupling for the introduction of each amino acid into the peptide is as follows: (1) three washes (10 ml / gram of resin) of three minutes each, with chloroform; (2) separation of the BOC group by treatment twice for twenty minutes at a time, with 10 ml / gram of resin, of a mixture of 29 percent trifluoroacetic acid, 48 percent chloroform, 6 percent triethylsilane, and 17 percent methylene chloride; (5) two washes (10 ml / gram of resin) of three minutes at a time, with chloroform;

(4) a three minute wash (10 ml / gram of resin) with methylene chloride; (5) three washes (10 ml / gram of resin) of three minutes at a time, with a mixture of 90 percent of t-butyl alcohol and 10 percent of alcohol-t-amyl

- 20 10 co; (6) three washes (10 ml / gram of resin) of three minutes at a time, with methylene chloride; (7) neutralization me. for three three-minute treatments each time with 10 ml / gram of 3 percent triethylamine resin in methylene chloride; (8) three washes (10 ml / gram of resin) of three minutes each time with methylene chloride; (9) three washes two (10 ml / gram of resin) three minutes at a time with a mixture of 90 pon percent t-butyl alcohol and 10 percent t-amyl alcohol; (10) three washes (10 ml / gram of resin) of three minutes each time with methylene chloride;

(ll) the addition of 1.0 mmol / gram resin of the pro-categorized amino acid and 1.0 mmol / gram of N, N'-dicyc.ohexyl - 'carhodiimide (DCC) resin in 10 ml / gram of methylene chloride resin, mixing afterwards for 120 minutes; (12). Three washes (10 ml / gram of resin) of three minutes each time with cLe methylene chloride; (13) three washes (10 ml / gram of resin) of three minutes at a time, with a mixture of 90 percent t-butyl alcohol and 10 percent thiyl amyl alcohol; (14) three washes (10 ml / gram of resin) of three minutes each time with methylene chloride; (15) neutralization by three three-minute treatments each time with 10 ml / gram of 3 percent resin of triethylamine in methylene chloride; (16) three washes (10 ml / gram of resin) of three minutes each with methylene chloride; (17) three washes (10 ml / gram of 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 of resin) of three minutes each with methylene chloride no; (19) three washes (10 ml / grams of resin) of three meats each with dimethylformamide; (20) Addition of 1.0 mmol

Ί ¡

- 2-11 gram of resin of the protected amino acid and 1.0 mtnoles / grnmo of resin of Ν, Ν'-dicyclobexylcarbodiimide (DCC) in 10 ml / gram of resin of a mixture of DMF in 1: 1 ratio and chloru

<td> 1</td><td>ro of methylene followed by mixing for 120 minutes; (twenty-one)</td>

<td>⁵ 10</td><td>three washes (10 ml / gram of resin) of three minutes each Y one with DMF; (22) three washes (10 ml / gram of resin) of three minutes each with methylene chloride; (2J) three washes (10 ral./gram of resin) of three minutes each with a mixture of 90 percent t-butyl alcohol and 10 percent percent t-amyl alcohol; (24) three washes (10 mi./gram</td>

<td>í</td><td>resin) of three minutes each with methylene chloride; (25) neutralization by three treatments of three minutes each with 10 ml / gram of resin of three percent</td>

<td> 15</td><td>of triethylamine in methylene chloride; (26) three washes (10 ml / gram of resin) of three minutes each with methylene chloride; (27) three washes (10 ml / gram &amp; e resin) of</td>

<td> 1 . 20</td><td>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 of resin) of three minutes each one with methylene chloride.</td>

<td>i i 25</td><td>The aforementioned treatment is used by adding from. each of the amino acids with the exception of the asparagine and glycine residues. The addition of the rest of glycine Use only steps 1-18. The rest of asparagine is incorporated through its active ester p-nitrophenyl. To the</td>

<td></td><td>to do so, the previous step (11) was modified to the following sequence of three stages: (a) three washes (10 mi./gram resin) three minutes each time with dimethylformamide;</td>

<td>³⁰</td><td>(b) addition of 1.0 mmol / gram of p-nitro- ester resin Nt-butyloxycarbonyl-L-asparagine Phenolic in 10 ml./gra-</td>

22nd resin of a 1: 3 mixture of dimethylformamide and methylene chloride, then mixing for? 20 minutes; and (c) three washes (10 ml / gram of resin) three minutes at a time with dimethylformamide. Also, step (2) above was modified to the use of the p-nitrophenyl ester of Ht-butyloxycarbonyl-L-asparagine in a 5: 1 mixture of dimethylfor msmide and methylene chloride, then mixing for 720 minutes.

The finished peptide resin was dried under vacuum. A portion of the product was hydrophilized, refluxing, for 72 hours in a mixture of hydrochloric acid and dioxane. The amino acid analysis of the resulting product gave the following results, using lysine as a reference:

z

Asn, ^ 1.04; 2Thr, 2.68; Ser, 1.08; Val, 1.12; Gly, 1.04; 5Phe 3.87; 2Xys, 2.00, Trp, 0.75

Tryptophan was determined by a 21-hour hydrolysis of a sample of the product in the presence of dimethyl sulfoxide and thioglycolic acid. The cysteine ​​was not determined since it is destroyed by the method of analysis.

Example 5

D-VALIL-GLICIL-L-CISTEINIL-L-LISIL-L-ASPARAGINIL-L-EEHILALA.

NIIr-L-PENIL-ALANIL-Ir-TRIPTOFIL-L-LXSIL-L-TREONIL-L-PENILALARIL-L-TREONIL-L-SERIL-L-CISTEINA.

To a mixture of 5_;ml. of anisole and 5 mi. of ethyl mercaptan, 2,828 grams (at a substitution level of 0.150 mmol / gram) of the protected tetradecapeptide resin of Example 2 were added. The mixture was cooled in liquid nitrogen, and 56 ml was added by distillation. of liquid hydrofluoric acid. The resulting mixture was allowed to warm to OSC., And stirred for 2 hours. Then the

<td> -</td><td> -25 -</td>

<td> 1</td><td>hydrofluoric acid by distillation, and added ether or The remaining mixture. The mixture was cooled to OBC., And the solid</td>

<td>s</td><td>The resulting was collected by filtration and washed with ether The product was dried, and the unprotected tetradecapeptide was extracted from the resin mixture using acetic acid 1 mo lar, and a small amount of glacial acetic acid. The acetic acid solution was lyophilized immediately to dryness in the dark. The resulting white solid was suspended in a mixture of 10 ml. 0.2 molar acetic acid desga</td>

<td> 10</td><td>Sified and 4 mi. of glacial acetic acid. The resulting suspension was heated; However, e3. solid did not dissolve</td>

<td> 15</td><td>completely. The insoluble portion was filtered and the filtrate colorless, opaque, was applied to a Sephadex G-25 E- column The chromatographic conditions were: solvent, acid 0.2 molar degassed acetic; column size 7-5 x 150 cm temperature, 2620., flow rate 629 mi./hour; volume of the fraction, 22.0 mi. The absorbance graph at 280 mp of each fraction i</td>

<td> 20 1</td><td>tion versus the fraction number, indicated a high maximum followed by a shoulder. Ultraviolet spectroscopy revealed that the main maximum was the product. The fractions that se-, combined and their elution volumes, were the following</td>

<td> 25</td><td>tes: fractions 224-240 (4906-5280 mi., maximum = 5O5²* ®1). This collection of fractions does not include the shoulder if-</td>

<td> 30</td><td>guide Ultraviolet spectroscopy indicated that 175 mg were present. of the product (yield = 24.8%). An Ellman degree of an aliquot indicated a contsnidc sulphydryl free of 95.6% of theory. Example 4</td>

_24

OXIDATION OF D-VAiA-SQMATOSTATINA

The solution of the reduced D-Val ^ -somatostatin (374 ml., Theoretically 175 mg.) Of Example 3, was diluted with 147 ml. of 0.2 molar acetic acid and 29 &amp; 7 mi. of distilled water to achieve a concentration of 50 / ig / ml. Concentrated ammonium hydroxy 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 Ellmkn titration indicated that the oxidation was complete.

'! The mixture was concentrated in vacuo to a volume of 10 ml. The concentrate was diluted with 10 ml of glacial acetic acid, and then desalified on a Sephadex G-25 Fe column. Chromatographic conditions were as follows._Fe: solvent, degassed acetic acid and j / o \ column size 5-0 x 90 was .; temperature, 2620., flow rate, 24-6 mi./hour; volume ^ of the fraction, 16.4 mi.

The absorbance graph at 280 ιημ for each fraction versus the fraction number indicated two large maximums. The first maximum represented the aggregate forms of the product, and the second maximum represented a monomeric product. The material represented by the second maximum / "fractions 4-9-64 (787-1050 mi./7 was collected and lyophilized to dryness in the dark. The resulting solid was dissolved in 15 ml. Of degassed 0.2 molar acetic acid and was applied to a column of Sephadex G-25 G. The chromatographic conditions were: solvent, acetic acid, degassed 0.2 molar; column size, 5-0 x 150 cm; temperature, 2520 .; flow rate, 4? 5 mi./hour; volume of the fraction, 16.6 mi.

The absorbance graph at 280 mu, for each fraction

-25 10

IS tion versus the fraction number showed a high maximum. Lfi ultraviolet spectroscopy indicated that the main part of the maximum was ^ the product. Fractions 157-172 (elution volumes of 2590-2855 ml., Maximum = 2667 ml.) Were combined and freeze dried to dryness in the dark. The ultraviolet spectroscopy indicated 95 mg. of the desired product (yield of the reduced form = 54 · 5%) ·

A portion of the resulting solid was dissolved in 5 ral of 50% acetic acid and recroraatographed on a Sephadex G-25 F column. Chromatographic conditions were: solvent, 50% degassed acetic acid; co lunjna size, 2.5 x 180 era .; temperature, 26SC .; flow rate,

I '

53.'2 mi./hour; volume of fraction 8.87 mi.

The graph of the absorbance at 280 ιημ of each fraction versus the fraction number indicated a large maximum. Ultraviolet spectroscopy indicated the largest portion of the maximum product. Fractions 56-69 (488-552 mi., Maximum cb 505 mi.) Were combined and freeze dried to dryness in the dark. 'og

Optical rotation / * 7 = -42.1 (1 percent acetic acid).

Amino acid analysis: Val, 0 -.- 98; Gly, 1.01; 2Cys, 1.81; 2Lys, 1.99; Asn, 0.95; 5Phe, 2.94; Trp, 0.80; 2Thr, 1.91; Ser, 0.85.

The above results are expressed as ratios at (Gly + Lys) / 3 = 1.0. The following three 21-hour hydrolysis were carried out:

(1) In the presence of dimethyl sulfoxide to oxidize cysteine ​​to cysteic acid. , (2) Thioglycolic acid removed.

wwwmr '' ** '

-26 (5) Without eliminator or oxidant.

All of the above values ​​are average of the three hydrolysis, except the following:

Cys and Ser; only (1) and (5);

Trp; only (2);

Phe; only (2) and (3).

D-Val was tested¹-somatostatin in dogs for in vivo hybridization of gastric acid secretion. In six dogs with chronic fistula and Heidenhain pouch, gastric secretion of HC1 was induced by infusion of the gastrin terminal C tetrapeptide at 0.5 jug '/ kg-hr. Each dog served as its own control, receiving on a separate day only tetrapeptide peptide. The next day, the six dogs received the tetrapeptide and after one hour of stable state secretion of HC1, the D-Val ^ -somatostátina s 0.75; ig / kg-hr was infused. for an hour. The collection of gastric acid samples was continued for an additional 1.5 hours at 15 minute intervals. The samples were titled.

...

at a pH of 7 with an automatic titrator. The maximum inhibitory effect of D-Val ^ -somatostatin was extrapolated against the dose response curve of somatostatin, and the relative potency of the analogue, with respect to that of somatostatin, is expressed as a percentage of activity. D-Val ^ -somatostatin inhibited stable state acid secretion induced by gastrin C-terminal tetrapeptide in 85 · 1 + 6.0% average interpretation error. This effect is equivalent to that of 0.935 Pg / kg-'tn ?. of somatostatin. Its relative activity with respect to that of somatostati'ns in this way is 125%.

D-Val ^ -somatostatin was also tested for

-27 I its action on the motility of the intestines in conscious dogs. Three dogs were used that have catheters inside ducts of tubular organs placed in the antrum, duodenum and pylorus. Pressure changes in the intestinal duct were recorded in a Visicorder using miniature light beam galvanometry and strain gauges. After a stable state control was established, D-Val ^ -somatostatin was infused intravenously over a period of ten minutes. The compound initially increased the pressure inside the duct in the pylorus and then decreased while the pressure in the duodenum and the anustrum remained depressed throughout the test. In the minimum effective dose required to increase the pyloric pressure and to decrease the pressure of the antrum and duodenum, it was less than 0.125 jug / kg-10 minutes. This compares with an activity for somatostatin itself from 0.125 to 0.2 / ng / kg-10 minutes.

D-Val ^ -somato'statin was also shown to inhibit

SO i

pancreatic creation In three dogs that have both pancreatic and total gastric fistula, secretion of the pancreas was induced by infusion of secretin in 2 units / kghr .. and cholecystokinin at 0.45 units / kg-hr., And gastric HCl secretion by infusion of tetragastrin at 0.5 jug / kg-hr. After a stable response was established, each dog received D-Va ^ -somatostatin for one hour at 0.75 jug / kg-hr. The maximum inhibitory effect expressed as a percentage of change over the control for the total protein was -51%.

D-Val ^ -somatostatin was also tested for its activity with respect to the release of the hormone from

-28 10 development. The procedure used was carried out using normal, male Sprague-Dawley rats weighing 100-120 grams (Laboratory Supply Company, Indianapolis, target). The test is a modification of the method of E. Brazeau, V. Vale, and R. Guilleman, Endocrinology, 94 184 (1974). In this analysis, five groups of eight rats each were used. Sodium pentobarbital was intrinsperitoneally administered to all rats to stimulate the development of hormonal growth. One group served as the control and received only saline solution. Two of the groups received somatostatin, 1 to 2 jug / rat, subcutaneously, and the other at 50 jug / rat, subcutaneously. The other two groups received DVal ^ -sorphiatostatin, one at 2 jug / rat, subcutaneously, and the other at hoJüg / rat, subcutaneously. The concentration of developmental hormone sera was measured 20 minutes after simultaneous administration of sodium pentobarbital and the test compound. The degree of inhibition of serum hormone concentration was then determined with respect to the control group, and the relative activities of D-Val ^ -somatostatin and somatostatin itself were compared.

At a dose level of 2 jug / rat, D-Val ^ -somatosta tub inhibited the increase in the secretion of developmental hormone by 2% over the control, while somatosta tub produced a 44% inhibition. At a dose level of h-0 jug / rat, D-Val ^ -somatostatin inhibited the increase in the secretion of developmental hormone by 73% over the control, while somatostatin itself produced a 79% inhibition. Λ

D-Val ^ -somatostatin was tested for its activity.

-29 10 life in vivo in the inhibition of insulin and glucagon secretion by stimulation with L-alanine. They were fasting overnight, dogs of indefinite breed, of any sex. Control blood samples were obtained and then an intravenous infusion of saline, somatostatin or D-Val-somatostatin was initiated. After 30 minutes, L-alanine was additionally administered in intravenous form, for a period of 15 minutes. The solution infusion! saline, somatostatin or D-Val ^ somatostatin se

I · continue for 15 minutes after the end of the infusion of L-alanine. The infusion of L-alanine caused a sharp increase in the serum concentration of glucagon and insulin, which returned to the control concentration at the end of the infusion of L-alanine. From the * above-. /

that the minimum dose of D-Val ^ -somatostatin for glucagon inhibition is 0.06 to 0.11 jug / kg / min. and for insulin inhibition it is 0.006 to 0.03 jug / kg / min., while the minimum dose of somatostatin for glucagon inhibition is 0.10 to 0.12 zig / kg / min., and for insulin inhibition it is from 0.3 to 010 / ig / kg / min.

In summary, the present Invention Patent that is requested must fall under the following:

<img file="ES476901A1_D0007.tif" />

- 5010