FR2522656A1 - Somatostatin analogue peptide growth hormone inhibitors - inhibit insulin and glucagon secretion and are resistant to proteolytic hydrolysis - Google Patents

Abstract

Somatostatin analogues of formula (I) and their acid addn. salts are new. In (I) R is H, an aminoacid or a dipeptide e.g. Ala-Gly; X1 is L-Lys or a bond; X2 is L-Asn, Tau (i.e. insertion of taurine in peptide sequence or -NH-CH2-CH2-SO2-) or a bond; X3 is L-Phe or L-Asn; X4 is L-Phe, Tau or a bond; with the condition that one of X2 and X4 is Tau; X5 is L-Thr, L-Phe or a bond; X6 is L-Ser or a bond; TRP is L-Trp or D-Trp; and CYS is L-Cys or D-Cys. (I) control the secretion of growth hormone (GH) and may be used to treat acromeoalia. Their inhibitory effect on glucagon and insulin secretion is different and in glycemic regulation they are useful for control of insulin dependent and non-insulin dependent sugar diabetes. They have low toxicity, relatively little effect on gastric secretion and are more resistant to enzymatic (proteolytic) hydrolytic degradation than somatostatin. They may be administered parenterally by intravenous, sub-cutaneous or intramuscular injection, usually in doses of 0.1-10 mg.

Description

et qui inhibe lessécrétions endogènes d'hormones de croissance (girl) d'insuline et de glucagon. Les abréviations utilisés sonL celles recommandées pour les aminoacides par la comlnission de nomenclature biochimique de 1'IUPAC (Journal of Biological Chemistry, 1972, 247, p.Somatostatin is a structural tetradecapeptide

and which inhibits the endogenous growth hormone (girl) secretions of insulin and glucagon. The abbreviations used are those recommended for amino acids by the Biochemical Nomenclature Comission of IUPAC (Journal of Biological Chemistry, 1972, 247, p.

977).

 This same peptide also significantly reduces the levels of digestive secretions. The denial of life of somatostatin is very short (about 4 minutes) because it is rapidly hydrolysed in vivo by proteolytic enzymes or degraded by aminopeptidases and carboxypeptidases.

 A very large number of analogues have been described by different authors in order to obtain either dissociations of biological activity or prolonged biological half-life.

dans laquelle R R désigne un Il, un acide aminé, un dipep-
tide comme Ala-Gly;
. X1 = (L)-Lys ou liaison simple
. X2 = (L)-Asn, Tau ou liaison simple
. X3 = (L)-Phe ou (L)-Asn
. X4 = (L)-Phe, Tau ou liaison simple l'un des deux groupements X2 ou X4 est obligatoirement Tau
. X5 = (L)-Thr, (L)-Phe ou liaison simple
X6 = (L)-Ser ou liaison simple
TRP = (L)-Trp ou (D)-Trp
. CYS = (1 )-Cys ou (D)-Cys. The products of the present invention correspond to the general formula

wherein RR denotes an II, an amino acid, a dipeptide,
tide like Ala-Gly;
. X1 = (L) -Lys or single bond
. X2 = (L) -Asn, Tau or single bond
. X3 = (L) -Phe or (L) -Asn
. X4 = (L) -Phe, Tau or single bond one of the two groups X2 or X4 is obligatorily Tau
. X5 = (L) -Thr, (L) -Phe or single bond
X6 = (L) -Ser or single bond
TRP = (L) -Trp or (D) -Trp
. CYS = (1) -Cys or (D) -Cys.

The Tau symbol represents the group that results from the insertion of Taurine into a peptide sequence, either
- NH - CH2 - CH2 - SO2
It is a sulfonamide type structure.

 These products are characterized by a better resistance to proteolysis in particular if the presence of a non-natural amino acid Taurine that proteolytic enzymes can not recognize and possibly through the introduction of amino acids of the D series.

- By dissociation of biological activity compared to somatostatin taken as a reference product both in terms of the inhibition of the 3 basic hormones involved in diabetes and the level of inhibition of digestive secretions.

The preferred products of the present invention are represented by the following formulas

C 14.462

CM 14.461

C 14.462

CM 14.468

CM 14.470

C 14.473

CM 14.471

C 14.473

CM 14.500

 These compounds are always used in the form of an addition salt such as hydrochloride, sulfate, phosphate, acetate, maleate, citrate, benzoate, succinate, malate, ascorbate, etc. These salts can be obtained by dissolving the compound in water. in the presence of two equivalents of the acid and then subjecting the assembly to lyophilization. The preferred addition salt, however, remains acetate.

General principles of the synthesis:
They are synthesized according to a mixed process between solid phase synthesis and solution synthesis.

The coupling operations between 2 amino acids are carried out in the liquid phase, that is to say in solution in a polar solvent such as dimethylformamide (DMF). The product of the reaction is isolated by adding a third solvent to the reaction medium which insolubilizes it and thus makes it possible to isolate it by filtration.

 The crude product thus obtained is then subjected to solid phase washes using appropriate solvents in which it is insoluble. For this purpose, a protecting group of the carboxylic acid function is used on the starting cysteine (cysteine 14) which insolubilizes the products in the usual washing solvents (water, alcohols, ethers, ethyl acetate, etc.), while maintaining a sufficient solubility in polar solvents such as dimethylformamide thus making it possible to perform the coupling operations in the liquid phase.

 The protective group which makes it possible to apply this strategy is the acid-stable 2- [4- (phenylazo) -benzylsulfonyl] ethyl ester (OPSE), which can be cleaved in a basic medium according to the removal process.

 The application of this technology to somatostatin synthesis has been described in Biorganic Chemistry 8, p. 429-442, (1979).

 The serine and threonine involved in these syntheses were used without protection of the hydroxyl functions in their side chair.

 The amine in # of the side chain of lysine is protected during the phases of elongation of the peptide by the 2- (methylsulfonyl) -ethoxycarbonyl (weave) stable in acidic medium and cleavable in basic medium.

 The thiol function of cysteine carries the protective group acetamido methyl (Acm) which is stable in basic or acidic medium. Deprotection is in this case ensured by treatment with a heavy metal salt such as silver or mercury.

 During the elongation phases, the temporary protection of amines at the m-position of the carboxyl is provided by the tert-butyloxycarbonylamino (Boc) protecting group. Thus each coupling operation will be followed by a selective deprotection operation of the Boc. This deprotection is ensured by treatment with trifluoroacetic acid and is also carried out in solution. Here also the deprotected product will be isolated from the reaction medium by adding a third solvent which insolubilizes it. However, in order to minimize the amounts of solvent used, it will be desirable to concentrate the reaction medium beforehand.

 After the elimination phases, the protected polysaccharide obtained will be released from the side chain protections of the lysines and the terminal carboxylic function by a basic treatment using sodium hydroxide or barite in a suitable solvent based on dimethylformamide.

 The peptide thus obtained always has the thiols of the cysteines protected by the acetamidomethyl group.

 It will be possible at this level, depending on its degree of purity, to be used, such as for the final oxidation stage, or else subjected to thorough purification by the countercurrent distribution technique or else by partition chromatography.

Finally, the final cyclization of the product by creating a disulfide bridge between the two cysteines will be ensured by the following operations
deprotection of the acetamidomethyl groups
two cysteines by a heavy metal salt (nitrate
money for example)
- elimination of the heavy metal by a treatment with
hydrogen sulphide
- outgassing and dilution to the air
solution thus obtained
- addition of this solution gradually and at PH
controlled at Wl oxidation medium constituted by a
ammonium acetate buffer containing the oxy agent
which is potassium ferricyanide.

This operation will always be carried out in
conditions of high dilution (2 liters per g of
treated product) in order to minimize the risks
of polymerization, as a consequence of an ocular thermal oxidation.

 The final product is then stripped of the complex ions from the oxidation phase by passing adequate overesin and then desalted and concentrated by fixation followed by elution with acetic acid on another resin.

 The crude product of evaporation should be subjected to gel permeation chromatography in dilute acetic acid in order to rid it of polymers that have formed during the oxidation phase.

 If after this gel permeation operation, the purity of the product proves to be insufficient, it may be subjected to other purification techniques such as partition chromatography or passage over ion exchange resin.

 As regards the introduction of Taurine in sequence, a peptide fragment containing this taurine is first prepared and then coupled to the rest of the molecule.

que 1 on couple sur 1' heptapept ide

For example, in the case of CM 14.462 the tetrapept ide is prepared

that 1 couple on the heptapept ide

Z-Tau-Phe-Phe-OCH3
Le tripeptide protégé est soumis ensuite à une saponification Z-Tau-Phe-Phe-OC113

Taurine is introduced from the benzyloxycarbonyl-taurine chloride chloride (Z Tau-Cl) according to the reaction sequence
Z-Tau-Cl + H-Phe-Phe-OCH3

Z-Phe-Phe-Tau-OCH 3
The protected tripeptide is then subjected to a Z-Tau-Phe-Phe-OC113 saponification.

sous la forme activée

sur ce tripeptide conduisant au tétrapeptide attendu

Z-Tau-Phe-Phe-OH
The benzyloxycarbonyl (Z) group is removed by vigorous acid treatment (H Br / CH 3 CO 2 H).
We then couple the

in the activated form

on this tripeptide leading to the expected tetrapeptide

 The following nonlimiting examples illustrate the syntheses that led to the preparation of the compounds of the present invention.

Symbols and abbreviations used
Z: benzyloxycarbonyl

Boc:

OHBT: hydroxybenzotriazole

Acm:

MSc:
ONSu:
OPse:
We p :

O-Tcp:

Me: CII3
DMF: dimethylformamide
IFA: Trifluoroacetic acid or trifluoroacetate
THF: tetrahydrofuran
TLC: silica thin-layer chromatography Merck
60F 254
HPLC: High performance liquid chromatography
BEW1: Chromatographic eluent: butanol (72), CH3CO2H
(7) and H20 (21)
BPEW1: """: butanol (50), pyri
dine (12), CH3CO2H (12),
H2O (25)
EBPEW1: """: ethyl acetate (42),
butanol (25), pyri dine (21), acic acid
tick (6), water (10)
EPAW1: """ethyl acetate (63), pyridine (210, acid
formic (10), water (6)
EPAS2: "" ethyl acetate (70),
pyridine (16), Acid
formic (8), water (6)
Truth: CHCl3 (95), MeOH (5),
CH3CO2H (3)
The amino acid analysis of the various intermediates mentioned in the following examples was carried out.

EXAMPLE 1 Preparation of CM 14.458

reaction scheme:

Preparation of (A)
1). Z-Tau-Ph3-OMe
16 g of phenylalanine methyl ester hydrochloride are dissolved in water and neutralized to fold 7 with 6.3 g of sodium hydrogen carbonate. After evaporation to dryness, the mixture is taken up in ethyl acetate, filtered, dried and evaporated at room temperature.

 The residue is taken up in 20 ml of tetrahydrofuran (THF) and added to a solution of 7, - Tau Cl. Tau Cl is a product described (Biochimica Biophysica Acta, 1955, 18, 353).

 After one night of reaction at room temperature, a white precipitate consisting of phenylalanine methyl ester hydrochloride is formed. The product is filtered. The solution is evaporated to dryness, taken up in water and then extracted with ethyl acetate. The solution is then washed with potassium hydrogen sulphate solution, potassium sulphate solution, sodium hydrogen carbonate solution and sodium chloride solution. After drying and evaporation to dryness, it is recrystallized from a solvent mixture: benzene and pentane.

 12.73 g of 1 is obtained; yield 89%, mp 93; TLC / Truth Rf: 0.53.

2). HBR, H-Tau-Phe-OMe
17.9 g of the product 1 prepared above are dissolved in 30.4 ml of a mixture of acetic acid and 33% hydrobromic acid. After 15 minutes at room temperature, evaporated to dryness and precipitated with ethyl ether.

 14.28 g of the expected product 2 are obtained, ie a yield of 91.4%. This product is very hygroscopic.

3). Boc-Phe-Phe-OMe-Tau
14.5 g of 2 prepared above are dissolved in 100 ml of DMF. To the solution is added 15.7; of Boc-Phe-ONp, 5.51 g of hydroxybenzotriazole (OHBT) and 8.7 ml of N-ethylmorpholine. The reaction lasts overnight at a pH of between 6 and 7. The mixture is evaporated to dryness and then taken up with ethyl acetate. Potassium sulphate solution and water are washed with a solution of potassium sulphate; it is dried, evaporated to dryness and then taken up with the minimum of warm ethyl acetate. It is precipitated by addition of hexane. The precipitate is filtered off, it is then washed with hexane and then with ethyl ether.

 After drying, 16.80 g of 81% yield is obtained; TLC / Truth Rf: 0.43.

4). TFA, H-Phe-Tau-Phe-OMe
13.97 g of 3 are dissolved in 84 ml of TFA.

After 15 minutes of reaction at room temperature, evaporated to dryness, precipitated with hexane to give 14 g of 4; 100% yield -; TLC / Truth Rf: 0.13.

5) .; Boc-Asn-Phe-Phe-OMe-Tau
In 100 ml of DMF was dissolved 14 g of 4 prepared above to which 9.66 g of Boc-Asn-ONp, 4 .6 g of HOBT and G, 6 ml of N-ethylmorpholine were added. After 2 hours of reaction at a pH of between 6 and 7, the mixture is evaporated to half, precipitated with 4 volumes of water and then washed successively with a solution of potassium acid sulphate, a potassium sulphate solution, water, a solution of sodium hydrogen carbonate, water and ethyl ether to 1% ethyl acetate, again ethyl ether and then dried.

17 g of 5 are obtained; 100% yield;
TLC / Truth Rf 0.33.

6). Boc-Asn-Phe-Phe-OH-Tau
17 g of 5 are dissolved in 255 ml of dioxane, 59.5 ml of normal sodium hydroxide are added. After 30 minutes at room temperature, evaporate to dryness. It is taken up in 60 ml of water, where it is acidified to pH 3.5. A precipitate is formed which is filtered off, washed with water and dried.

13 g of 6 are obtained; yield 78.2%
TLC / Truth Rf: 0.65.

7). TFA, H-Asn-Phe-Tau-Phe-OH
13 g of 6 described above are dissolved in 65 ml of TFA. After 20 minutes of reaction at room temperature, evaporated to dryness and then precipitated with ethyl ether and dried.

12.58 g of 7 is obtained, giving a yield of 94.7%
TLC / BEW1 Rf: 0.33.

de OHBT et 5ml de N éthylmorpholine. Après 3 heures à pH compris entre 6 et 7, on évapore a sec puis on amène à pli = 4,5, on. fait précipiter par l'acétate d'éthyle, on lave et on seche par l'éther éthylique.In 100 ml of DMP, 12.5 g of 7 are dissolved and 1 g, 5 ml of water and then 8.35 g of water are added.

of OHBT and 5ml of N-ethylmorpholine. After 3 hours at pH between 6 and 7, the mixture is evaporated to dryness and then brought to pH = 4.5. precipitated with ethyl acetate, washed and dried with ethyl ether.

12.41 g of 8 is obtained; yield 70.5%; TLC / BEW1 Rf: 0.44

12.8 g of 8 prepared above are dissolved in 60 ml of TFA, after 20 minutes at room temperature and evaporated. An oil is formed. Precipitated with ethyl ether to obtain 14.37 g of 9; 100% yield;
TLC / BPEW1 Rf: 0.40.

2,34 g de OHBT et 4,29 ml de N éthylmorpholine. Après 3 heures de réaction à pH compris entre 6 et 7, on évapore la solution de moitié puis on amène à pH = 3,5 par addition d'une quantité suffisante d'acide acétique.13 g of 9 prepared above are dissolved in 91 ml of DMF and 15 ml of water. 7.8 g of

2.34 g of OHBT and 4.29 ml of N-ethylmorpholine. After 3 hours of reaction at pH between 6 and 7, the solution is evaporated in half and then brought to pH = 3.5 by addition of a sufficient amount of acetic acid.

The product precipitates by the addition of ethyl ether; Weight: 16.84g; yield 97.3%, CCM / BPEW1
Rf: 0.62.

 Fifteen grams of the above prepared was dissolved in 70 ml of TFA. After 20 minutes at room temperature, an oil is obtained from which a precipitate is formed by the addition of ethyl ether.

13.86 g of 11 is obtained; 93% yield
TLC / BPEW1 Rf 0.38.

12.55 g of it are dissolved in a mixture of 83 ml of DMF and 13 ml of water. 3.4 g of Boc-Ala are added.
Gly-O-Tcp, 1.75 g of OHBT and 2.88 ml of N-ethylmorpholine.

After 3 to 4 hours of reaction at pH between 6 and 7, the mixture is evaporated to half and then brought to pH = 3.5. The precipitate obtained by addition of ethyl acetate is washed with ethyl acetate and then acidified to pH 3.5. The product 12 obtained is the product (-A) of the reaction scheme. We obtain 13.45 S, a yield of 97.1%; CCM / BEW1
Rf: 0.28.

Preparation of (B)

(J. Amer. Chem., 1972, 94, 5456) dans 6 l de pyridine anhydre, puis on dissout 608 g de Pse OH et on refroidit la solution à 0 C.700 g of

(J. Amer Chem., 1972, 94, 5456) in 6 l of anhydrous pyridine, then 608 g of Pse OH are dissolved and the solution is cooled to 0 ° C.

 In 800 ml of anhydrous pyridine, 500 g of dicyclohexylcarbodiimide are dissolved. This mixture is added slowly (in 7 hours) to the preceding solution while maintaining the temperature between 0 ° C. and 50 ° C. with stirring.

 After stirring overnight at 0 ° C. to 5 ° C., the dicyclohexylurea formed is filtered off. The solvent is evaporated.

The oily residue formed is poured into 15 l of a water and ice mixture with stirring. The product crystallizes, is washed several times with a saturated solution of sodium hydrogen carbonate and then with water and dried. M.p. 75-77 ° C; TLC / BEW1 Rf 0.69; [α] 25 = - 16 (c = 1, acetic acid).

sont ajoutés rapidement à
une solution de 1 ml d'acide chlorhydrique concentré dans
100 ml d'acide formique à température ambiante. Après
10 minutes le solvant est évaporé sous vide. Le résidu
huileux repris dans 15 ml de méthanol est versé dans 150ml
d'éther anhydre agité à 0 C. Le produit formé est filtré, rincé à l'éther et séché sous vide en présence d'anhydride
phosphorique.5.78 g of

are added quickly to
a solution of 1 ml of concentrated hydrochloric acid
100 ml of formic acid at room temperature. After
10 minutes the solvent is evaporated under vacuum. The residue
oily taken up in 15 ml of methanol is poured into 150ml
anhydrous ether stirred at 0 C. The product formed is filtered, rinsed with ether and dried under vacuum in the presence of anhydride
phosphoric.

M.p. 162-1640C; CCM / BEW1 Rf:

 5.15 g of 14 are dissolved in 60 ml of anhydrous pure DMF to which 1.15 g of N-ethylmorpholine are added.

After cooling to -100 ° C., 2.05 g of N-butoxycarbonyl serine are added, then 2.47 g of dicyclohexylcarbodiimide and 2.70 g ofOHOH dissolved in 20 ml of DMF are added dropwise.

 After 1 hour at -10 ° C., it is slowly allowed to return to room temperature. After 6 hours, the DMP is evaporated under vacuum and the residue is taken up in 200 ml of ethyl acetate, the dicyclohexylurea formed is filtered and the solid is washed with aqueous solutions of potassium hydrogen sulfate, potassium sulphate , water, 5% sodium hydrogen carbonate, water.

After drying over anhydrous magnesium sulfate, the solvent is concentrated to 50 ml. After one night at 0 ° C., the product precipitates, it is filtered, rinsed with ethyl acetate, anhydrous ether and then dried under vacuum. 5.3 g is obtained, a yield of 80%. M.p. 111-113 ° C; TLC / BEW1 Rf: 0.63: [?] 25 = - 25 (c = 1, methyl alcohol).

 38.5 g of 15 are added rapidly to 300 ml of TFA previously cooled with OOC. After a few minutes, the acid is evaporated under vacuum, the residue is taken up in anhydrous ether, then filtered and rinsed several times with anhydrous ether and dried.

38.5 g of 16 is obtained, ie a yield of 97%; TLC / BPEW1 Rf 0.60.

 6.80 g of 16 are dissolved in 100 ml of pure DMF.

While cooling with the ice cream, 2.52 nil of N ethylmorpholine and 3.79 g of Boc are added successively.
Thr-Onsu. After 2 hours at room temperature, the DMF is evaporated at 30 ° C., the product solidifies in the presence of ice, it is filtered under vacuum and then washed with water, with a solution of potassium acid sulphate and potassium sulphate at pH = 2, with water, with a solution of 5% sodium hydrogen carbonate and 2 times with water.

 After drying in air and then on phosphorus pentoxide, 7.5 g of 17 is obtained, ie a yield of 9804.

Melting point: 136-138 ° C; TLC / BEW1 RF: 0.69.

est obtenu à partir de 17 en opérant selon la méthode décrite pour la préparation de 16.

18).

is obtained from 17 by operating according to the method described for the preparation of 16.

7.81 g of 18 are dissolved in 90 ml of anhydrous DMF and treated at 0 ° C. with 2.52 ml of N-ethylmorpholine and then 4.64 g of Boc-Phe-ONp.

 After a night in the refrigerator, the DMF is evaporated to 305C empty, the product obtained is washed with water, an aqueous solution of potassium sulphate and potassium acid sulphate, water, an aqueous solution of sodium hydrogen carbonate and water. After drying, it is washed again with isopropanol and ether.

 011 thus obtains 8.22 g, ie a yield of 90%.

Melting point: 153-155 ° C; TLC / BEW1 Rf 0.75; [α] 25 = - 16 (c = 1, acetic acid).

20). TFA, H-Phe-Thr-Ser-Cys-OPse is obtained at
MA from 19 following the procedure described for the preparation of 16.

est obtenu à partir de 20 par addition de Boc-Thr-ONsu selon le mode opératoire décrit pour la préparation de 17.TLC / BPEW1 Rf: 0.65; [?] =? 1805 (c = 1, methanol).

is obtained from 20 by addition of Boc-Thr-ONsu according to the procedure described for the preparation of 17.

TLC / BEW1 Rf 0.65; Melting point: 169 - 172 C.

 2 g of 22 are obtained from 2.03 g of 21 according to the procedure described for the preparation of 16.

TLC / EPAW1 Rf: 0.50.

10.29 g of 22 are dissolved in 100 ml of anhydrous DMF and treated at 0 ° C. with 2.52 ml of N-methylmorpholine and 6.2 g of

After 15 to 20 hours at room temperature, the solvent is evaporated under vacuum. The mixture is stirred in the presence of ice, the precipitate formed is filtered and then washed with water, with an aqueous solution of potassium hydrogen sulfate and sodium sulfate. potassium, with water, twice per 100 ml of isopropanol and finally abundantly with ether and then with acetone.

est obtenu à partir de 23 en suivant le mode opératoire décrit pour la préparation de 16.TLC / BEW1 Rf: 0.57: [α] 25 = -19.5 (c = 1 acetic acid.)

is obtained from 23 following the procedure described for the preparation of 16.

TLC / EPAW1 Rf: 0.50.

13.075 g of 24 are dissolved in 130 ml of anhydrous DMF and treated in an ice-bath with 2.5 ml of
N-ethylmorpholine, 5.11 g of Boc-Trp-Onp and 2.70 g of hydroxybenzotriazole.

 After about an hour at room temperature the reaction is complete, the solvent evaporated, the residue taken up in the ice solidifies gradually; it is finely ground, filtered, washed thoroughly with water and then after light drying twice with 120 ml of warm isopropanol and then with 100 ml portions of warm acetone until satisfactory purity on TLC.

12.6 g of the product having the following characteristics are thus obtained
TLC / EPAW2 Rf: 0.50; [α] 25 = - 17.5 (c = 1, acetic acid).

26) B The following mixture is prepared with ice-cream Lain: 130 ml TFA, 13 ml ethane dithiol and 6 ml anisole.

 13.3 g of 25 are rapidly added with stirring and stirring is continued for 20 minutes at room temperature. After evaporation under vacuum, the residue is taken up twice in 50 ml of isopropanol which is then evaporated under vacuum, taken a second time by the same solvent from which it is filtered after trituration and finally washed with isopropanol ether and then with ether alone and dried.

 13.4 g of the salt of (B) are thus obtained.

 TLC / BEW1 Rf: 0.36.

 Preparation of (C).

 27). In 90 ml of DMF is dissolved 6 g of (A) to which is added 8.16 g of (B), 2.16 g of OHBT and 1.62 g of dicyclohexylcarbodiimide. The pH is between 6 and 7, left at room temperature overnight then the volume is evaporated to a tenth, precipitated with ethyl acetate, filtered and the filtrate is taken up in ethyl ether. Filtered again and then taken up with a little methyl cyanide and then with ethyl ether.

 There is thus obtained 11.57 g of (C), a yield of 91%; TLC / EBPEW1 Rf 0.85.

Preparation of (D).

 11.5 g of (C) are dissolved in 57 ml of methylene chloride and then 6 ml of anisole and 6 ml of ethanedithiol, 57 ml of TFA are added. After 20 minutes of reaction, an oil is obtained; a precipitate is formed by adding 4 volumes of ethyl ether.

There is thus obtained 11.33 g of 28, a yield of 98%; TLC / BPEW1 Rf: 0.58.

 5 g of 28 are dissolved in 125 ml of DMF and 15 ml of ethyl alcohol. The mixture is cooled to +5 ° C. 89 ml of barium hydroxide are added at a temperature of between 15 ° C. and 200 ° C., the addition being cured for not more than 30 minutes. At the end of the reaction, carbon dioxide is bubbled to pH 7. A precipitate is formed, after centrifugation, evaporated to dryness and then precipitated with 5 volumes of ethyl acetate and washed with sodium acetate. 'ethyl. Weight 2 g; yield 55%; TLC / BPEW1 Rf: 0.32.

 Preparation of (D): CM 14.458.

 39). 2 g of 29 are dissolved in 200 ml of a 50% degassed acetic acid solution. At room temperature, under nitrogen, protected from light, 10 ml of a normal solution of silver nitrate are introduced. It is left overnight at room temperature in the absence of light under nitrogen. The solution is then evaporated to dryness under nitrogen, the residue is taken up in ether and then in 250 ml of water. The mixture is centrifuged, decanted and the solid is taken up in 250 ml of water and 250 ml of degassed DMF. A stream of hydrogen sulphide is passed through for a few minutes, then centrifuged, decanted and the black pellet washed.

 The washings are combined, degassed, 3 1 of 0.2 N acetic acid solution is added thereto. The solution obtained (4.2 l) is added over 16 hours under stirring and under nitrogen at pH = 6.9. to a solution having the following composition: 1.5 l of water, 950 g of potassium ferricyanide and 3750 g of ammonium acetate. The pH is maintained at 6.9 by addition of a solution of ammonia for 2 hours. The mixture is left stirring for 2 hours and the solution is then added with concentrated acetic acid. It is desalted by passing through Biorad Ag 3x4 resin and then fixed on Biorex 70 resin, these dcw; resins being in acid form. It is washed and then eluted with 50% acetic acid.

 After evaporation to dryness, the residue is taken up in ether, the precipitate is filtered off and the excess of ammonium acetate is then sublimed. 1.46 g of crude product are obtained.

Purification is performed first on
Sephadex G 25 (molecular sieving) and then partition chromatography on Sephadex G 25, eluent: butanol (4), acetic acid (1), water (5). 194.7 mg of pure product is obtained, TLC / BPEW1 Rf: 0.21; [α] 25 = ~ 32 (C = 1, acetic acid a 590).

EXAMPLE 2 Preparation of CM 14.461
Reaction scheme

Preparation of (A)
1). Z-Tau-Phe-Phe-O CH3
75 g of Boc-Phe-Phe-OMe are dissolved in 300 ml of a solution of 2.5 N hydrochloric acid in ethyl acetate. After 15 minutes a precipitate is formed, added with ethyl ether, filtered and dried.

60.24 g of H-Phe-Phe-OMe is obtained, ie a yield of 9850.

This product is taken up in a mixture volume for volume water and TIIF. It is neutralized with 15 g of sodium hydrogen carbonate, after 20 minutes of reaction, evaporated to dryness and then taken up in TIF, 21.1 g are added.
Z-Cl-Tau. The reaction is slightly exothermic, a white precipitate forms.

 After one night at ambient temperature, the mixture is filtered, evaporated to dryness and the residue is taken up in ethyl acetate. Potassium sulphate solution, potassium sulphate solution and then sodium chloride solution are washed with potassium sulfate solution. Dry, evaporate to dryness and then take up the residue with pentane.

 37.37 g of 1 are obtained, that is to say a turnover of 89%; TLC / Truth Rf: 0.5.

2). Z -Tau-Phe-Phe-OH
37 g of Z-Tau-Phe-Phe-OMe dissolved in 370 ml of dioxane are deprotected in 148 ml of normal sodium hydroxide.

After 30 minutes at room temperature, it is evaporated to dryness, the residue is taken up in water and then acidified with an acetic acid solution. A gum is formed which is extracted with ethyl acetate and then dried over magnesium sulfate. It is evaporated to dryness under vacuum, the residue is taken up in pentane and then evaporated to dryness under vacuum.

 31 g of product 2 are obtained, ie a yield of 86%; TLC / Truth Rf: 0.31.

 3). HBR, H-Tau-Phe-Phe-OH is obtained from C 2 according to the process described for Compound 2 of Example 1.

27.55 g of 3 is formed, representing a yield of 98.6%
TLC / BEW1 Rf: 0.30; TLC / BPEW1 Rf 0.60.

et 1,5 g de OIIBT. Le PH est amené à une valeur comprise entre 6 et 7 par addition d'une quantité suffisante do N éthyl- morpholine. Après 4 heures de réaction, on évapore sous vide saris chaufrer jusqu'à obtention d'une huile. On diluc daiis un volume égal d'eau puis on amène à pll 4,5.5 g of the compound 3 described above are dissolved in a solution comprising 40 ml of DMF and 6.5 ml of water to which 5.7 g of

and 1.5 g of OIIBT. The pH is brought to a value between 6 and 7 by the addition of a sufficient amount of N-ethyl morpholine. After 4 hours of reaction, the mixture is evaporated under vacuum and heated until an oil is obtained. It is diluted with an equal volume of water and then brought to pH 4.5.

It is extracted with ethyl acetate and then the organic phase is washed with a potassium hydrogen sulfate solution, a potassium sulphate solution, dried over magnesium sulphate and then evaporated to dryness, the residue is taken up in pentane. then evaporate to dry again.

We obtain 8.05 g, a yield of 100%
TLC / BEW1 Rf: 0.67.

8 g of this product are prepared from 8 g of 4 according to the process described for compound 9 of Example 1. The yield is 98%; TLC / BEW1 Rf: 0.26.

7 g of this product are prepared from 8.2 g of 5 according to the process described for compound 10 of Example 1. Yield 71%; TLC / BEW1 Rf: 0.57.

6.36 g of this product are prepared from 7 g of 6 according to the process described for compound 11 of Example 1.

 This compound is (A) of the reaction scheme.

5.36 g of 7 dissolved in 30 ml of DE1F and 4 ml of water.

2.52 g of Boc-Ala-Gly-O-Tcp and 0.86 g of OHBT and then a sufficient amount of N-ethylmorpholine are added to bring the medium pest between 6 and 7.

 After 4 hours of reaction, it is evaporated to dryness under vacuum and then taken up with acidified ethyl ether.

A solido is obtained which is dried after spinning weight 5.98 g; 100% yield; TLC / BEW1 Rf 0.49.

 Preparation of (B).

 The preparation of this compound is described in Example 1.

 Preparation of (C).

9). 2 g of (A) are dissolved in 30 ml of DMP, 2.9 g of (D) are added and then neutralized by addition of N-ethylmorpholine. To the solution is added 0.92 g of
BOP and 0.26 ml of N-ethylmorpholine; after 2 hours, 0.2 g of BOP and 0.05 ml of N-ethylmorpholine were added again. After wetting overnight at room temperature, filter and precipitate with ethyl acetate. After wringing, the precipitate is washed with ether, with water and then with ether.

 2.42 g of 9 is obtained, ie a yield of 54.1%.

Preparation of (D).

2.19 g of this product are obtained from (C) according to the process described in Example 1 for the preparation of 16; 99% yield.

 1.48 g of this product are obtained from 2 g of 10 according to the process described in Example 1 for the preparation of 17; 100% yield.

12). CM 14.461 = (D)
This compound is prepared according to the method described for the last step of Example 1.

From 1,48 g of 11 mi gets 0.670 g of
CM 14.461, representing a yield of 52%; TLC / BPEW1 Rf: 0.53; [α;] 25: - 32 (C = 1, 5% acetic acid).

EXAMPLE 3 CM 14.462.

Reaction scheme

(D) = CM 14.462
Preparation of (A).

 Compound 3 of Example 2 is first prepared: HBR, H-Tau-Phe-Phe-OH. 20 g of this product are dissolved in 160 ml of DMF and 26 ml of water. 19 g of Boc-Cys-ONp, 6 g of OHBT and a sufficient amount of N-ethylmorpholine are added to the solution to bring the pH to between 6 and 7. After 3 to 4 hours of reaction, the mixture is evaporated to dryness. under strain and then acidified to pH = 3.5. Then taken up with ethyl acetate, the liquid phase is washed with water, with a solution of potassium sulphate, a solution of sulphate of potassium and a solution of sodium chloride. It is dried over magnesium sulphate and then evaporated to dryness and the residue is taken up in ethyl ether.

 Thus 21.84 g of 1 is obtained, ie a yield of 78.6%; TLC / BEW1 Rf 0.77.

 Preparation of (B).

2). Preparation of

This compound is identical to compound 24 described in Example 1.

 6 g of EC 14.335 are dissolved in 61 ml of DMF.

1.16 ml of N-ethylmorpholine, 2.34 g of Boc (D) Trp-ONp and 1.22 g of OHBT are added. The pH is adjusted between 6 and 7 by addition of N-ethylmorpholine. After 3½ hours of reaction, it is evaporated in half under vacuum and then precipitated by the addition of 3 volumes of cold water. After wringing, the solid is washed successively with water, potassium hydrogen sulfate solution, potassium sulfate solution, water, sodium hydrogen carbonate solution and water.

 After drying, the mixture was washed again with 75 ml of isopropanol, diethyl ether, 50 ml of ethanol, ethyl ether, 40 ml of acetone and again with ethyl ether.

Thus 4.86 is obtained; of 3, a yield of 72%; TLC / BEW1 Rf: 0.68.

3.4 g of 3 are dissolved in a solution of 14 ml of TFA, 13 ml of anisole and 1.4 ml of ethanedithiol. After 20 minutes at room temperature, precipitate with isopropanol
3.59 is obtained from 4, ie a yield of 100%;
TLC / BEW1 Rf 0.48.

Preparation of (C)
(C) is obtained by addition of (A) and (B) according to the procedure described in Example 2 for the preparation of compound 9.

3.10 g of (C) are obtained, ie a yield of 64%;
TLC / BEW Rf: 0.61.

Preparation of (D).

 This product is obtained from (C) following the procedure described in Example 1 for the preparation of compound (C).

From 3.8 g of (C) there were obtained 3.63 of 5, giving a yield of 94.8%; TLC / BEW1 Rf 0.76.

 Deprotection of lysine to obtain 6 from 5 is carried out according to the procedure described in Example 1 for the preparation of compound 6.0n. 3.07 g of 6 is obtained from 3.6 g of 5. yield is 100%; CCM / BEW1; Rf 0.36.

7). (D) = CM 14.462
The cyclization of the peptide 6 and its purification are carried out according to the procedure described for the last step of Example 1 (preparation of D).
TLC / BPEW1 Rf: 0.59; [α] 25 = - 48 (C = 1, 5% acetic acid).

EXAMPLE 4 CM 14.468

Reaction scheme

(D) = CM 14.468
Preparation do (A).

 1). This product is the one described in 1 in Example 3.

Preparation of (B).

35 ml of DES to which was added 2.57 g of Boc-Phe-ONp and 1.05 g of OHBT and a sufficient amount of N-ethyl morpholine to bring the fold between 6 and 7. After 4 hours of reaction at room temperature, It is evaporated to dryness and then precipitated with 5 volumes of ice-water and washed with a solution of potassium acid sulphate, a solution of potassium sulphate, a solution of sodium hydrogen carbonate, a solution of potassium sulphate and a solution of sulphate. potassium acid, water and pentane acidified with 1% acetic acid.

After drying, 4.18 g of product is obtained, giving a yield of 97%; TLC (N butanol 96, acetic acid 1, water 1) Rf: 0.77.

The 4.18 g dc 2 obtained above are dissolved in 21 ml of TPA. After 20 minutes at room temperature, the mixture is evaporated to dryness and then the residue is taken up in pentane and dried; Weight 4.18 g; yield 99 ', u'; TLC / (methyl trichloride 90, methyl alcohol 20, acetic acid 03) Rf: 0.09.

 This compound is obtained from 3 by following the procedure described for the preparation of 2.

 3.69 g of i are obtained, ie a yield of 78%.

TLC / (methyl trichloride 90, methyl alcohol 20, acetic acid 03) Rf: 0.61.

4.3 g of 5 are obtained from 4.3 g of 4 by dissolving in 19 ml of TFA and then evaporating at room temperature after 20 minutes at room temperature, the residue being taken up in ethyl ether. Yield 98.4%
TLC / (methyl trichloride 90, methyl alcohol 20, acetic acid 03) Rf: 0.18.

 This compound is obtained from 5 by addition of Boc-Thr-ONSu following the procedure described for the preparation of 2.

Starting from 3.2 g of 5, 2.91 g of 6 is obtained, ie a yield of 83%; TLC / (butyl alcohol 96, acetic acid 01, water 03) Rf: 0.83.

4,16 g de ce produit sont obtenus à partir dc 3,7 g dc 7 par addition de

selon le procédé décrit pour la préparation de 2; rendement 88,7% ; CCM/ (mélange de solvants cité pour 3) Rf : 0,87.

3.8 g of this compound are obtained from 6 (3.7 g) in the same manner as for the preparation of 5. Yield 99.8%; TLC / (solvent mixture cited for 3) Rf: 0.43.

4.16 g of this product are obtained from 3.7 g of 7 by addition of

according to the process described for the preparation of 2; yield 88.7%; TLC / (solvent mixture cited for 3) Rf 0.87.

4 g of 9 are obtained from 8 (4 g) according to the procedure described for the preparation of 98.9% yield; TLC / (solvent mixture cited for 3)
Rf: 0.14.

10 is obtained from 9 by addition of
Boc- (D) Trp-ONp according to the procedure described for the preparation of 2.

4 g of 9 gives 4.1 g of 10; 90% yield;
TLC / (solvent mixture cited for 3) Rf: 0.82.

To 4 g of the above-mentioned solution in 20 ml of methylene chloride, 2 ml of anisole, 2 ml of ethanedithiol and 20 ml of TFA are added. After 20 minutes at room temperature, evaporated, taken up with water and washed and dried to obtain 4 g of 11; 98% yield
TLC / (in the solvent described for 3) Rf: 0.30.

 Preparation do - (C).

 12). This product is obtained by addition of (A) and (B) according to the method described for the preparation of the compound (C) of Example 2.

 3.75 g of 12 was obtained from 2.12 g of (A) and 3.5 g of 11, giving a yield of 77.9%; TLC / (in the solvent mixture described for 3). Rf: 0.66.

Preparation of (D).

 13 is obtained in a yield of 100% according to the process described for compound 16 of Example 1.

TLC / BPEW1 Rf 0.72.

1.3 g of 14 is obtained from 2 g of 13 by following the procedure described for compound 17 of Example 1. 1.3 g of product; CCM / BPEW1
Rf: 0.51.

 15). (D) = CM 14.468.

 This product is prepared from 14 according to the procedure described in Example 1 for the final step of the preparation of (D).

0.843 g of CM 14.468 is obtained; CCM / BPEW1
Rf 0.77; [α] 25 = - 54.4 (C = 1, 5% acetic acid)
EXAMPLE 5 CM 14.470
Reaction scheme

(D) = CM 14.470
Compound (A) is identical to that described in Example 3.

Compound (B) is prepared from TFA, H-Phe
Cys-OPse by successive additions of protected amino acids
Acin according to the method described in Example 4.

 011 then prepares (C) and then (D) following the procedures described above.

(D) = CM 14.470; TLC / BPEW1 Rf: 0.74 [α] 25 = - - 76 (C = Li acetic acid)
EXAMPLE 6: CM 14.471
Reaction pattern.

(D) = CI 14.471
The compound (A) is identical to that described in Example 3.

 By addition of (B) prepared according to the methods described above, (C) and then (D) are obtained.

(D) = CM 14.471; CMM / BPEW1 Rf: 0.62.

EXAMPLE 7 CM 14.473
Reaction scheme

(D) = CM 14.473
(A) is prepared by successive additions of amino acid, starting from TFA, H-Phe-Phe-OMe.

 (B) is identical to (B) of Example 5.

By adding (A) and (B) and operating according to the methods described above, (C) is obtained, then (D);
TLC / BPEW1 Rf: 0.58.

EXAMPLE 8 CM 14.500
Reaction scheme

(D) = CM 18,500
(A) is obtained from Boc-Phe-Tau-Phe-OH by operating according to the methods described above.

 (ss) is identical to that of Example 6.

 Addition of (A) and (B) followed then (C) and then (D) following the procedures described above: [α] 20 = + 19.2 (C = 0.5, acetic acid).

 The peptides of the present invention as well as their pharmacologically acceptable salts play an active role in the control of growth hormone secretion in both animals and humans (acromegalic therapy).

 The differential inhibitory action of these products on insulin and glucagon production and in glyceric regulation makes them useful in the control of diabetes mellitus insulin-dependent or non-insulin-dependent.

 These molecules have little or no action on digestive secretions, another differentiating factor compared to the profile of native somatostatin.

 The products according to the invention have a low toxicity; in no case during the tests did the treated animals show any alarming signs.

 The dosage range clinical use of these substances varies with the desired therapeutic effects and with each paticnt, it is generally between 0.1 and 10 mg of active ingredient.

 The peptides of the present invention may be administered intravenously, subcutaneously or intramuscularly in solution in a solvent such as physiological saline (isotonic saline) or in phosphate buffer and always under rigorous sterility conditions; the saline solutions generally comprise from 1 to 5% by weight of the product according to the invention.

 The products according to the invention have been examined with regard to their biological activity.

Biological tests.

Inhibition of secretion of pancreatic hormones and growth hormone (GH) was determined in vivo in native Sprague Dawley male rats
Charles River from 200-to 220 g. Dosages of insulin, glucagon and growth hormone are carried out by radioimmunological techniques.

 Inhibition of acid gastric secretion in vitro was investigated from isolated gastric mucosa of guinea pig.

 In each case, the concentration of peptide inhibiting 50% of the hormonal secretion or gastric secretion is determined. In the following table the results are exlirimé compared to the somatostatin used as reference product. Since the somatostatin activity has been considered in each case to be 100, the figures for the peptides of the invention express the ratio of the activity of the peptide considered to the activity of somatostatin.

Inhibition of pancreatic hormone secretion of GH and acidic gastric secretion by four somatostatin derivatives in comparison with native somatostatin

<tb><SEP> Insulin <SEP> Glucagon <SEP> GH <SEP> Secretion
gastric <tb><SEP>
<tb> Somatostatin <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
<tb> CM <SEP> 14.461 <SEP> inactive <SEP> 20 <SEP> 162 <SEP># 0
<tb> CM <SEP> 14.462 <SEP> 30 <SEP> 44 <SEP> 174 <SEP> 11
<tb> CM <SEP> 14,470 <SEP> 24 <SEP> 63 <SEP> 136 <SEP>
<tb> CM <SEP> 14.471 <SEP> 72 <SEP> 157 <SEP> - <SEP>

Claims (7)

1. Saatostatic analogs characterized in that they correspond to the general formula wherein R denotes an II, an amino acid, a D-peptido such as Ala-Gly for example. X1 = (L) -Lys or single bond. X2 = (L) -Asn, Tau or single bond. X3 = (L) -Phe or (L) -Asn; . X4 = (L) -Phe, Tau or single bond one of the two groups X2 or X4 is obligatorily Tau; . X5 = (L) -Thr, (L) -Phe or single bond; , X6 = (L) -Scr or single bond, TRP = (L) -Trp or (D) -Trp. CYS = (L) -Cys or (D) -Cys, and the addition salts of these products with pharmaceutically acceptable acids. 2. Products according to claim 1, characterized in that the analog of somatostatin has the formula

 3. Products according to claim 1, characterized in that the somatostatin analogue has the formula

 4. Products according to claim 1, characterized in that the somatostatin analogue has the formula

S. products include revondication 1, characterized in that the somatostatin analogue has the formula

 6. Products according to claim 1, characterized in that the somatostatin analogue has the formula

  7.Products according to claim 1, characterized in that the somatostatin analogue has the following formula:

 8. Products according to claim 1, characterized in that the somatostatin analogue has the formula

 9. Products according to claim 1, characterized in that the somatostatin analogue has the formula

 10. Medicines useful in particular for the treatment of diseases result from a disturbance of the secretion of growth hormone, insulin or glucagon, characterized in that they contain at least one product according to one of claims 1 to 9. 11. Medicaments according to claim 10, characterized in that they are administered by intravenous, subcutaneous or intramuscular injections.