KR810000692B1 - Process for preparing somatostain analogs - Google Patents

Abstract

Somatostatin analogs(I) were prepd. by oxidn. of, III. Thus, Me3CO2C-Ala-Gly-Cys-(CH2C6H4OMe-P)-Lys(Ecl-2)-Asn-Phe-Trp-Lys(Ecl-2)-Thr(CH2Ph)-D-Phe-Thr(CH2Ph)-Ser(CH2PH)-Cys(CH2 C6H4O-Me-P)-OCH2Q(ZCl-2=CO2CH2C6H4Cl-2) was prepd. by the solid-phase method and treated with HF to give the corresponding unprotected linear tetradecapeptide which was oxidized to give I(X = D-Phe) (II). II at 50 μg inhibited the increase in serum GH of rats by 70 %.

Description

Method for preparing somatostatin homologue

The present invention relates to a method for preparing tetra decapeptides of the following structural formula (I).

Somatostatin (known as somatostatin releasing inhibitor) is a tetra decapeptide having the structure

It is known that such tetra decapeptides isolate from the hypothalamic extract of sheep and are active in inhibiting the secretion of growth hormone (GH), also known as somatostatin.

See P. Brazeau. W. Vale, R. Burhus, N. Ling, M. Butcher, J Rivier, and R. Guillemin, Science, 179, 77 (1973)], except that U.S. Patent No. 3,904,594 describes natural hormone 3-14 as well as natural somatostatin. Other two classes of compounds with dodeca peptides labeled by position are described.

Moreover, compounds called as D-Ala ¹ -somatostatin have already been described in the literature.

(See Ferland et al., Molecular and Cellular Endcorinology, 79 88-88 (1976).)

의 활성을 나타낸다. 본 발명의 화합물인 D-Val¹-소마토스타틴은 2개의 수소가 메틸그룹에 의해서 치환된 D-Ala¹-소마토스타틴과는 다르다. D-Vla¹-소마토스타틴의 약리적 활성에 관한 몇 가지를 제외하고는 이의 효과는 D-Ala¹-소마토스타틴과 유사하다. 이와 같이, 이것은 생체내에서 위산 분비 저해물질이 천연 호르몬보다도 효과가 적었다.Although the structural stereoisomer of natural L-Ala ¹ -somatostatin, D-Ala ¹ -somatostatin is a natural somatostatin that inhibits gastric acid secretion in vivo,

Indicates activity. The compound of the present invention, D-Val ¹ -somatostatin, differs from D-Ala ¹ -somatostatin in which two hydrogens are substituted by methyl groups. Except for a few things regarding the pharmacological activity of D-Vla ¹ -somatostatin, its effect is similar to that of D-Ala ¹ -somatostatin. As such, it was found that gastric acid secretion inhibitors were less effective than natural hormones in vivo.

These results indicated that D-Val ¹ -somatostatin had surprising activity when compared with conventionally similar compounds.

The physiologically active tetradecapeptides of formula (I) include nontoxic acid salts thereof. Its structure is different from the structural formula of somatostatin substituted with D-varin at position 1 instead of L-alanine. For convenience, the tetra decapeptide of formula (I) may be referred to as D-Val ¹ -somatostatin.

및 이의 약학적으로 가능한 무독성 산부가염, 및 중간물질로서 다음 구조식(II)의 화합물을 제조하는 방법을 관한 것이다.As such, the present invention is a structural formula

And pharmaceutically possible non-toxic acid addition salts thereof, and methods of preparing compounds of formula (II) as intermediates.

이다.Wherein R is hydrogen or α-amino arc, R ¹ is hydrogen or thioprotecting group, R ₂ is hydrogen or ε-amino protecting group, R ₃ and R ₄ are hydrogen or hydroxy protecting group, R ₅ is hydrogen Or formyl, X is hydroxy or

to be.

Here, the resin is polystyrene.

경우에는 R, R₁, R₂, R₃, 및 R₄는 수소 이외의 것이다.Provided that when X is hydroxy, R, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each hydrogen and X is

In the case, R, R ₁ , R ₂ , R ₃ , and R ₄ are other than hydrogen.

Structural formula (I)

The novel tetradecapeptides of the formula (III) are HD-Val-Gly-L-Cys-L-Asn-L-Phe-L-Phe-L-Trp-L-Lys-L-Thr-L-Phe-L The corresponding straight chain tetradecapeptides of -Thr-L-Ser-L-Cys-OH are prepared by reaction with an oxidizing agent. The reaction is the conversion of two sulfhydryl groups to disulfide bridges.

Pharmaceutically possible non-toxic acid addition salts include organic and inorganic acid addition salts such as hydrochloric acid, sulfuric acid, sulfonic acid, taric acid, fumaric acid, hydrobromic acid, glycolic acid, citric acid, maleic acid, phosphoric acid, succinic acid, acetic acid, Addition salts prepared from nitric acid, benzoic acid ascorbic acid, P-toluene sulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and propionic acid. Preferably the acid addition salt is prepared from acetic acid. These salts are prepared by conventional methods.

RD-Val-Gly-L-Cys (R ₁ ) -L-Lys (R ₂ ) -L-Asn-L-Phe-L-Phe-L-Trp (R ₅ ), an intermediate of formula (II) -L-Lys (R ₂ ) -L-Thr (R ₃ ) -L-Phe-L-Thr (R ₃ ) -L-Ler (R ₄ ) -L-Cys (R ₁ ) -X Belongs to the range.

Preferred intermediates are as follows:

Intermediates of the above formulas contain protecting groups for amino, hydroxy and thio (sulfohydryl) functional groups. The protecting group has two properties. First, the protecting group prevents the reaction residues of the molecular sieve from reacting under conditions that cause the cleavage of other reaction residues. Second, the protecting group can be easily removed by recovering the original reactive electricity under reaction conditions that do not unnecessarily affect other parts of the molecular sieve. Groups useful for this purpose, ie protecting amino, hydroxy and thio groups, are known to those skilled in the art.

Several documents describe how to use such groups. One example is Protective Groups in Organic Chemistry J.F.W. McOmie, Editor, Plenum Press, New York, 1973.

In the above structure defined as intermediate, R represents an α-amino hydrogen or an α-amino protecting group. α-amino fraction R is known to those skilled in the art. Many of these are Jay. W., Barton's Mac Omie, described in detail in Chapter 2.

Such protectors include the following.

Benzyloxycarbonyl, P-chlorobenzyloxycarbonyl, P-bromobenzyloxycarbonyl, O-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, O-bromobenzyloxycarbonyl, P-methoxybenzyloxycarbonyl, P-nitrobenzyloxycarbonyl, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, 2- (P-biphenyl Reel) isopropyloxycarbonyl (B9OC), adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl, triphenylmethyl (trityl) and P-toluenesulfonyl.

The α-aminoprotecting group defined as R is preferably t-butyloxycarbonyl.

R ¹ represents a protecting group for the hydrogen or sulfhydryl substituent of the sulfhydryl group of cysteine. Protectors like these are. G. Huh? buoy. are. Lao and W. G. Mac Omie of Rhodes, described in Chapter 7.

Such protecting groups include P-methoxybenzyl, benzyl, P-tolyl, benzhydryl, acetamidoethyl, trityl, P-nitrobenzyl, t-butal, isobutyloxymethyl, as well as most trityl derivatives Etc. Such additional groups are described in the following references. See, Houben-Weyl, Methodes der Organischen Chemie, Synthesevon Peptiden, Vols, 15/1 and 15/12 (1974), Stuttgart, Germany.

Preferably the sulfhydryl protecting group defined by R ¹ is P-methoxy benzyl.

R ₂ represents hydrogen of the ε-amino functional group of the lysine residue or a conjugated ε-amino protecting group. Such groups are described in the literature mentioned in the α-amino protecting groups. Examples of such groups are as follows.

Benzyloxycarbonyl, t-butyloxycarbonyl, t-amyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, P-methoxybenzyloxycarbonyl, P-clotobenzyloxycarbonyl, P-bromobenzyloxycarbonyl, O-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, O-bromobenzyloxycarbonyl, P-nitrobenzyl Oxycarbonyl, isopropyloxycarbonyl, cyclohexyloxycarbononyl, cycloheptyloxycarbonyl and P-toluenesulfonyl.

The method for preparing tetra decapeptides of formula (I) includes the periodic cleavage of α-aminoprotecting groups from terminal amino acids present in the peptide chain. As such, the only restriction on the ε-amino protecting group on the lysine residue is that it should not cleave under the conditions used to selectively cleave the α-amino protecting group. Appropriate choices of α-amino and ε-aminoprotecting groups are well known to peptide chemists of ordinary skill in the art and depend on the cleavage of certain protecting groups. Groups such as 2- (P-biphenyl) isopropyloxycarbonyl (B9OC) and trityl are very unstable and can cleave in the presence of a mild acid. Mild strong acids, such as hydrochloric acid, trifluoroacetic acid, or boron trifluorite in acetic acid, are known as t-butyloxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl and P-methoxybenzyloxycarbonyl It is necessary to split other groups.

Stronger acid conditions are required to cleave other protecting groups such as benzyloxycarbonyl, halobenzyloxycarbonyl, P-nitrobenzyloxycarbonyl, cycloalkoxycarbonyl and isopropyloxycarbonyl. Cleavage of these latter groups requires extreme acidic conditions such as hydrobromic acid, hydrofluoric acid or boron trifluoro acetate in trifluoroacetic acid. Of course, many unstable groups can split under strong acid conditions. Appropriate Selection of Such Amino Groups Use of an α-amino functional group, which is more unstable, as an ε-amino protecting group under cleavage conditions intended to selectively remove only an α-amino functional group. In the present specification, R ₂ is preferably 0-chlorobenzyloxycarbonyl or cyclopentyloxycarbonyl, and in this connection, an α-amino protecting group for use in each amino acid added to the peptide is t-butyloxycarbonyl. desirable.

The R ₃ and R ₄ groups represent hydroxyl groups or protecting groups for alcoholic hydroxyls of threonine and serine, respectively. The majority of these protection groups are described in Chapter 3 of Macomie of Seeds, Bey and Resaries. Such typical protecting groups include, for example, C ₁ -C ₄ -alkyl such as methyl, ethyl and t-butyl; P-methoxybenzyl; Substituted benzyl, such as P-nitrobenzyl, P-chlorobenzyl and 0-chlorobenzyl; C ₁ -C ₃ alkanoyl such as formyl, acetyl and propionyl; Triphenylmethyl (trityl) and the like. In the case of R ₃ and R ₄ protecting groups, it is preferable to select benzyl as the protecting group in both cases.

R ₅ group represents hydrogen or formyl and is defined as> NR ₅ , which is a tryptophan residue. The use of such protecting groups is optional, and therefore R ₅ is appropriately hydrogen (N-unprotected) or formyl (N-unprotected).

Group X represents the terminal carboxyl of the tetradecapeptide chain, which may be hydroxyl, in which case it is defined as the free carboxyl group. In addition, X represents the linked solid resin of the carboxyl of the peptide terminal. This solid resin can be represented by the following structural formula.

In the above, when X is hydroxyl, R, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each hydrogen.

R, R ₁ , R ₂ , R ₃ , and R ₄ represent a protecting group when X is a solid resin support.

The following abbreviations are mostly well known and commonly used in the art.

Ala-Alanine Lys-Lysine

Asn-asparagine Phe-phenylalanine

Cys-cysteine Ser-serine

Gly-Glycine Thr-Threonine

Trp-Tryptophan CBzOC-o-chlorobenzyloxycarbonyl

Val-varin CPOC-cyclopentyloxycarbonyl

DCC-N, N'-dicyclohexylcarbodiimide BzL-benzyl

DMF-N, N'-Dimethylformamide For-Formyl

BOC-t-butoxycarbonyl

BpOC-2- (p-biphenylyl) isopropyloxycarbonyl

PMB-p-methoxybenzyl

The choice of a particular protecting group used to prepare the compound of formula (I) is well known to those skilled in synthetic peptide chemistry, but the series of reactions performed depends on the choice of a particular protecting group. In other words, the choice of protecting group should be chosen that is stable under the conditions of the reactants or the reaction.

For example, as mentioned above, the specific protecting group used should be inert under the conditions used to cleave the α-amino protecting group of the terminal amino acid residues of the peptide to couple the amino acid to the peptide chain. It is important to select a protecting group that does not affect the peptide chain and is easily removed once the synthesis of the desired tetradeca peptide product is complete. All such problems are well known to peptide chemists of ordinary skill in the art.

As described above, the tetradeca peptide of formula (I) can be prepared by solid phase synthesis. This synthesis involves making a peptide chain starting at the C-terminus of the peptide. In particular, cysteine is first linked to the resin by reacting an amino-protected, S-protected cysteine with a chloromethylated resin or hydroxy methyl resin at its carboxyl function. The preparation of hydroxy methyl resins is described in the references. See, Bodanszky te al., Chem. Ind (London), 38 1587-98 (1966).] Chloromethylated resins are very useful commercially. [See, Lab Systems, Inc., San Mateo, California.] The protected cysteine is first converted to its hereditary salt in order to link the C-terminal cysteine to the resin. This salt is then reacted with the well by the method described in the references. [See, B.F. Gisin, Helv. Chim. Acta 56, 1476 (1973).] Optionally, cysteine can be bound to the resin by activating the carboxyl functionality of the cysteine molecular sieve by conventional methods. For example, cysteine can be reacted with a resin in the presence of a carboxyl group activating compound such as N, N'-dicyclohexyl carbodiimide (DCC).

Once the free carboxyl cysteine is properly linked to the resin support, each amino acid is added stepwise to the N-terminal peptide chain to generate a peptide. As such, the specific sequencing involved involves cleaving the α-amino protecting group from the amino acid represented by the N-terminal peptide and subsequently coupling another amino acid to the free and reaction line N-terminal amino acid. Cleavage of the α-amino protecting group is performed by forming respective acid addition salts in the presence of hydrobromic acid, hydrochloric acid, trifluoroacetic acid, p-toluene sulfoic acid, benzenesulfonic acid, naphthalenesulfonic acid and acetic acid. Another method of removing amino protecting groups is boron trifluoride. For example, boron trifluoride diethyl etherate in glacial acetic acid converts an amino-protected peptide fraction to a BF ₃ complex and treats the foreign substance with a base such as aqueous potassium bicarbonate solution to convert to a deprotected peptide fraction. As long as only the N-terminal α-amino protecting group can be removed without damaging other protecting groups present in the peptide chain, any of the above methods may be performed. In this regard, a method of removing the N-terminal protecting group using trifluoro acetic acid is preferable. Generally this reaction is carried out in the range of 0 ° C to room temperature.

After the N-terminus removal operation is completed, the products obtained are generally obtained in the form of acid addition salts, which are then converted to the free terminal amino compounds by treatment with tertiary bases such as weak bases, specialpyridine, triethyl amine and the like.

The peptide can now react with the following amino acids. This can be done by introducing a variety of certified technologies. In order to couple subsequent amino acids to the N-terminal peptide chain, amino acids with free carboxyl groups and protected with α-amino groups and other active groups are used. This amino acid is subjected to the conditions under which the carboxyl group is active in the coupling reaction. Such activation techniques introduced in this synthesis include methods for converting amino acids into mixed anhydrides. In this way, the free carboxyl groups of the amino acids are activated by reaction with other acids (especially carbonic acid in acid chloride form). Examples of acid chlorides that form suitable mixed anhydrides that are available are chloroformate, phenylcroloformate, secondary-butyl chloroformate, isobutyl chloroformate, and pivaloyl chloride.

Another method of activating the carboxyl group of an amino acid in order to carry out the coupling is to convert the amino acid into an active esdero derivative. Examples of such active esters are 2,4,5-trichlorophenyl esters, pentachlorophenyl esters, P-nitrophenyl esters, esters of 1-hydroxy benzotriazole, esters of N-hydroxy succinimide. Another method of coupling the C-terminal amino acid to the peptide fractions is a coupling reaction in the presence of equimolar amounts of N, N'-dicyclohexylcarbodiimide (DCC). The latter method is better for preparing tetradecapeptides of formula (II).

Once the desired amino acid linkage is obtained, the obtained peptide can be removed from the resin support. This is done by treating the resin supported tetradecapeptides to be protected with hydrogen fluoride. Treatment with hydrogen fluoride releases the peptide from the resin, but it also removes the α-amino protecting group of the N-terminal amino acid as well as the remaining protecting groups present in the active groups on the peptide chain. The reaction is carried out in the presence of anisole when removing the protecting group with hydrogen fluoride and cleaving the peptide from the resin. It has been found that addition of anisole can prevent alkylation of amino acids present in the peptide chain. Or the cleavage reaction is preferably carried out in the presence of ethyl mercaptan. Ethyl mercaptan protects the indole ring of the tryptophan group and promotes the conversion of protected cysteines to thiol form. In addition, when R ₃ is formyl, ethyl mercaptan promotes hydrogen fluoride cleavage of the formyl group.

The product obtained by completion of the cleavage reaction is a straight chain peptide containing 14 amino acid residues. In order to obtain the product of formula (I), it is necessary to convert two sulfhydryl groups, cysteinyl moieties, to the disulfide bridge in the cysteinyl moiety under a condition affecting its oxidation, and to treat it with a straight tetradecapeptide. Do. This can be done by treating a thin solution of the linear tetradecapeptide with various oxidants such as, for example, iodine and potassium ferricyanide. Air can be used as the oxidant and the pH of the mixture is generally about 2.5 to 9.0, preferably about 7.0 to 7.6. When air is used as the oxidant, the concentration of the peptide solution is generally about 0.4 mg of peptide per ml of solution and is generally about 50 mg / ml.

Compounds of the structural formula can be administered to warm-blooded animals, including the human body, in several ways, such as oral, sublingual, subcutaneous, intramuscular, intravenous and other suitable methods of administration. Each of these compounds, although not to the same extent, is active in inhibiting the release of growth hormone. This inhibitory effect is to treat excess secretion of somatotropin, which causes juvenile diabetes and acromegaly. The compound also has other physiological effects, which inhibit gastric acid secretion and are effective in treating ulcers, inhibiting exocrine secretion of the colon and treating colitis, inhibiting the secretion of insulin and glucagon, reducing gastrointestinal motility, Used in the treatment of radiation medicine. Preferably, the daily dose of sublingual or oral administration is about 1 mg to 100 mg / kg. In general, the dose of intravenous, subcutaneous or intramuscular administration is between 10 mg and 1 mg / kg and in particular between 50 mg and 100 mg / kg. The range of dosage will vary depending on the severity of the condition or condition being treated.

The compound of formula (I) may be mixed with a pharmaceutical carrier to administer, for example, as a tablet or capsule. Magnesium carbonate or lactose, which is an inert diluent or carrier, uses a common disintegrating agent, ie, corn starch and arginine acid or glidant, ie, magnesium stearate. The amount of carrier or diluent is 5 to 95% and particularly 50 to 85% of the final composition. Suitable fragrances are used in the final preparation to suit the mouth upon administration.

Suitable carriers, such as isotonic saline and phosphate buffers, are used for intravenous administration of the compound of formula (I).

The following example illustrates the preparation of the compound of formula (I) and its intermediates.

Example 1

N-t-butyloxycarbonyl-L-cysteinyl (S-P-methoxybenzyl) methylated polystyrene resin

Chlorochlorine in 500 ml of N, N-dimethyl formamide (DMF) containing cesium salt of Nt-butyloxycarbonyl- (SP-methoxybenzyl) cysteine (prepared from 9.06 g (26.5 mmol) of free acid) Methylated polystyrene resins {Labsystem. Inc., 0.75 mmol / gram) is added 51.0 g. The mixture is stirred for 6 days at room temperature. The resin is then filtered and subsequently washed three times with 90% DMF and 10% water, three times with 95% ethanol and three times with DMF. To a resin suspended in 500 ml of DMF was added a 10.5 g solution of cesium acetate. The mixture is stirred for 6 days at room temperature. The resin is then filtered and washed successively once with DMF, three times with a mixture of 90% DMF and 10% water, three times with 95% ethanol, three times with methylene chloride, three times with 95% ethanol and three times with chloroform. . Fine particles are removed by suspending the resin four times in chloroform and each time along the liquid. The resin is then dried at 40 ° C. under vacuum overnight to give 44.8 g of the title product. Amino acid analysis revealed 0.25 mmol of cysteine per gram of resin. This cysteine is defined as cysteine acid obtained by carrying out acid hydrolysis using a 1: 1 mixture of dioxane and concentrated hydrochloric acid with a small amount of dimethyl sulfoxide.

Example 2

Nt-Butyloxycarbonyl-D-baryl-glycyl-L- (RP-methoxy-benzyl) cysteinyl-L- (N ^E -O-chloro benzyloxycarbonyl) -lysyl-L-asparaginyl -L- phenyl Ara carbonyl -L- phenyl Ara carbonyl -L- (formyl) - tryptophyl -L- (N ^E -O--chloro-benzyloxycarbonyl) - Rai chamber -L- (O- benzyl) Tre O'Neal -L- (phenylalanyl) -L- (O-benzyl) threonyl-L- (O-benzyl) seryl-L- (SP-methoxybenzyl) cysteinylyl methylated polystyrene resin.

The product of Example 1 (7.0 g) was placed in a reaction vessel, a Beckman 990 automated peptide synthesizer, and the remaining 12 of the 13 remaining amino acids were placed in the autosynthesizer. The resulting protected tridecapeptide resin is divided in equal proportions and the final residue is put on one side. The sequence of amino acids used is as follows.

(1) N-t-butyloxycarbonyl- (0-benzyl) -L-serine;

(2) N-t-butyloxycarbonyl (0-benzyl) -L-threonine;

(3) N-t-butyloxycarbonyl-L-phenylalanine;

(4) N-t-butyloxycarbonyl- (0-benzyl) -L-threonine;

(5) N ^r -t-butyloxycarbonyl-N ^E -0-chlorobenzyl-oxycarbonyl-L-lysine;

(6) N ^r -t-butyloxycarbonyl- (N-formyl) -L-tryptophan;

(7) N-t-butyloxycarbonyl-L-phenylalanyl;

(8) N-t-butyloxycarbonyl-L-phenylarmyl;

(9) N-t-butyloxycarbonyl-L-asparagine, P-nitrophenyl ester;

(10) N ^r -t-butyloxycarbonyl-N ^E -0-chlorobenzyloxycarbonyl-L-lysine;

(11) N-t-butyloxycarbonyl- (S-P-methoxybenzyl) -L-cysteine;

(12) N-t-butyloxycarbonyl-glycine; And

(13) N-t-butyloxycarbonyl-D-varin.

The order of desorption, neutralization, coupling and recoupling for introducing each amino acid into the peptide is as follows.

(1) Wash with chloroform (10ml / g resin) three times for 3 minutes each.

(2) treatment with a mixture of 29% trifluoroacetic acid, 48% chloroform, 6% triethylsilane, and 17% methylene chloride twice each at 10 ml / g resin ratio for 20 minutes to remove the BOC group;

(3) Wash twice with chloroform (10 ml / g resin) for 20 minutes.

(4) Wash once with methylene chloride (10 ml / g resin) for 3 minutes.

(5) Wash three times for three minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin).

(6) Wash with methylene chloride (10 ml / g resin) three times for 3 minutes each.

(7) Neutralize by treating with 3% triethylamine (10 ml / g resin) dissolved in methylene chloride three times for 3 minutes each.

(8) Wash with methylene chloride (10 ml / g resin) three times for 3 minutes each.

(9) Wash three times for 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 mL / g resin).

(10) Wash with methylene chloride (1.0 mmol / g resin) three times for 3 minutes each.

(11) Add 1.0 mmol / g resin of protected amino acid dissolved in 10 ml / g resin of methylene chloride and 1.0 mmol / g resin of N, N'-dicyclohexyl carbodiimide (DCC) and mix for 120 minutes do.

(12) Wash with methylene chloride (10 ml / g resin) three times for 3 minutes each.

(13) Wash three times for 3 minutes each with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin).

(14) Wash three times with methylene chloride (10 mL / g resin) for 3 minutes.

(15) Neutralize by treating with glycine and asparagine residues (10 ml / g) for 3 minutes each.

(16) Wash three times for 3 min with methylene chloride (10 mL / g resin).

(17) Wash three times for three minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 ml / g resin).

(18) Wash three times for 3 minutes with methylene chloride (10 ml / g resin).

(19) Wash three times for 3 minutes with DMF (10 mL / g resin).

(20) To the 10 ml / g resin of DMF and methylene chloride and 1: 1 mixture was added 1.0 mmol / g resin protected amino acid and N, N'-dicyclohexylcarbodiimide (DCC) of 1.0 mmol / g resin. Then mix for 120 minutes.

(21) Wash three times with DMF (10 mL / g resin) for 3 minutes.

(22) Wash three times with methylene chloride (10 mL / g resin) for 3 minutes.

(23) Wash three times for 3 minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 mL / g resin).

(24) Wash three times with methylene chloride (10 mL / g resin) for 3 minutes.

(25) Wash three times for 3 minutes with 3% triethylamine (10 mL / g resin) in methylene chloride.

(26) Wash three times with methylene chloride (10 mL / g resin) for 3 minutes.

(27) Wash three times for 3 minutes with a mixture of 90% t-butyl alcohol and 10% t-amyl alcohol (10 mL / g resin). And

(28) Wash three times with methylene chloride (10 mL / g resin) for 3 minutes.

The above processing sequence is used to add each amino acid other than glycine and asparagine residues. Addition of glycine is performed only 1-18. Aspartic acid residues merge via their P-nitrophenyl active esters. In this process, the above process (11) is modified in the order of the following three steps:

(a) Wash three times with DMF (10 mL / g resin) for 3 minutes.

(b) 1.0 mmol / g resin of P-nitrophenyl ester of N-t-butyloxycarbonyl-L-asparagine is added to 10 ml / g resin of 1: 3 mixture of DMF and methylene chloride and mixed for 720 minutes. And

(c) three washes for 3 minutes with DMF (10 mL / g resin). In addition, the above (20) process is modified by mixing for 720 minutes using N-t-butyloxycarbonyl-L-asparagine P-nitrophenyl ester in a 3: 1 mixture of DMF and methylene chloride.

The final peptide-resin is dried in vacuo. The product is hydrolyzed by refluxing for 72 hours in a 1: 1 mixture of concentrated hydrochloric acid and dioxane. Results based on lysine of amino acid degradation of the resulting product are as follows: Asn, 1.04; 2 Thr, 2.68; Ser, 1.08; Val, 1.12; Gly, 1.04; 3 Phe, 3.87; 2 Lys, 2.00, Trp, 0.75.

Tritopane is determined by hydrolyzing the product for 21 hours with each other in the presence of dimethyl sulfoxide and thioglycolic acid. Cysteine cannot be determined because it is destroyed by elemental analysis.

Example 3

D-Varyl-Glysyl-S-Cysteinyl-L-lysyl-L-asparaginyl-L-phenylalanyl-L-phenyl-alanyl-L-trippophyll-L-lysyl-L-tre 2.828 g of the protected tetradecapeptide-resin of Example 2 (substituted 0.150) in a mixture of 5 ml of onyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteineanizol and 5 ml of ethyl mercatan Millimoles / g) is added. The mixture is cooled in liquefied nitrogen and 56 ml of hydrogen fluoride is distilled off. The resulting mixture is warmed to 0 ° C. and stirred for 2 hours. Hydrogen fluoride is then distilled off and ether is added to the remaining mixture. The mixture is cooled to 0 ° C. and the resulting solid is collected by filtration and washed with ether. This product is dried and the deprotected tetradeca peptide is extracted from the resin mixture using 1M acetic acid and a small amount of glacial acetic acid. This acetic acid solution is immediately nucleophilized and dried in the dark. The resulting white solid is suspended in a mixture of 10 ml of degassed 0.2 M acetic acid and 4 ml of glacial acetic acid. The resulting suspension is heated. However, the solid does not dissolve completely. The insoluble portion is filtered off and the clear colorless filtrate is placed on a Sephadex G-25F column. The conditions of the chromatography were as follows: solvent, degassed 0.2 M acetic acid; Column size, 7.5 × 150 cm; temperature, 26 ° C.

Outflow rate, 620 ml / hr

Fraction volume, 22.0ml

Absorption at each fraction 250 mμ for each fraction shows a broad peak with the following shoulder.

The U.V spectrum shows that the main part of the peak is the product. The recovered fraction and its elution amount are as follows. Fractions 224 to 240 (4906 to 5280 ml, peak = 5504 ml) The recovered fraction does not include subsequent shoulders.

U.V spectrum shows the presence of 175 mg of product. (Yield = 24.8%) Elman's titration was 93.6% of the theoretical content of free sulfhydrin.

Example 4

Oxidation on D-Val ¹ -Somatostatin

The reduced solution of D-Val ¹ -somatostatin (374 ml, 175 mg theory) of Example 3 was diluted with 0.2 mol of 147 ml of acetic acid and 2967 ml of distilled water to a concentration of 50 µg / ml. Concentrated ammonium hydroxide is added to adjust the pH of the mixture to 6.7. After Elman titration of the solution, the oxidation was completed, followed by stirring for 64 hours at room temperature in a dark place.

The mixture is concentrated in vacuo to about 10 ml. This concentrate is diluted with 10 ml of glacial acetic acid and then the salt is removed on a Sephatex G-25F column. Chromatography conditions are as follows.

Solvent, degassed 50% acetic acid;

Column size, 5.0 × 90 cm;

Temperature, 26 ℃

Outflow rate, 246 mL / hour;

Fraction volume, 16.4 ml

Absorption at each fraction 280 μm for the fraction number results in a broad peak. The first peak appears in the form of product recruitment and the second peak represents the product which is a monomer. The material indicated as the second peak (fractions 49 to 64 (787 to 1050 ml)) is recovered and freeze-dried in the dark. The resulting solid is dissolved in 15 ml of degassed 0.2 M acetic acid and added to a Sephadex G-25F column. Chromatographic conditions were as follows: solvent, degassed 0.2 M acetic acid;

Column size, 5.0 × 150 cm;

Temperature, 26 ° C .;

Outflow rate, 475ml / hr

Fractional volume, 16.6ml

Absorption at 280 m [mu] m of each fraction for fraction number is represented by one broad peak. The U.V spectrum shows that the main part of the peak is the product. Fractions 157 to 172 (2590 to 2855 ml of discharge capacity, peak = 2667 ml) were recovered and freeze-dried in the dark. U.V spectrum showed 95 mg of the desired product (yield from reduced form = 54.3%).

The resulting solid portion is dissolved in 5 ml of 50% acetic acid and chromatographed again on a Sephadex G-25F column. Chromatography conditions are as follows.

Solvent, degassed 50% acetic acid;

Column size, 2.5X180 cm

Temperature, 26 ° C .;

Outflow rate, 53.2 mL / hour;

At 280 mμ of each fraction for fraction volume, 8.87 ml, fraction number, the absorption appears as one broad peak. U.V spectrum shows that the main part of the peak is the product. Fractions 56 to 60 (488 to 532 mL, peak = 505 mL) were recovered and freeze dried in the dark.

Radiance [α] ²⁶ _D = -42.1 (1% acetic acid)

Amino acid analysis, Val, 0.98; Gly, 1,01; 2 Cus, 1.81; 2 Lys, 1.99; Asn, 0.95; 3 Phe, 2.94; Trp, 0.80; 2 Thr, 1.91; Ser, 0.85.

The results are expressed as the ratio (Gly + Lys) /3=1.0. It is hydrolyzed for 21 hours under the following three conditions.

(1) in the presence of dimethyl sulfoxide + cysteine oxide + cysteinic acid

(2) thioglycolic acid

(3) without remover or oxidizer.

All the above values are the average of three hydrolysis except the following.

Dys and Ser; (1) and (3)

Trp; (2)

Phe; (2) and (3)

D-Val ¹ -somatostatin was tested in dogs for gastric acid secretion in vivo.

In six dogs with chronic ulcers, gastric acid secretion is induced by injecting 0.5 μg / kg-hr of gastrin with C-terminal tetrapeptide. Each dog used as a control recovers only tetrapeptides. In addition, tetrapeptides were recovered from 6 dogs, and hydrochloric acid was secreted after 1 hour, and then D-Val ¹ -somatostatin was injected at 0.75 µg / kg-hr. Recovery of gastric acid continues for 1.5 hours at 15 minute intervals. This is titrated to qH 7 using an automatic titrator.

The maximum inhibitory effect of D-Val ¹ -somatostatin is extrapolated to the dose-response curves of somatostatin and shows relative activity similar to that of somatostatin in% activity. D-Val ¹ -somatostatin inhibits acid secretion induced by the C-terminal tetrapeptide of gastrin by 85.1 ± 6.0% (standard deviation). This effect corresponds to the effect of 0.935 μg / kg-hr of somatostatin. Thus, the effect on the activity of somatostatin is 125%. D-Val ¹ -somatostatin was also tested for intestinal motility in conscious dogs.

Three dogs with intraluminal enema located in the lumen, duodenum and pylorus were used. Intestinal pressure changes are recorded on a Visi-corder using a stress meter and a reduced light beam galvanos meter. After conditioning, D-Val ¹ -somatostatin is injected intravenously over 10 minutes. This compound initially increases the pressure in the tourism in the pylorus, while decreasing the pressure in the duodenum and river. The minimum effective amount required to increase the pressure in the pylorus and the minimum effective amount required to reduce the pressure in the duodenum and cavity is <0.125 μg / kg-10 minutes. This is compared to the activity for somatostatin itself is from 0.125 to 0.52 μg / kg-10 minutes.

D-Val ¹ -somatostatin also shows inhibition of pancreatic fluid. In three dogs with both pancreas and gastric fistula, the pancreatic sinus was induced by injection of 2 Units / kg-hr of secretin and 0.45 unit / kg-hr of crecistokinin. Intestinal HCl secretion is injected by injecting [mu] g / kg-hr of tetra gastrin. After induction, each dog is administered D-Val ¹ -somatostatin at 0.75 µg / kg-hr for 1 hour. The highest inhibitory effect, expressed as the rate of change for total protein, was -51%.

D-Val ¹ -somatostatin was also tested for its activity with respect to growth GH secretion. Tests were performed using normal Sprague-Dawley rats (females, 100-120 g thick). These tests are Phi, Brazeu, W. Veil and. It is an improved method. (See Endocrinology, 94 184 (1974)). Here, five groups of eight rats each were used. Sodium pentobarbital was administered intraperitoneally in all mice to stimulate growth hormone secretion. One group was used as a control. In both groups, somatostatin was administered subcutaneously at 2 μg / rat in one group and 50 μg / rat in the other, and in the other two groups, 2 μg / rat in subcutaneous injection. The ratio of D-Val ¹ -somatostin is administered and the other is administered at a rate of 50 µg / rat The serum concentration of growth hormone was measured for 20 minutes after the simultaneous administration of sodium pentobarbital and the test compound. The degree of inhibition of growth hormone concentration is determined by comparison with the control group and the relative activity of D-Val ¹ -somatostatin and somatostatin itself is compared: At the dose of 2 μg / rat, D-Val ¹ -somatostatin While Matostatin inhibits 44% of the inhibitory effect on growth hormone secretion by 2%, at a dose of 50 μg / d, D-Val ¹ -somatostatin secretes about 73% of growth hormone in comparison to the control. , Whereas somatostatin itself shows 79% inhibition.

D-Val ¹ -somatostatin was performed in vivo for the inhibition of glucagon and insulin secretion stimulated with L-Aranine. Do not feed positive (positive) mongrel overnight. A control warm blood sample is obtained and injected intravenously with Dasarin, somatostatin or D-Val ¹ -somatostatin. After 30 minutes, L-Aranine is administered intravenously for 15 minutes. Injection of sarin, somatostatin, or D-Val ¹ -somatostatin is continued for 15 minutes after the L-alanine injection. Infusion of L-Aranine sharply increases the serum concentrations of glucagon and insulin, which can control the concentration with L-Aranine. From the above, the minimum dose of D-Val ¹ -somatostatin for inhibition of glucagon is 0.06 to 0.11 µg / kg / min, and 0.006 to 0.03 µg / min for inhibition of insulin. On the other hand, the minimum dose of somatostatin for inhibition of glucagon is 0.10 to 0.12 µg / kg / min and 0.03 to 0.10 µg / kg / min for inhibition of insulin.

Claims (1)

A method for preparing a compound of formula (I) or an acid addition salt thereof, characterized by reacting tetradecapeptipe, a straight chain of formula (III), with an oxidizing agent.