Somatostatin analogs and intermediates thereto

The present invention relates to a process for the preparation of a tetradecapeptide used as an intermediate in. synthesis of valuable analogues of soma-_gtostatined, of formula I:

H - D - Val - Gli - L - Cis - L - Lis - L - Asp - L - Fen - L - Fen - L - Tri - L - Lis - L - Tre - L - Fen - L - Tre-L- Serum- _{in the}

- L - Cis - OH (JJ in which the amino acids of the tetradecapepiidic chain represented by the usual abbreviations are Val - valine, Gli - gli- _lgDinner (giicocol), Cis - cysteine, Lys - lysine, Asp - asparagine, Phen - phenylalanine, Tri - tryptophan, Tre - teronine and Ser - serine.

A tetradecapeptide is known with ₂the formula L - Ala - Gli - L - Cis - L. -Lis-LrAsp * I.-Feai - L - End - L - Tri -L-Lis-L - Three - L - End - L-Three-L- Ser - L - Cis - GH, (in which Ala represents, alanine and, the connection between the residues of ₂5 cysteine ​​represents a "disulfide bridge" called sonaatostatin or inhibitor of the formation of somatothyrin. This tetradecapeptide was isolated from hypothalamic ovine extracts,_3θbeing sold is active in inhibiting tetradeapeptide sequestration ^fjrnn nn ho io ί. j \ ή. 'i) IAf'^! Growth hormone creation, also known as the denomination.

Somatostaiin · natural is known as a general class of other compounds having the dodecapepidic sequence represented by positions 31–14 of the natural hormone.

The compound designated as D-Ala ^ romatostatin is also known. D-Ala'-S'omatostatin, although structurally a stereoisomer of natural L-Alal-somatostotin, exhibits approximately half the natural somatostatin activity in inhibiting gastric acid secretion in vivo.

The process, according to the invention, allows the preparation of tetradeapeptides of the formula I above, in that it treats a compound with the general foripula il: '.4 -:, c ..- j.

R - D - Val - Gl i - L - CisiRi), - L

- Lis ^ R?) - L - Asp - L - End - L - End - L -FrifRâl- - L - LisțR¹) - L - Tre4R,>

- L - Fen - L - TrelRî) - L - SeiȚRj - L - Cis (Ri) - X (ΙΪ) wherein R represents a group ® // n-anîWW ^ piiotecioar.e, Ri; represents the group (31079

PRICE <LEi 16.35 · <· β .Ο 1 - AÎȚ33J03 ί 91079 _. .. f-- Γ-4 Λ, £

2Î ίΠθ J> J? ',] ήµ — 1 .Λ ..' 'V * · **. .Vfll, tp! I epsilon-amino-protectors, - Ry and R * represent each »; .p gfima, ltfotectQaE & _ for bidrpxiltri * ilbSottV BWrenfteBiW-tY to a forniir group and X represents a group of the formula ________ 5 —O — CH:

¹-R^{R <}zr ··. In which JR · represents a polystyrene residue with hydrofluoric acid at a temperature of about 0^pC, then the reaction product is separated from the reaction mixture in a known manner. '15

The following is an example of an embodiment of the invention.

A. Preparation of methylated N-tert-butyloxycarbonylL-cysteinyl - (Sp-methoxybenzyl) -polystyrene 20

To 500 ml of N, N-dimethylformamide containing the N-phosphate-butyloxycarbonyl - (S - p -methoxybenzyl) - cistern (prepared from 9.06 g (26.5 mmol free acyl)) salt is added 51.0 g of chloromethylated polystyrene resin (0.75 mmol / g). The mixture is stirred at room temperature for six days. The resin is filtered and then washed successively three times with a 90% Ν, Ν-dimethyl- mixture. ... 30 formamide and 10% water, three times with 95 ° ethanol /₀ and three times with N, N-dimethylformamide. To the resin suspended in 500 ml of Ν, Ν-dimethylIformamide is added a solution of 10.5 g of cesium acetate. The mixture is stirred for six days at room temperature. The resin is filtered and washed successively, once with aqueous solution of N, N-dimethylformamide, three times with a mixture of 90% N, N-40 dimethylformamide and 10% water, three times with 95% ethanol, three either with dichloromethane, three times with 95% ethanol and three times with chloroform. The fine particles are removed by suspending the resin in the chloroform four times, extracting the liquid each time. Thereafter, the resin was dried in vacuo at ȚO'C overnight to obtain 44.8 g of N -ether-butyloxycarbonyl-L-cis-50-teinyl - (Sp-methoxybenzyl) -methylated polystyrene. Analysis of amino acids determined 0.25 mmol of Cis per gram of resin. Cysteine ​​was determined as cystic acid from an acid hydrolysis carried out with a 1: 1 mixture of dioxane and concentrated hydrochloric acid to which a small amount of dimethylsulfoxide was added.

B. Preparation of N-tert-butyloxycarbonyl-60

D - Valyl-glycyl - L - ( Sp-methoxybenzyl) cysteine ​​- L - (N - epsilon - o - chloro -. <Benzyloxycarbonyl) - lysyl - L - asparaginyl - L - phenylalanyl - L - ienylalanyl L - (formyl) - tryptophil - L -, (N - epsi- 65: j:^r u 'i. »8 * Λ 5 τ '» f. 9

Ion - o - chloro - benzyloxycarbonyl) - lysyl - L - (O-benzyl) -treonyl-L- phenylalanyl - L - (O-benzyl) -treonyl-L- (O-benzyl) -seryl - L - (Sp- methoxybenzyl) - methylated cysteinyl polystyrene M

An amount of 7.0 g of the product from point A above is placed in the reaction vessel of a Beckman 990 automatic peptide synthesizer and twelve of the remaining thirteen amino acids are added using the automatic synthesizer. The resulting protected threecapeptide resin is divided into two equal portions and the residue ÎINAT half-translates into one of the portions. The amino acids used and the sequence of their use are as follows: (1) N-'f-butyloxycarbonyl (O-benzyl) - L - serine, (2) N-iron-butyloxycarbonyl - (O-benz yl) -L-threonine;

(3) N-tert - butyloxycarbonyl-L-phenylalanine; (4) N - tertiary - butyloxycarbonyl (O-bands!) - L - threonine; (5) N-d / fo / er (-butoxycarbonyl - N - epsilon - or chlorobenzyloxycarbonyl - L - lysine ·, (6) No / fo-butyloxycarbonyl - (N-formyl) L-tryptophan; (7) N-tert-butyloxycarbonyl - L - phenylalanine; (8) N-tert-butyloxycarbonyl - L - phenylalanine; (9) p-nitrophenyl ester N-tert - butyloxycarbonylL-asparagine; (10) N-cilfa - tert - butyloxycarbonyl - N - epsilon-o-chlorobenzyloxycarbonyl - L - lysine; (11) N-iron-butyloxycarbonyl - (Sp-methoxybenzyl) - L cysteine; (12) N-third-butyloxycarbonylglycine and (13) N-fert-butyloxycarbonyl D-valine. The sequence of deprotection, neutralization, coupling and replication for the introduction of each amino acid into the pepftide is as follows: (1) three washes (IO ml / gram of resin) for three minutes each with chloroform; (2) removal of the hierf-butyloxycarbonyl group by treatment twice for 20 minutes with 10 ml / g of resin mixture of 29% trifluoroacetic acid, 48% chloroform, 6% triethylsilane and 17% dichloromethane; (3) two washes (10 ml / g of resin) of 3 niin with chloroform; (4) a wash (10 ml / g of resin) for 3 minutes with dichloromethane; (5) three washes (10 ml / g of resin) for 3 minutes each with a mixture of 90% alcohol (-butyl and 10% tertamyl alcohol; (6) three washes (10 ml / g of resin) each 3 mm with dichloromethane; (7) neutralization by three treatments, of 3 min each with 10 ml / g of triethylamine resin 3% in dichloromethane; (8) three washes (10 ml / g of resin) of 3 min each with dichloromethane; (9) three washes (10 ml / g of resin) of 3 minutes each in a mixture of 90% alcohol (-butyl and 10% alcohol-amyl alcohol; (10) three washes (10 ml / g of resin) of 3 min each with dichloromethane; (11) adding 1.0 mmol / g of protected amino acid resin and 1.0 mmol / g of N, N'-dicyclohexyl = carbediamide resin in 10 ml / g of dichloromethane resin followed by stirring for 120 min; (12) three washes (10 ml / g of resin} of 3 min each with dichloromethane 7 (Î3) three washes (10 ml / g of resin) of 3 min each with a 90% ferf-butyl aqueous mixture and 10% alcohol Herbal alcohol (14) three washes (10 m / g resin)¹ was 3 min each with dichloromethane; (15) neutralization by three treatments of 3 min each with 10 ml / g of resin with a 3% triethylamine solution * in dichloromethane; (16) three washes (IO ml / g of resin) · each 3 min with dichloromethane; (17) three^:washes (IO ml / g of resin) of 3 min each with a mixture of 90% tert-butyl alcohol and 10% alcohol / erf-amyl; (18) three washes (10'ml / g of resin) of 3 min each with dichloromethane? (19) three washes (10 ml / g of resin) of 3 min each with Ν, Ν-dimethylforin amide; (20) adding 1.0 mmol / g of resin from the protected amino acid and 1.0 mmol / g of N'-dicyclohexylcarbodiimide resin in_;10 ml / g of 1: 1 mixing resin of N, M ~ dimethylformamide and dichloromethane followed by mixing for 120 min / (21) three washes (10 ml / g of resin), of 3 min each with Ν, Ν dimethylformamide; (22) three washes (10 ml / g of resin; resin) of 3 min each with dichloromethane; (23) three washes (10 ml / g of resin) each with 3 mixtures; LM) a /_a tert -butyl alcohol and 10% * tert-amyl alcohol? ^J(24) three washes, {10; «Ml / g of resin) for 3 min each with dichloromethane; (25) neutralization by? .treatment of 3 min each 3 W 10'AieilMg; 3% triin solution resinManine in dichloromethane_; (26) three wash-¹lilies (T0 ml / g of resin) of 3 mini bottles each with dichloride; (27) three drops of water (10 ml / g of resin) of 3 ml each with a mixture of 90% tert-butiUc alcohol and 10% iron-emyl alcohol and (28) three washes (10 ml / g of resin) of sample: 3 min each with dichloromethane.

The above treatment sequence was used to add each amino acid except glycine and asparagine residues. Only the phases were used to add the glycine residue. The asparagine residue was incorporated via its active p-nitrophenyl ester. In this purpose, the above step (1) is modified in the following three-phase sequence: (a) three washes (10 ml / g of resin) of 3 min each with Ν, Ν-dimethylformamide; (b) addition of 1.0 ml of N-yesterday-butyloxyparbonyl p-nitrophenyl ester resin; -asparaginel · in 10 ml / g of 1: 3 mixture of N, N-dimethylformamide and dichloromethane, followed mixing for 720 min and (c) · three washes (10 ml / g resin) of 3 min each with Ν, Ν-dimethylformamide. Also, the above phase (20) was modified by using the p-nitrophen9fc ester of N-tert -butyloxycarbonyl-L-asparagine in a 3: 1 mixture of N, N-dimethylphonamide and dichloromethane, followed by mixing for d. P20 min.

The finished peptide resin sd is dried in vacuo. A portion of the product is hydrophobed by refluxing for 72 hours in a mixture of hydrochloric acid and dioxane Amino acid analysis of the non-given product¹ the following results, they were used as standard: Asp, 1.04; 2Tre, 2/68 '; Ser, 1.08 ^ Val, l, tftc2; Gli, 1.C4; 3Fen, 3.87; 2Lis, 2.00; Three, 0.73. The tryptophan was determined by *; tr it_;- hydrolysis for 21 ha of one; "Ιοε-. three, of. product _ in the presence of ^ idiquetjlsulfoxide and thioglycolic apid. The cistern was not determined because it is destroyed by this method of analysis.

C. Preparation of D-valyl-glycyl - L - cisfernyl - L - JisM -L- asp & ragin / J - L - terrilalanil - L - ieniiayanil-L-lriptofyPL-lysis] - L - -trons¹! -L- Hypalanil-L-lreonylL-serieJ-L-cysteine

To a mixture of 5 ml of anisole and 5 ml of ephimdrcaptan is added 2,828 g (at a <substitution level of 0.150 mmol / g) protected tetradecapeptide resin from point B. The mixture is cooled to liquid nitrogen and added to distraction. 56 ml db liquid hydrofluoric acid. The result antestecoS is allowed to warm to 0^?C and stir for 2 hours. Then remove it by distillation of the hydrofluoric acid and add ether to the remaining mixture. The mixture is cooled to 0^cC and the resulting solid is collected by filtration and washed with ether. The product is dried, and the tetradecapepffcida -.prepared-is extracted from the mixture of the resin fdto9iB <l · atetic-iecetic IM' and a bribe (Amount of acid of <$ e »Hc glacial. The acetic solution is then lyophilized immediately to dry in The resulting white solid is suspended in a mixture of 10 ml of degassed 0.2M acetic acid and 4 ml of glacial acetic acid. The resulting suspension is heated, although the solid does not completely dissolve. The insoluble portion is removed by filtration and the filtrate op ^ c, Colorless is applied on a column with Sefadex ion exchange resin. Chromatography conditions are: solvent, 0.2M degassed acetic acid; . column dimensions, 7.5χΐ50 cm? temperature, 2B⁼C; flow rate, 629 ml / h; volume of the fraction, 22.0 ml.^r η

The absorption at 280 sqm a, each fraction graphically represented according to the number of fractions indicated a broad peak followed by. a shoulder. Ultraviolet spectroscopy showed that the main tip is the product. The fractions seem to have been combined and their effluent volumes are 224-240 · (4 906-528Q ml, peak = = 5 064 ml). This collection of fractions does not include the following shoulder. Spectroscopy in ultraviolet showed the presence of 175 mg of product (yield = «24.8¼). Ellman titration of an aliquot obtained a content of free suhydryl groups of 93.6% theoretically. The product can be further oxidized to D-wave¹-somatostatin.

The compound of formula I is obtained, by the process according to the invention, from compounds of general formula II:

R - D - Val - Gli - L - Cis (Ri) - L - Lis (R?) - L - Asp - L - End - L - End - L Tri (R0 - L - Lis (R-) - L - Tre (R₃) - L - End - L - Three (R₃) - L - Ser (R4) - L - Cis (Ri) X (II)

Preferred compounds of formula II are: H - D - Val - Gli - L - Cis - L - Lis - L - Asp - L - Fen - L - Fen - L - Three - L - Lis - L - Three - L - Fen - L - Three - L - Ser - L - Cis - OH and

N - (tert-butyloxycarbonyl) -D-Val-Gli-L- (p-methoxybenzyl) - Cis - L - (o - chlorobenzyloxycarbonyl) - Lis - L - Asp - L-Fen-L-Fen-L- (formyl ) Tri - L - (o - chlorobenzyloxycarbonyl) - Lis - L - (benzyl) Tre - L - Fen-L- (benzyl) Tre - L - benzyl) - Ser - L - (p-methoxybenzyl) Cis-Oj- ^ /resin

The above formulas defining starting materials of formula II also include the protecting groups for the amino, hydroxy and thio (suhydhydryl) functions. The properties of a protection group are twofold. First, the protecting group prevents a reactive moiety from a molecule from reacting when the molecule is subject to conditions that would normally cause the active moiety to react. secondly, the protection group can be easily removed for restoring the active moiety of the molecule under conditions that do not produce undesirable effects on it; the other portions of the molecule. The groups that can be used for these purposes, respectively for the protection of the amino, hi8 droxy and thio groups, are known by the specialist.

In general formula II, R represents either an α / ία-amino-hydrogen or an α / ία-amino-protecting group. The affaamino-protecting groups that may be used are, for example, the benzyloxycarbonyl, prichlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, methoxybonyloxycarbonyl, 2,4-dichlorobenzyloxybonyloxycarbonylbonyloxycarbonylbonyloxycarbonyl, butyloxycarbonyl, tertiary - .amyloxycarbonyl, 2- (p-biphenylyl) - isopropyloxycarbonyl, adam * tiloxycarbonyls, isopropyloxycarbonyl, cyclopentyloxycarbonyl, cycloheptyloxycarbonyl, triphenylmethyl (trityl) and p · toluenesulfonyl. Preferably, the affa-amino-protecting group defined by the radical R is the tert-butyloxycarbonyl group.

The radical R 1 represents either the hydrogen atom of the sulfhydryl group of the cistern or a protecting group of the sulfhydryl substituent. Suitable protecting groups for sulfhydryl are, for example, p-methoxybenzyl, benzyl, p-tolyl, benzhydryl, acetamidomethyl, trityl, p-nitrobenzyl, tert-butyl, isobutoxymethyl, as well as trityl derivatives. Preferably, the protecting group of the sulfhydryl group, defined, by the radical R 1, is a β-methoxybenzyl group.

Radical R? represents either the hydrogen atom of the epsilon-amino group of the ltsin moiety, or a non-protecting epsilon-ami group. Examples of such protection groups are most of the protective groups of the c / fa-amino group, presented above. Typical groups for this purpose are the benzyloxycarbonyl groups, lys (-butox ycarbonyl, tert - amyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, p - methoxybenzyloxycarbonyl, p - chlorobenzyloxycarbonyl p - bromobenzyloxycarbonyl, 2,4-chlorobenzylbycarbonylbycarbonylbenzobyl, 2,4-chlorobenylcarbenzyl, o - bromobenzyloxycarbonyl, p - nitrobenzyloxycarbonyl, isopropyloxycarbonyl, cyclohexycaroxycarbonyl, cycloheptyloxycarbonyl and; p-Loluensulfonyl. _b

As seen from the example₆In one embodiment, the process for preparing the tradecapeptide of formula I involves ^ the periodic cleavage of the α / α-amino-g protecting group of the terminal amino acid in the peptide chain, Thus, the only limitation on the identity of the epsilon-aminoprotective group of the lysine moiety is that: · it does not: or cleaved under the conditions used for the selective cleavage of the a / a * amino-protecting group. The choice - adequate -¹· protection of the protection groups a / fa-âiriittO-,.

and epsilon-am ino is known to a specialist and depends on the relative ease with which one protection group or another may be cleaved. Thus, the 2- (p-biphenyl) -isopropyloxycarbonyl and trityl groups are very labile and may split even in the presence of a weak acid. For cleavage of other protecting groups, such as iron (-butoxycarbonyl, tert-amyloxy carbonyl, adamantyloxycarbonyl and p-methoxybenzyloxycarbonyl, an acid having a moderate strength, such as hydrochloric acid, trifluoroacetic acid or trifluoride in boron acid, is required. . For the cleavage of other protecting groups, such as beta-zyloxycarbonyl, halogen benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl and isopropyloxycarbonyl, strong acids are required. The cleavage of the latter protecting groups requires drastic acid conditions, such as the use of hydrobromic acid, hydrofluoric acid or boron trifluoroacetate in trifluoroacetic acid. Of course, under these strong acidic conditions, the weaker protection groups will be split. The proper choice of the amino-protecting groups involves the use of a protection group for the more off-amino function than that for protecting the epsilon-amino function under cleavage conditions that selectively remove only af / a-amino-protecting groups. In this context, R? it is preferably an oclorobenzyloxycarbonyl or cyclopentyloxycarbonyl group and the α / c-aminoprotective group of all other amino acids that are added to the peptide chain is preferably the (er) -butoxycarbonyl group.

Groups R3 and R * represent the hydroxyl salt of the hydroxyl group or a protecting group of the hydroxyl alcohol group of threonine or serine. Typical protecting groups for this purpose are, for example, C 1-4 alkyl groups, such as methyl, ethyl and / erf-butyl, benzyl, substituted benzyl, such as p-methoxybenzyl, p-nitrobenzyl, p- chlorobenzyl and o-chlorobenzyl, C1 -C3 alkanoyl, such as formyl, acetyl and propionyl, triphenylmethyl (trityl). Preferably, when R3 and R <represent protection groups, these groups are both bands.

Group R5 represents hydrogen * ¹ or ⁴formyl and defines the remainder - NRs to a tryptophan residue. The radical tfOrmil serves as a protection group. The use of such a protecting group is optional, as Rs may thus represent either hydrogen (N-unprotected) or formyl (N-protected).

lo

The X-group of the tetradecapeptide chain may be hydroxyl, in which case it defines a free carboxylic group or may represent a solid resin support to which the 'carboxy-terminal moiety of the peptide binds during synthesis- The solid resin is represented by the formula':

- ^ / resin

<img file="RO81079A_D0001.tif" />

When X represents a hydroxyl group, each of the radicals R, Ri, Ra, R3, R4 and R₅ represents hydrogen, When X represents a solid resin carrier, each of the radicals R, R₁₍ r₂₍R₃ and R * represent a protection group.

Although the choice of particular protection groups for the preparation of the compound of formula I remains a matter of the competence of the specialist, it must be recognized that the sequence of reactions to be performed raises the problem, the choice of particular protection groups. In other words, the chosen protection group must be stable both in the reactants and in the conditions used in the next steps of the reaction sequence. For example, as discussed to some extent above, the group, the 'particular protection paw used' must remain intact under the conditions used for cleavage of the alpha-amino-protecting group of the remaining amino.₍terminal acid of the peptide fragment * which is prepared for coupling the next amino acid fragment to the peptide chain. It is also important when choosing the protection group that / it remains intact during ·¹* building the peptide chain and being able to ', without being easily removed at the end of the tetrad-ecapeptidic-wanted product synthesis. The appropriate choice of the above mentioned protection groups can be easily carried out by the specialist.^f

From the above discussion¹ it results' paths; The xetiadecapeptide of formula Ϊ can be prepared by solid phase synthesis. This synthesis involves a sequential construction of the peptide chain starting from the terminal carbon atom of the peptide. Thus, cysteine ​​is first linked by its carboxylic function to the resin; by the reaction between the aminoprojected cysteine. and S-protected "with a chloromethylated resin. or a hydroxymethylated resin. The preparation of the hydroxymethylated resin is known [Bodanszky et al., Chem. Ind. (London), 38, pp. 1597 ... 98 (1966)].

83 &lt;W î υ

The dormant resin is also commercially available.

In order to make the connection between the terminal carbon atom of the cistern and the resin, the protected cysteine ​​is first converted to its cesium salt. This salt is then treated with the resin [according to the process described by BF, Gisin in Helv. Chyme. Acta, 56, p. 1476 (1973)]. Alternatively, cysteine ​​may be linked to the resin by activating the carboxylic function of the clsteinene molecule, by applying known techniques. For example, cysteine ​​may be treated with the resin in the presence of a compound that activates the carboxylic acid, such as, for example, N_fN-diciclohexilcarbodiimlda.

Once the cysteine ​​with the free oarboxylic group is appropriately bound to the resin support, the step-by-step addition of each amino acid follows. terminal nitrogen atoms of the peptide chain. Necessarily, the following reaction sequence involves cleavage of the u / toamlno-protecting group of the amino acid representing the terminal nitrogen atom of the peptide fragment, followed by coupling the next amino acid residue to the next free terminal nitrogen atbm. Splitting of the α / α-amino group can be performed in the presence of an acid, such as hydrobromic acid, hydrochloric acid, trifluoroacetic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and acetic acid, with the formation of salts. with the appropriate acid addition. Another process for splitting the aminoprotective group is based on the use of boron trifluoride. For example, diethyl etherate of trifluoride. boron in glacial acetic acid converts the amlno-extended peptide'.c fragment into a BF3 complex - which can then be unblocked by treatment with a base, such as an aqueous solution; of potassium bicarbonate. Any of these processes can be used with the weighting of the cleavage of the N-terminal alloy / amino-protecting group without destroying any other protection group in the peptide chain. For this reason, it is preferable to split the N-terminal protection group using trifluoroacetic acid. In general, the splitting will be performed at a temperature between about O '&lt;C and the room temperature.

After the N-terminal cleavage, the resulting product is found as an acid addition salt used to cleave the protecting group. The product can then be converted! To a coafupus with free and terminal group by treatment with a weak &amp; weak base_T typically tertiary amines, such as pyridine or triethylamine.

The peptide chain is now ready for the reaction as the next amino acid. This reaction is performed by using the known technique. For the coupling of the following amino acid to the N-terminal peptide chain, an amino acid with a free carboxylic group, appropriately protected in the α / fa-ammo group as in any other active residue, is used. The amino acid exits' then subjected to conditions that activate the car function (boxed for the coupling reaction. One such activation technique that can be used in synthesis is to transform the amino acid into a mixed anhydride. In this regard, the free carboxylic function of the anuno acid is activated by the reaction with another acid, usually with an oarboxybc acid in the form of its acid chloride. Examples of acid acids which may be used for the formation of mixed anhydrides are ethyl mofoflOtate, phenyl claroformate, sec-butyl chloformate, isobutyl chloroformate and pi-vaioyl chloride.

An aHa method of activating the carboxylic function of the amino acid for reagent coupling is to transform the years of the noacid into an active ester. Also active esters are Estonians 2_r4_#5-trichlorophenyl Hc, pentachlorophenyl ester, p-nitrophenyl ester, 1-hydroxybenztriazole ester and N-hydroxysuccinimide ester. Such a method for effecting the coupling of the terminal carbon atom of the amino acid to the peptide fragment consists in performing the coupling reaction in the presence of at least an equimolar amount of N, N'-dtoictote3 ^; caflrÎwxiilinl «icU * This last preferred eote method loses its preparation. tetredeoapeptidetor in? which, X represents a resin group

After preparing the desired sequence a. laxainoacizhor, the resulting peptide can * be removed from the resin support. This is achieved by treating the tetradeoapeptide on resin substrate, protected with hydrofluoric acid. Treatment with hydrofluoric acid causes the breakdown of the> resin peptide and at the same time the cleavage of all the remaining protection groups on the reactive residues of the peptide chain as well as of the c / fa-atnino-protecting group of the terminal nitrogen atom. When using hydrofluoric acid as breast 81079

Providing the peptide on the resin as well as removing the protecting groups, it is preferred that the reaction be carried out in the presence of anisole. It has been observed that the presence of anisole inhibits possible alkylation of amino acid residues in the peptide chain. It is also preferable that the cleavage be carried out in the presence of ethylmercaptan. Ethylmercaptan serves to protect the indolic cycle of the rest of the tryptophan and, in addition, facilitates the conversion of the blocked tanks to their thiol forms. Also, when Rs is formyl, the presence of ethylmercaptan facilitates the cleavage of the hydrofluoric acid by the group, mil.

After the cleavage reaction is complete, the obtained product of formula I is a linear chain peptide containing 14 amino acid residues. This compound represents an intermediate for obtaining a tetradecapeptide linked by a disulfide bridge between the cysteinyl residues. For this purpose, the linear tetradecapeptide obtained may further be subjected to oxidation for the preparation of some somatostatin analogues.

The process according to the invention has the advantage that it is possible to obtain, by a simple way, an intermediary necessary to obtain important pharmaceutical products.