FI64575C - PROCEDURE FOR THE FRAMEWORK OF ANIMAL THERAPEUTIC ANALYSIS SOMATOSTATINANALOG - Google Patents

Description

ΓΒΐ (11) ANNOUNCEMENT / »COC

LJ '' UTLÄGGN1NGSSKR1FT '3 c (4¾ P'tait1 "^ y ^ (51) Kv.lk. ^ Int.CI.3 G 07 C IO3 / 52 FINLAND - FINLAND (21) P« * nttlhik «mui - Pu * nt «ntöknlnj Τ8ΐΐ83 (22) H« k * ml * p »lvl - An * ekninf $ d * j l8.OU.78 (23) AlkupSIvl - Giltifh * t * d» j 18.Ol.78 (41) Tullut to the public - Bllvlt offtntllg 22 10.78

National Board of Patents and Registration of the Republic of Finland

Patent · och registerstyrelsen 'Antttkan utiagd oeh utljkrKt «n publktnd jx.u0.u3 (32) (33) (31) Requested Privilege — Begird prlorltet 21. OU. 77 01.02.78 USA 789 ^ 73, 87U173 (71) Eli Lilly and Company, 3Ο7 East McCarty Street, Indianapolis,

Indiana 46206, USA (72) James Edwin Shields, Indianapolis, Indiana,

The present invention relates to a process for the preparation of a therapeutically useful somatostatin analogue. The present invention relates to a process for the preparation of a therapeutically useful somatostatin analogue. pt icliä HD- Vai-G ly- L-Cys- L-Lys-L-Asn-L-Phe-L-Phe-D-Trp-L-Lys-L.-Thr-L-Phe-L-Thr- L-Sep-L-Cys-OH (Formula I), as well as its pharmaceutical hazards Lomia acid addition salts.

Somatostatin, also known as a somatotropin release inhibitor, is a tetradecapeptide of the formula L-A1a-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe-L-Trp-L-Lys-L -Thr-L-Phe-L-Tnr-L-Ser-L-Cys-OH. This tetradecapeptide has been isolated from sheep hypothalamic extracts and has been found to inhibit the secretion of growth hormone, also known as somatotropin. See. closer? Brazeau, W. Vale, R. Burgus, N-Ling, M. Butcher, J. Rivier, and R. Guiilemin. Science, 179, 77 (1973).

In addition, this compound, conveniently called D-Trp somatostatin, has been previously described by Brown et al., Endocrinology, 9-8, No. 2, 336-343 (1976).

2 64575

The formula of the biologically active tetradecapeptide of formula I is set forth above and also includes its non-toxic acid addition salts. The structure of the new compound differs from the structure of somatostatin in that the D-tryptophan residue is in position 8 instead of the L-tryptophan residue and the D-valine residue at position 1 in place of the L-alanine residue. For convenience, can be marked

Ί B

tetradecapeptides of formula I as follows: D-Val, D-Trp - somatostatin.

In the preparation of a compound of formula I, the intermediate RY-Gly-L-Cys (R ) -L-Thr (R 2) -L-Phe-L-Thr (R 2) - L-Ser (R 2) - L-Cys (R) -X, formula II wherein Y is D-Val, R is hydrogen or an itino-amino protecting group, R 1 is hydrogen or a thio function protecting group, R 2 is hydrogen or an E-amino function protecting group,

R 9 and R 6 each represent hydrogen or a hydroxy protecting group,

Rt- is hydrogen or formyl, and X is hydroxy or / _____. resin in which the resin is polystyrene, provided that when X is hydroxy R, R 1, R 2, R 3> R 2 and R 6 each represent hydrogen, and when X is <~ "" ~ \, resin so R, R 1, R 2, and R 2 are each other than hydrogen.

A novel tetradecapeptide of formula I is prepared by administering the corresponding rectilinear tetradecapeptide of formula III, Y-Gly-L-Cys-L-Lys-L-Asn-1-Phe-L-Phe-D-Trp-1. .-Lys- h-Thr- 1, -

J-I

Phe-L-Thr-L-Ser · -L-Cys-OH, where Y is D-VuL, reacts with acids I 64575. This reaction converts the two sulfhydryl groups into a disulfide bridge.

Pharmaceutically acceptable non-toxic acid addition salts include organic and inorganic acid addition salts, for example, prepared with the following acids: hydrochloric acid, sulfuric acid, sulfonic acid, tartaric acid, fumaric acid, hydrobromic acid, acetic acid, glycolic acid, citric acid, citric acid, maleic acid, maleic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and propionic acid. The acid addition salts are most preferably prepared with acetic acid. All of the above salts can be prepared by conventional methods.

The above formula, which represents intermediates, contains protecting groups for amino, hydroxy and thio (sulfhydryl) functions.

The properties of the protecting groups presented here are twofold. First, the protecting group prevents the reactive portion of the molecule in question from reacting when the molecule is exposed to conditions that could cause the otherwise active moiety to cleave. Second, the protecting group is such that it can be readily removed by restoring the original active moiety under conditions that would not adversely affect other portions of the molecule. The groups used for such purposes, i.e. the groups used to protect amino, hydroxy and working groups, are well known to those skilled in the art. Even entire works have been made to explain and review the use of such groups. One such work is the textbook Protective Groups in Organic Chemistry, J. F. W. McOmie, published by Plenum Press, f 'New York, 1973. /

In the above formula defining intermediates, R represents either a ^ -amino-hydrogen or a -amino-protecting group. Those skilled in the art of peptide chemistry are well acquainted with the protecting groups for the t-amino function represented by R. Several of them are detailed in McOmie, supra, Chapter 2, published by J. W. Barton. Examples of such protecting groups include: “64575 benzyloxycarbonyl, E-chlorobenzyloxycarbonyl, E-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyloxy-benzyloxycarboxy-bryloxy] -carbonyloxycarbonyl, o-chlorobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl -butyloxycarbonyl (BOC), t-amyloxycarbonyl, 2- (E-biphenylyl) isopropyloxycarbonyl (BpOC), adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cyclohexyl, cycloheptyl, cycloheptyl The amino protecting group represented by R is most preferably t-butyloxycarbonyl.

represents either hydrogen in the sulfhydryl group of cysteine or a protecting group of a sulfhydryl substituent. Several such protecting groups are disclosed in McOmie, supra, Chapter 7, R.G. Hickey, V.R. Rao and W.G. Rhodes. Suitable examples of such protecting groups include E-methoxybenzyl, benzyl, E-tolyl, benzhydryl, acetamidomethyl, trityl, E-nitrobenzyl, t-butyl, iso-butyloxymethyl, as well as any of several trityl derivatives. More such groups can be found, for example, in Houben-Weyl, Methoden der Organischen Chemie, "Synthese von Peptide", parts 15/1 and 15/2, (1974). Stuttgart, Federal Republic of Germany. The sulfhydryl-protecting group represented by R 1 is most preferably E-methoxybenzyl.

R 1 represents either hydrogen in the E-amino function of the lysine residue or a suitable protecting group for the E-amino function. Examples of such groups are most of those mentioned above which are suitable for use as protecting groups for the o <amino function. Typical such groups are benzyloxycarbonyl, t-butyloxycarbonyl, t-amyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, E-methoxybenzyloxycarbonyl, E-chlorobenzyloxycarbonyl.i, e-bromobenzyl, 2,6-dichlorobenzyl oxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, o-bromobenzyloxycarbonyl, E-nitrobenzyloxycarbonyl, isopropyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl and p-cycloheptyloxycarbonyl.

As will be seen below, the process for the preparation of tetrade capeptides of formula I involves periodic repetitive cleavage of the terminal amino acid; x, -aminofunction ion protecting group of the peptide chain. The only limitation, thus, with respect to the identity of the protecting group of the ε-amino function in the lysine residue, is that it does not cleave under conditions in which the <X-amino protecting group is selectively cleaved. The appropriate choice of protecting groups for the β-amino function and the β-amino function is well known to those skilled in the art and depends on the relative ease with which a protecting group can be cleaved. Thus, for example, groups such as 2- (p-biphenylyl) large -propyloxycarbonyl (BpOC) and trityl are very labile and can be cleaved using a very weak acid, and a fairly strong acid such as hydrochloric acid, trifluoroacetic acid or boron trifluoride in acetic acid is required for cleavage of other groups such as t-butyloxycarbonyl, t-butyloxycarbonyl, Even stronger acidic conditions are needed to effect cleavage of certain other protecting groups, such as benzyloxycarbonyl, halobenzyloxycarbonyl, p-nitrobenzyloxyocarbonyl, cycloalkyloxycarbonyl, and isopropyloxycarbonyl, and isopropyloxycarbonyl. very strong acidic conditions, such as the use of hydrogen bromide, hydrogen fluoride or boron trifluoroacetate in trifluoroacetic acid. Naturally, all the most labile groups also cleave under stronger acidic conditions. Thus, the appropriate choice of amino-protecting group means that a more labile group is used as the protecting group for the α-amino function than for the protection of the ε-amino function, and also associated with cleavage conditions designed to selectively remove only the <X-amino function.

In this regard, 1 * 2 is most preferably o-chlorobenzyloxycarbonyl or cyclopentyloxycarbonyl, and with them is most preferably selected for use as a protecting group for the o <-amino function at each amino acid to be added to the peptide chains, t-butyl.oxycarbonyl.

Groups and. represent the hydroxyl group of hydrogen or the alcohol hydroxyl protecting group of threonine and serine, respectively. Several such protecting groups are disclosed in McOmie, supra, Chapter 3, C.B. Reese. Typical such protecting groups include, for example, C 1 -C 4 alkyl such as methyl, ethyl and t-butyl; 6,64575 benzyl, substituted benzyl such as E-methoxybenzyl, E-nitrobenzyl, E-chlorobenzyl and o-chlorobenzyl; C 1 -C 6 alkanyl such as formyl, acetyl and propionyl; triphenylmethyl (tri-style). When Rg and are protecting groups, benzyl is most preferably selected as the protecting group in each case.

The group represents either hydrogen or formyl and defines the moiety ^ NR <. tryptophan residue. Formyl acts as a protecting group. The use of such a protecting group is optional and therefore R 1 may suitably be hydrogen (unprotected N) or formyl (protected N).

Group X represents the carboxyl terminus of the tetradecapeptide chain; it may be hydroxyl, which defines a free carboxyl group. In addition, X represents a solid resin support moiety to which the terminal carboxyl of the peptide is attached during its synthesis. This solid resin can be represented by the formula · = · resin -0-CH_— · 2 S ✓ • - ·

In all of the above compounds, R, R 1, R 9,

R 9, R 10 and R 11 are each hydrogen when X is hydroxyl. When X represents a solid resin support moiety, R, R 1, R 8, R 8 and R 2 are each protecting groups.

The following abbreviations, most of which are well known-- '' and and commonly used in the art, are hereinafter used:

Lower Alanine

Asn - Asparagine

Cys - Cysteine

Gly - Glycine

Lys - Lysine

Phe - Phenylalanine

Ser - Serine

Thr - Threonine

Trp - Tryptophan

Vai - Intermediate 7 64575 DCC - N, Ν'-Dicyclohexylcarbodiimide DMF - N, N-dimethylformamide BOC - t-butyloxycarbonyl PMB - E-methoxybenzyl CBzOC - o-chlorobenzyloxycarbonyl CPOC - Cyclopentyloxycarbonyl

Bzl - Benzyl

For - Formyl j

BpOC - 2- (p-biphenyl) isopropyloxycarbonyl

Thus, although a skilled chemist working in the field of synthetic peptides will be able to select the protecting groups to be used in the preparation of the compounds of formula Ij, it is good to know that the reactions to be performed! the sequence requires the selection of specific protecting groups. In other words, the protecting group selected must be stable both to the reagents and to the conditions prevailing in the successive steps of the reaction sequence. As already stated somewhat above, a particular protecting group must be one that remains intact under the conditions used to cleave the <X amino function protecting group from the terminal amino acid residue of the peptide fragment when preparing the subsequent incorporation of the next amino acid fragment into the peptide chain. It is also important to select as the protecting group one that remains intact during the construction of the peptide chain and that is easily removed. when supplementing the synthesis of the desired tetradecapeptide compound. All these facts are known to a skilled peptide chemist. /

The starting tetradeca · peptide of formula III can be prepared by solid phase synthesis.

This synthesis involves the construction of a peptide chain periodically starting from the C-terminus of the peptide. More specifically, the cysteine of its carboxyl function is first attached to the resin by reacting the 'amino-protected, S-protected cysteine with a chloromethylated resin or hydroxymethyl resin. The preparation of hydroxymethyl resin has been described by Bodanszky et al. Chem. Ind. (London), 3_8, 1 597-98 (1966). Chloromethylated resin is commercially available from Lnb. Systems, Inc. San Matero, California.

ö 64575

Once the C-terminal cysteine is attached to the resin, the protected cysteine is first converted to its cesium salt. This salt is then reacted with the resin according to the method described by B.F. Gisin, Helv. Chim. Acta, J36, 1476 (1,973). Alternatively, cysteine can be attached to the resin by activating the carboxyl function of the cysteine molecule using readily known techniques. For example, the cysteine can be reacted with a resin with the aid of a carboxyl group activating compound such as N, N'-dicyclohexylcarbodiimide (DCC).

After the free carboxyl-containing cysteine is suitably attached to the resin support moiety, the remainder of the peptide construction sequence comprises the periodic addition of each amino acid to the N-terminal portion of the peptide chain. Therefore, each private sequence involves cleavage of the * amino function protecting group from the amino acid representing the N-terminal portion of the peptide fragment, followed by sequential addition of the next amino acid residue to the N-terminal amino acid, which is then free and reactive. Cleavage of the o * amino function can be performed using an acid such as hydrobromic acid, hydrochloric acid, trifluoroacetic acid, n-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and acetic acid to form the corresponding acid addition salt. Another method that can be used to cleave the amino protecting group is the use of boron trifluoride. For example, boron trifluoride diethyl etherate in glacial acetic acid converts an amino-protected peptide fragment to a BFg complex, which can then be converted to an unprotected peptide fragment by treatment with a base such as aqueous potassium bicarbonate. All of these methods can be used as long as it is known that the chosen method must be able to cleave the protecting group of the N-terminal ° t-amino function without removing other protecting groups in the peptide chain. Therefore, it is most preferred that the cleavage of the N-terminal protecting group be performed using trifluoroacetic acid. Cleavage is usually performed at a temperature between 0 ° C and room temperature.

When the N-terminal cleavage is performed, the resulting compound is then normally in the form of an acid addition salt of the acid used in the cleavage of the protecting group. After that it can be changed! 9,64575 / to a compound having a free amino terminus by treatment with a dilute base, most typically a tertiary amine such as pyridine or triethylamine.

The peptide chain is then ready to react with the next amino acid in sequence. It can be done with any of the known techniques, of which there are several. When the next amino acid in sequence is attached to an N-terminal peptide chain, an amino acid having a free carboxyl but whose amino function is suitably protected as well as any of its other reactive moieties is used. The amino acid is subjected to conditions in which its carboxyl function is activated for the coupling reaction. One such activation technique that can be used in the synthesis is the conversion of an amino acid to a mixed anhydride. The free carboxyl function of the amino acid is then activated by reacting with another acid, most typically a carboxylic acid in the form of its acid chloride. Examples of such acid chlorides which can be used to form suitable mixed anhydrides include ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate and pivalyl chloride.

Another method by which the carboxyl function of an amino acid can be activated to effect a coupling reaction is to convert the amino acid to a reactive ester derivative. Examples of such reactive esters are 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, ε-nitrophenyl ester, an ester formed from 1-hydroxybenzotriazole and an ester formed from N-hydroxycyclic imide. Another method of effecting the incorporation of a C-terminal amino acid into a peptide fragment is to perform the coupling reaction in the presence of at least an equimolar amount of β, β'-dicyclohexylcarbodiimide (DCC). This latter method is preferred for the preparation of a tetradecapeptide of formula II wherein X is • - · resin

-0-QW- / '/ S

\ / χ · = · 10 64575

When the desired amino acid sequence is complete, the resulting peptide can be removed from its resin support moiety. This is done by treating the protected tetradecapeptide with a resin support moiety with hydrogen fluoride. Hydrogen fluoride treatment cleaves the peptide from the resin; however, in addition, it also cleaves all remaining protecting groups of the reactive moieties in the peptide chain, as well as the N-terminal amino acid (Xrmino-functional protecting group. When hydrogen fluoride is used to cleave the protecting groups, it is most preferred to carry out the reaction in the presence of anisole. It has also been found to inhibit the potential alkylation of some amino acid residues in the peptide chain. cleavage with hydrogen fluoride.

When the cleavage reaction is completed, 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 straight-chain tetradecapeptide under oxidative conditions in which the two sulfhydryl groups in the molecule, one in each cysteine moiety, are converted to disulfide-silia. This is accomplished by treating a dilute solution of the linear tetradecapeptide with any oxidizing agent, for example, iodine or potassium ferricyanide. Air can also be used as the oxidizing agent, with the pH of the mixture usually being 2.5 to 9.0 and most preferably about 6.2 to 7.2. When air is used as the oxidizing agent, the concentration of the peptide solution usually does not exceed about 0.4 mg peptide / ml per solution, and usually about 50 pg / ml.

The compound of formula I can be administered as a medicament to warm-blooded mammals, including humans, and in particular can be used to smooth smooth muscle.

In particular, the gas Lroin Les Linear region can be relaxed.da given 1 fi racket parenterally Li small amounts of U-Val, D-Trp -somaLos-tutin. This effect, which results in a reduction in intestinal motility, is not due to its advantageous hypogonetic gas irradiation in 11 64575 testicular X-rays. In addition, this compound can be used to treat spastic colon, pylorus spasm, and other spastic conditions of the gastrointestinal tract, as well as to treat ureter and gallbladder colic.

When smooth muscle relaxation is desired, the compound is usually administered in a dose of about 0.1 μg to 3 μg per kilogram of the recipient's body, and preferably about 3 pg to 1.5 pg per kilogram of body weight. The drug is administered parenterally and may be intramuscularly, subcutaneously or intravenously; most preferably, the compounds are administered intravenously or intramuscularly.

Parenteral unit dosage forms are usually prepared using a compound with a pharmaceutical carrier such as isotonic saline, isotonic glycine, lactose, mannitol, dilute acetic acid, bacteriostatic water, or, for example, water containing about 1% benzyl alcohol and phosphate. , as well as with suitable combinations containing any standard carrier. The carrier is usually present in an amount of about 25: 1 to 1000: 1 by weight relative to the active ingredient.

The compound, depending on the carrier and the concentration used, can either be suspended or dissolved in a suitable sterile solvent, of which water is preferred. When solutions are prepared, the compound and carrier can be dissolved in the chosen solvent, the solution is filtered and placed in a suitable small vial or ampoule, and the vial or ampoule is sealed. Excipients such as a topical anesthetic or a buffer may preferably be dissolved in the solvent. To improve stability, the compound can be dissolved in water with a carrier and the aqueous solution placed in a small vial and then lyophilized. The dry lyophilized solid is then sealed in a vial and provided with an accompanying vial containing a solvent to reconstitute the mixture before use. Parenteral suspensions may be prepared in substantially the same manner except that the compound is suspended in the carrier instead of being dissolved.

The compound of formula I is also effective, although not necessarily to the same extent, in inhibiting growth hormone isolation. This inhibitory effect is useful in cases where the subject is in need of therapeutic treatment due to excessive somatotropin secretion associated with adverse conditions such as juvenile onset diabetes and acromegaly. This compound also has other physiological effects, such as inhibition of gastric acid secretion, which is useful in the treatment of gastric ulcer; as well as inhibition of pancreatic exocrine secretion, which is potentially useful in the treatment of pancreatitis, and inhibition of insulin and glucagon secretion. This compound can be administered in a variety of ways, such as orally, sublingually, subcutaneously, intramuscularly, and intravenously. For sublingual and oral administration, the dose is most preferably about 1 mg to 100 mg / kg body weight. Intravenous, subcutaneous or intramuscular dosage in these latter indications is usually about 1 pg to 1 mg / kg body weight, and most preferably about 50 pg to 100 pg / kg body weight. It will be appreciated that the dosage may vary widely depending on the particular condition as well as the severity of the condition.

The compound of formula I may be administered orally or sublingually as a medicament together with a pharmaceutical carrier, for example in the form of tablets or capsules. Inert diluents or carriers, for example magnesium carbonate or lactose, may be used in combination with conventional disintegrants, for example maize starch and alginic acid, and lubricating agents, for example magnesium stearate. The amount of carrier or diluent is stylistically about 5-95% of the final mixture and most preferably 50-85% of the final mixture. Suitable flavoring agents may also be used in the final preparation to make the mixture taste better.

When a compound of formula I is administered parenterally, suitable carriers may be used, such as any of those described above in connection with the use of these compounds for the relaxation of smooth muscle.

i3 64575

The following examples illustrate the preparation of a compound of formula I and its intermediates.

Example 1 Nt-butyloxycarbonyl-L-cysteinyl (Sp-methoxybenzyl) -methylated polystyrene resin in 1000 ml of N, N-dimethylformamide (DMF) containing the cesium salt of Nt-butyloxycarbonyl- (Sp-methoxybenzyl) -cysteine ( prepared from 17.5 g of free acid), 100 g of chloromethylated polystyrene resin (Lab. Systems, Inc., 0.75 mmol Cl / g) was added. The mixture was stirred at room temperature for five days. The resin was then filtered and washed alternately three times with a mixture of 85% DMF and 15% water and DMF alone, and then twice with DMF. The resin was suspended in 1000 ml of DMF and a solution of 16 g (83.4 mmol) of cesium acetate was added. The mixture was stirred for 9 days at room temperature. The resin was then filtered and washed alternately three times with a mixture of 85% DMF and 15% water and DMF alone. The resin was washed with CHCl 3 and then suspended four times in CHCl 3 in a separatory funnel and the liquid was withdrawn each time to remove fine particles. The resin was filtered, washed with 95% ethanol and then alternately three times with benzene and 95% ethanol. The Ijtrt was then dried in vacuo at 30 ° C to give 115.3 g of the title compound. Amino acid analysis showed 0.254 mmol Cys / g per resin. Cysteine was determined as cysteic acid by acid hydrolysis performed using a 1: 1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethyl sulfoxide had been added.

i4 6 4 5 7 5

Example 2 Nt-butyloxycarbonyl-D-valyl-glycyl-L- (S-? -Methoxy Benzyl) cysteinyl---L- (N * -o-chlorobenzyloxycarbonyl) -lysyl-L-asparaginyl-L- phenylalanyl-L-phenyl and methyl-D-tryptophyl-L-CN (-o-chlorobenzyloxycarbonyl) lysyl-L- (O-benzyl) threonyl-L-phenylalanyl-L- (O-benzyl) threonyl- L- (O-Benzyl) seryl-L- (S-? -Methoxybenzyl) cysteinyl methylated polystyrene resin

The product obtained in Example 1 (5.0 g) was placed in a Beckman reaction vessel 990, an automatic peptide synthesizer, and twelve of the remaining thirteen amino acids were added using an automatic synthesizer. The resulting protected tridecapeptide resin was divided into two similar portions and the final residue was placed in one of these portions. Amino acids were used according to the following sequence of use: (1) N-t-butyloxycarbonyl- (O-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, N-t-butyloxycarbonyl-N, N-chlorobenzyloxycarbonyl-L-lysine; (6) N-t-butyloxycarbonyl-D-tryptophan; (7) N-t-butyloxycarbonyl-L-phenylalanine; (8) N-t-butyloxycarbonyl-L-phenylalanine; (9) N-t-butyloxycarbonyl-L-asparagine, E-nitrophenyl ester; (10) NA-t-butyloxycarbonyl-N, -o-chlorobenzyloxycarbonyl-L-lysime; (11) N-t-butyloxycarbonyl- (S-E-methoxybenzyl) -L-cysteine; (12) N-t-butyloxycarbonylglycine; and (13) N-t-butyloxycarbonyl-D-valine. The sequence of deprotection, neutralization, attachment, and reattachment, according to which each amino acid is attached to the peptide, is as follows: 1) three washes (10 ml / g of resin) for three minutes, each performed with chloroform; 2) Removal of the BOC group by treatment twice for 20 minutes, each time using (10 ml / g resin) a mixture of 29% trifluoroacetic acid, 48% chloroform, 6% triethylsilane and 17% methylene chloride; 3) two washes (10 ml / g resin) for three minutes, each with chloroform; 4) one wash (10 ml / g resin) for three cm minutes of methylene chloride I1a; 5) washings (10 ml / g resin) for three minutes, each using a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol; 6) three washes (10 ml / g resin) for three minutes, each with methylene chloride; 7) neutralization by three treatments, for three minutes each and using (10 ml / g resin) a mixture of 3% triethylamine in methylene chloride; 8) three washes (10 ml / g resin), each for three minutes, with methylene chloride; 9) Three washes (10 ml / g resin), each for three minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol; 10) three washes (10 ml / g resin) for three minutes each with methylene chloride; 11) 1.0 mmol / g of resin protected amino acid and 1.0 mmol / g of resin N, N'-dicyclohexylcarbodiimide (DCC) in 10 ml / g of resin methylene chloride are added, followed by stirring for 120 minutes ; 12) three washes (10 ml / g resin), each for three minutes with methylene chloride; 13) three washes (10 ml / g resin) for three minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol; 14) three washes (10 ml / g resin) for three minutes each, with methylene chloride; 15) neutralization by three treatments, each for three minutes, using 10 ml / g of resin in 3% triethylamine in methylene chloride; 16) three washes (10 ml / g resin) for three minutes, each with methylene chloride; 17) three washes (10 ml / g resin) for three minutes, each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol; 18) three washes (10 ml / g resin) for three minutes, each with methylene chloride; 19) three washes (10 ml / g resin) for three minutes, each with DMF; 20) 1.0 mmol / g of resin protected amino acid and 1.0 mmol / g of resin n "n'-dicyclohexylcarbodiimide (DCC) in 10 ml / g of resin are added in a 1: 1 mixture of DMF, and methylene chloride followed by stirring for 120 minutes, 21) three washes (10 ml / g resin), each for three minutes with DMF, 22) three washes (10 ml / g resin), each for three minutes with methylene chloride, 23) three washing (10 ml / g resin), each for three minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol, 24) three washes (10 ml / g resin) for three minutes each with methylene chloride, 25) neutralization with three treatment, each for three minutes using 10 ml / g of resin in a mixture of 3% ie 64575 triethylamine in methylene chloride; 26) three washes (10 ml / g of resin), each for three minutes with methylene chloride; 27) three washes (10 ml / g of resin); g of resin), each for three minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol, and 28) k we are washing (10 ml / g resin), each for three minutes with methylene chloride.

The above sequence treatment was used to add every amino acid except glycine and asparagine residues. Glycine addition was performed using steps 1-18 only. The asparagine residue was added via its active ε-nitrophenyl ester. Thus, step 11) above was converted to the following 3-step sequence: a) three washes (10 ml / g resin), each for three minutes with DMF; b) 1.0 mmol / g of resin N-t-butyloxycarbonyl-L-asparagine p-nitrophenyl ester in 10 ml / g of resin was added in a 1: 3 mixture of DMF and methylene chloride, followed by stirring for 720 minutes; and c) three washes (10 ml / g resin), each for three minutes using DMF. Similarly, step 20) above was modified to use N-t-butyloxycarbonyl-L-asparagine E-nitrophenyl ester in a 3: 1 mixture of DMF and methylene chloride, followed by stirring for 720 minutes.

'. The finished peptide-resin was dried in vacuo. The product was hydrolyzed under vertical condenser conditions for 72 hours in a 1: 1 mixture of concentrated hydrochloric acid and dioxane. Amino acid analysis of the resulting compound gave the following results: Asn 1.00; 2 Thr 2.18; Ser 0.95; Gly 1.00; Or 0.99; 3 Phe 3.45; 2 Lys 2.00.

Example 3 D-Valyl-glycyl-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 7.2 ml of anisole and 7.2 ml of ethyl 1 imercaptan was added 3.914 g (substitution level 0.155 mmol / g) of the protected tetradecapeptide resin of Example 2. The mixture was cooled in liquid nitrogen, and 80 ml of liquid hydrogen fluoride was added by distillation. The resulting mixture was allowed to warm to 0 ° C and stirred for 17 hours. Hydrogen fluoride was then removed by distillation. Ether was added to the residue and the mixture was cooled to 0 ° C.

The resulting precipitate was collected by filtration and washed with ether.

The product was dried and the unprotected tetradecapeptide was extracted from the resin mixture using 1 M acetic acid and 50% acetic acid. The acetic acid solution was then immediately lyophilized to dryness in the dark.

The resulting pale yellow precipitate was suspended in a mixture of 10 ml of 0.2 M degassed acetic acid and 4 ml of glacial acetic acid. The resulting suspension was heated slightly with 6 mL of 50% acetic acid until a clear yellow solution formed and this solution was applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent, 0.2 M degassed acetic acid; column size 7.5 -150 cm; temperature 26 ° C; flow rate 1,670 ml / h; fraction volume 25.05 ml.

The absorbance of each fraction at 280 nm showed one broad peak per fraction number, followed by a shoulder. U.S. spectroscopy showed that the majority of the peak was product. The fractions that were pooled and their flow rates are as follows:

Fractions 207-233 (5160-5837 ml, peak = 5515 ml).

These combined fractions did not include the back of the shoulder. UV spectroscopy showed that 470 mg of product was present, (yield = 46.4%). Ellman titration showed that the amount of free sulfhvJ groups was 95% of theory.

Example 4 1 8

Oxidation to D-Val, D-Trp-somatostatin 1 8

A solution of reduced D-Val, D-Trp somatostatin (677 ml) from Example 3 was diluted with distilled water to a concentration of 50 pg / ml. Concentrated ammonium hydroxide was added to adjust the pH of the mixture to 6.7. The solution was stirred at 4 ° C in the dark for 64 hours, after which time Ellman titration indicated that the oxidation was complete.

The mixture was concentrated in vacuo to a volume of 45 mL, and 45 mL of glacial acetic acid was added. The mixture was then absorbed onto a Sephadex G-25 F column. The chromatographic conditions were as follows: solvent of 50% degassed acetic acid, column size 5, ϋ x 215 cm, temperature 66 ° 0, flow rate 148 ml / h, fraction volume 17.3 m].

18 64 575

Absorption at 280 mm for each fraction number showed two broad peaks. The first peak represented aggregated forms of the compound and the second peak represented a monomeric compound. The material from the second peak was recovered / fractions 116-155 (2,000-2,685 mL) _7. UV spectroscopy showed that the sample contained 279 mg of compound (yield = 59, * +%). The solution was lyophilized to dryness in the dark.

The resulting white solid was rechromatographed in two portions of approximately the same size. The first portion was dissolved in 25 mL of 50% degassed acetic acid and absorbed onto a Sephadex G-25 F column. The chromatographic conditions were: a solution of 50% degassed acetic acid, column size 5.0 x 215 cm, temperature 26 ° C, flow rate 148 ml / h, fraction volume 17.3 ml.

Absorption at 280 nm for each fraction number showed two broad peaks. A conservative division was made from the second peak. Fractions 119-125 (flow volumes 2,128-2,256 ml) were combined. UV spectroscopy showed that this sample contained 65.3 mg of compound. The solution was lyophilized to dryness in the dark to give the desired compound.

The second portion was chromatographed in the same manner as the first and with the same results. The two correct products were combined to give a total of 125 mg by UV spectroscopy (45.2% yield of pure product). The combined product was dissolved in 15 mL of 0.2 M degassed acetic acid and applied to a Sephadex G-25 F column. The chromatographic conditions were: solvent 0.2 M degassed acetic acid, column size 5.0 x 150 cm, temperature 26 ° C, flow rate 466 ml / h, fraction volume 16.3 ml.

Absorption at 280 nm for each fraction number showed one broad peak. UV spectroscopy showed that most of the peak was an excellent product. Fractions 160-180 (2,592-2,934 ml, peak = 2,685 ml) were combined and lyophilized to dryness in the dark. UV spectroscopy showed that the product was 90.4 mg (71.7% yield).

Optical rotation δ 6 6 - -56.1 ° (1% acetic acid).

Amino acid analysis: Val 1.0; Gly 0.97; 2 Cys 1.62; 2 Lys 2.00; Asn 1.01; 3 Phe 2.87; Trp 1.02; 2 Thr 1.83; Ser 0.81.

is 64 57 5

The above results are expressed as Lys / 2 = 1.0.

All values are averages of two 21-hour hydrolyses without scavengers. Tryptophan was determined by UV spectroscopy (relative to Lys / 2), serine value was not corrected for losses during hydrolysis.

The above product contains minor impurities. If desired, the product can be further purified by subjecting it to a preparative high pressure liquid chromatography (HPLC) apparatus.

1 8

An alternative method for oxidizing reduced D-Val, D-Trp soma-X 8 'tostatin to D-Val, D-Trp somatostatin is

treatment with potassium ferricyanide. The oxidation is carried out in aqueous solution

with a pH adjusted to 6.7 as described above in this example. An aqueous solution of potassium ferricyanide is added to the mixture to give a final concentration representing approximately 3.3 times the concentration of reduced D-Val, D-Trp somatostatin. The solution is stirred in the dark at room temperature for about two hours. The completeness of the oxidation can be determined by Ellman titration.

X 8 D-Val, D-Trp somatostatin was tested in dogs for its in vivo inhibitory effect on gastric acid secretion. Six dogs with chronic fistula and Heidenhainpussi induced gastric HCl secretion by infusing gastrin C-terminal tetrapeptide at 0.5 pg / kg / h. Each dog was under its own control. After continuous constant secretion of HCl for one hour, D-Val, D-Trp somatostatin was infused for one hour at 0.15 pg / kg / h. The collection of gastric acid samples was continued for another 1.5 hours at 15 minute intervals. The samples were titrated to pH 7 with an auto-X 8 automatic titrator. The maximal inhibitory effect of D-Val-D-Trp somatostatin was extrapolated from the somatostatin dose-response curve, and the relative efficacy of the analog compound relative to the efficacy of somatostatin is expressed as a percentage effect.

X 8 D-Val, D-Trp somatostatin inhibited steady state acid secretion induced by gastrin C-terminal tetrapeptide by approximately 48.22 ± 6.45% (standard mean error).

This effect is equivalent to 0.175 ug / kg / h of somatostatin.

Thus, its effect is proportional to the effect of somatostatin J16%.

20 64575 1 8 A more fully purified sample of D-Val, D-Trp somatostatin administered at doses of 0.200, 0.156 and 0.138 pg / kg / h prevented continuous steady state acid isolation induced by gastrin C-terminal tetrapeptide, 77.63 , 71.57 and 67.8%. This represents an effect of 302-325% relative to the effect of somatostatin.

18 D-Val, D-Trp somatostatin was also tested for its effect on intestinal motility in conscious dogs. Three dogs with intraluminal catheters placed in the antrum, duodenum, and pylorus were used in the experiments. Pressure fluctuations in the lumen of the gut were recorded Visicorder device jännitysmittaimen BY, and a miniature light beam galvanometers. After a steady state check, the test compound was infused intravenously for 10 minutes. Initially, the test compound increased the intraluminal pressure in the pylorus and then decreased it, while the pressure in the duodenum and antrum remained low throughout the test. The minimum effective amount required to increase pylorus pressure and lower duodenal and antrum pressures is about 0.05 ng / kg to 10 min for 18 r D-Val, D-Trp somatostatin. This corresponds to the amount of somatostatin itself 0.125 to 0.25 ug / kg to 10 min.

18 D-Val, D-Trp somatostatin was also tested for its effect on growth hormone secretion. The procedure used was performed on standard Spraque-Dawley male rats weighing 100-120 g (laboratory Supply Company, Indianapolis, Indiana). The test is a modification of the method described by P. Brazil, W. Wale and R. Guillman, Endocrinology, 9_4 184 (1974). A total of five groups, eight rats each, were used in this experiment to test each compound. Sodium pentobarbital was administered intraperitoneally to all rats to stimulate growth hormone secretion. One group served as a control and received saline only. Two groups received somatostatin, one 2 pg / rat subcutaneously, and the other group SU 2 g / rat subcutaneously. The other two groups received the test compound, one 10 ug / rat subcutaneously and the other 0.4 pg / rat subcutaneously. Serum growth hormone concentrations were measured 20 min after simultaneous administration of sodium pentobarbital and test compound. The degree of inhibition of serum growth hormone concentration relative to the control group and the Lesti Compound and test compound are determined. The degree of inhibition of serum growth hormone concentration was determined relative to the control group and the relative efficacy of the test compound and somatostatin itself was compared.

At a dose level of 0.4 hg / rat and 10 μg / rat, D-Val1,8 * D-Trp somatostatin inhibited the growth of growth hormone secretion by 14%, and 42% over the control group. Somatostatin, at a dose level of 2 μg / rat, had no effect on the increase in growth hormone secretion, whereas at a dose of 50 μg / rat, it inhibited the increase in growth hormone secretion by 56% over the control.

1 o '

At a dose level of 0.4 μg / rat and 10 μg / rat D-Ala, D-Trp somatostatin inhibited the increase in growth hormone secretion by 54% and 91% over the control group. Somatostatin at the 2 pg / rat and 50 pg / rat dose levels inhibited the increase in growth hormone secretion by 40% and 87% over the control group.

Ί Ö

The efficacy of D-Val, D-Trp somatostatin to inhibit glucagon and insulin secretion in vivo after stimulation with L-alanine was tested. Normal, mixed-breed dogs of both sexes were allowed to infuse saline, somatostatin, or test compound. After 30 minutes, additional L-alanine was administered intravenously for 15 minutes. The infusion of saline, somatostatin or test compound was continued 15 minutes after L-alanine administration. Infusion of L-alanine caused a sudden increase in serum glucagon and insulin concentrations, which returned to control concentrations when L-alanine irradiation was complete. From the above, a minimum dose of 0.04 to 0.11 μg / kg / min for D-Val, D-Trp somatostatin, which inhibits glucagon secretion, and an insulin secretion inhibiting dose of less than 0.004 μg / kg / min were determined.

Pharmacological comparative tests

Somatostatin was used as a control. The D-Val, D-Trp somatostatin of the present invention-X 8 had the following activities compared to somatostatin:

Gastric acid secretion (dog, in vivo) 116% and more with purified sample 302-325%. Intestinal motility (dogs, in vivo), the minimal effective dose of 0.05 g / kg - compared to the value obtained for 10 minutes with somatostatin 0.125 - 0.25 / kg - 10 min. Inhibition of growth hormone release i ιητι (thigh) 0.4 pg / rat - 14% growth hormone secretion growth inhibition, 1 () up / rot la = 47% growth of hormone hormone