FR2523125A1 - Somatostatin analogues having different activity profiles - inhibit growth hormone, insulin and glucagon secretion, are resistant to proteolysis and have little effect on gastric secretion - Google Patents

Abstract

Somatostatin analogues of formula (I) and their acid addition salts are new. (where R is H, an aminoacid or a dipeptide e.g. Ala-Gly; X1 is (L) Phe, (D) Ala or bond; X2 is (L)Phe, (D) Phe or Gly; X3 is (L) Phe, (D) Phe or a bond and the Cys groups have (L) or (D) configuration). (I) regulate the secretion of insulin, glucagon and growth hormone (GH) and may be used to regulate glycemic levels in insulin dependent and non-insulin dependent diabetes and to control acromegalia. Due to the (D) aminoacids, (I) have better resistance to proteolysis than somatostatin and have prolonged activity. (I) have little action on secretion of gastric juices and low toxicity. The activity profile of the pcds. differ from that of somatostatin and suitable cpds. may be selected for the required activity. (I) are administered as 1-5% solutions by intravenous, intramuscular or sub-cutaneous injection ot give doses of 0.1-10 mg.

Description

Somatostatin analogues with modified biological activity and drugs containing them.

et qui inhibe les sécrétions endogènes d'hormones de croissance (GH), d'insuline et de glucagon. Les abréviations utilisées sont celles recommandées pour les aminoacides par la commission de nomenclature biochimique de l'lUPAC (Journal of Biological Chemistry, 1972, 247, page 977).Somatostatin is a structural tetradecapeptide

and which inhibits the endogenous secretions of growth hormones (GH), insulin and glucagon. The abbreviations used are those recommended for amino acids by the IUPAC Commission on Biochemical Nomenclature (Journal of Biological Chemistry, 1972, 247, page 977).

 This same peptide also significantly reduces the levels of digestive secretions. The biological half-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.

 Many analogs have been described by various authors in order to obtain either dissociations of biological activity or a prolonged biological half-life.

dans laquelle R R désigne un H, un acide aminé, un dipeptide comme Ala-Gly par
exemple; x1 = (L) Phe, (D) Ala ou liaison simple; * X2 = (L) Phe, (D) Phe ou Gly; . X3 = (L) Phe, (D) Phe ou liaison simple; . Cys = (L) Cys ou (D) Cys;
Ces produits se caractérisent en particulier - par une meilleure résistance à la protéolyse grâce à l'introduction
d'un ou plusieurs acides aminés de la série (D) en des points judi
cieusement choisis de la molécule de façon à conserver ou modifier
l'activité biologique de la somatostatine native.Ceci se traduit
dans les faits par l'obtention de molécules présentant un effet
retard prolongé; - par une dissociation d'activité biologique par rapport à la soma
tostatine prise comme produit de référence aussi bien au niveau
de l'inhibition des trois hormones de base impliquées dans le
diabète qu'au niveau de l'inhibition des sécrétions digestives.the products of the present invention correspond to the general formula

wherein RR denotes an H, an amino acid, a dipeptide such as Ala-Gly by
example; x1 = (L) Phe, (D) Ala or single bond; * X2 = (L) Phe, (D) Phe or Gly; . X3 = (L) Phe, (D) Phe or single bond; . Cys = (L) Cys or (D) Cys;
These products are characterized in particular - by a better resistance to proteolysis thanks to the introduction
of one or more amino acids of the series (D) at judi points
carefully chosen from the molecule so as to preserve or modify
the biological activity of native somatostatin.
in fact by obtaining molecules having an effect
prolonged delay; - by a dissociation of biological activity compared to soma
tostatin taken as reference product both at the level of
inhibition of the three basic hormones involved in the
diabetes in terms of inhibition of digestive secretions.

The products of the present invention are represented
by the following formulas
CN 14.479

CM 14.481

CM 14.480

CM 14.481

CM 14.482

CM 14.483

CM 14.498

CM 14.499

CM 14.501

 These compounds are always used in the form of an addition salt such as hydrochloride, sulfate, phosphate; acetate, maleate, citrate, benzoate, succinate, malate, ascorbate ...

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 synthesis.

 They are synthesized according to a mixed process between solid phase synthesis and solution synthesis. The coupling operations between two 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 washing operations using suitable 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 sufficient solubility in polar solvents such as dimethylformamide thus making it possible to perform the coupling operations in the liquid phase.

 The protective group that makes it possible to apply this strategy is 2- [4- (phenylazo) -benzylsulfonyl] ethyl ester (OPSE) which is stable in an acid medium and cleavable in a basic medium according to the elimination process.

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

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

 The lysine side chain amine is protected during the phases of elongation of the peptide by the 2- (methylsulfonyl) -ethoxycarbonyl (Mse) group which is stable in an acid medium and cleavable in a basic medium.

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

 During the elongation phases, the temporary protection of the amines at position a of carboxyl is provided by the tertiobutyloxycarbonylamino (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 too, the deprotected product will be isolated from the reaction medium by the addition of 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 elongation phases, the protected peptide obtained will be released from the protections of the lateral lysine and end carboxylic groups 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. He will be able at this level. depending on its degree of purity, it can be used as it is for the final oxidation stage or 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 of the two
cysteines by a heavy metal salt (silver nitrate
for example);
- elimination of heavy metal by a hydro treatment
sulfide gene;
- degassing and dilution away from the solution
thus obtained;
- addition of this solution gradually and pH con
trilled with an oxidation medium consisting of a buffer
ammonium acetate containing the oxidizing agent that is
potassium ferricyanide.

This operation will always be carried out in condi
dilution (2 liters per gram of product)
treated) in order to minimize the risk of polymerisation
as a consequence of intermolecular oxidation.

 The final product is then stripped of complex ions from the oxidation phase by passage on suitable resin, 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 stater insufficient, it may be subjected to other purification techniques, such as partition chromatography or passage over ion exchange resins ...

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

Symbols and abbreviations used.

Boc

CHBT: hydroxybenzotriazole

mAb:

ONSu

MSc:

ONSu

ONp

: deméthylformamide
TE : acide trifluoracétique ou trifluoracétate
CC!! : chromatographie sur couches minces de silice
Merck 60F 254
HPLC : chromatographie liquide haute performance
BPEW1 éluant de chromatographie butanol (50), pyridine (12),
CH3CO2H (12), H2O (25).ODSE:

We p

: demethylformamide
TE: trifluoroacetic acid or trifluoroacetate
DC !! : thin layer chromatography of silica
Merck 60F 254
HPLC: high performance liquid chromatography
BPEW1 butanol (50) chromatography eluent, pyridine (12),
CH3CO2H (12), H2O (25).

 The amino acid analysis of the various intermediates mentioned in the following examples was carried out.

EXAMPLE 1: CM 14.479

<tb> 1) <SEP> Boc-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm
<tb><SEP> 3.5 <SEP> g <SEP> of <SEP> TFA, <SEP> H- (D) <SEP> Cys-OPse <SEP> are <SEP> dissolved <SEP> in <SEP > 35 <SEP> ml
<tb><SEP> Acm
DMF to which was added 2.57 g of Boc-Phe-ONp and 1.05 g of OHBT and a sufficient amount of N-ethylmorpholine to bring the pH between 6 and 7. After 4 hours of reaction at room temperature evaporated to dryness and then precipitated with 5 volumes of ice water and washed with potassium potassium sulphate solution, potassium sulphate solution, sodium hydrogen carbonate solution, potassium sulphate solution, a solution of potassium sulphate and a solution of potassium sulphate. of potassium sulphate, water and pentane acidified with 1% acetic acid.

After drying, 4.18 g of product is obtained, ie a yield of 97%; TLC / (n-butanol 96, acetic acid 1, water 1);
Rf 0.77.

<Tb>

2) <SEP> TFA, <SEP> H-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm
<Tb>
The previously obtained 4.18 g of 1 are dissolved in 21 ml of TFA. After 20 min at room temperature, evaporated to dryness and the residue is taken up with pentane and dried; weight 4.18 g; 99% yield; TLC / (methyl trichloride 90, methyl alcohol 20, acetic acid 03), Rf: 0.09.

<Tb>

3) <SEP> Boc-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm
<Tb>
This compound is obtained from 2 by following the procedure described for the preparation of 1.

 3.69 g of 3 are obtained, ie a yield of 78;

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

<Tb>

4) <SEP> TFA, <SEP> H-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm
<Tb>
4.3 g of 4 are obtained from 4.3 g of 3 by dissolution in 19 ml of TFA, followed by evaporation to dryness after 20 min 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.

<Tb>

5) <SEP> Boc-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm
<Tb>
This compound is obtained from 4 by addition of
Boc-Thr-ONSu following the procedure described for the preparation of 1.

Starting from 3.2 g of 4, 2.91 g of 5 is obtained, ie a yield of 83%; TLC / (butyl alcohol 96, acetic acid 01, water 03) Rf: 0.83.

<Tb>

6) <SEP> TFA, <SEP> H-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm
<Tb>
3.8 g of this compound are obtained from 5 (3.7 g) in the same manner as for the preparation of 4. Yield 99.8%; TLC / (solvent mixture cited for 2), Rf: 0.43.

<tb> 7) <SEP> Boc-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<tb> 4.16 g of this product is obtained from 3.7 g

<tb> of <SEP> 6 <SEP> by <SEP> addition <SEP> of <SEP> Boc-Lys-ONp <SEP> according to <SEP><SEP> method <SEP> describes <SEP> for <SEP > the
<tb><SEP> Msc
<tb> preparation of 1; yield 88.7% TLC / (solvent mixture cited for 2), Rf 0.87.

<Tb>

8) <SEP> TFA, <SEP> H-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
4 g of 8 are obtained from 7 (4 g) according to the procedure described for the preparation of 4; yield 98.9%;
TLC / (solvent mixture cited for 2), Rf: 0.14.

<Tb>

9) <SEP> Boc- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
9 is obtained from 8 by addition of
Boc- (D) Trp-ONp according to the procedure described for the preparation of 1.

4 g of 8 give 4.1 g o; 90% yield;
TLC / (solvent mixture cited for 2), Rf: 0.82.

<Tb>

10) <SEP> TFA, <SEP> H- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
To 4 g of 9, suspended in 20 ml of methylene chloride, 2 ml of anisole, 2 ml of ethanedithiol and 20 ml of TFA are added. After 20 min at room temperature, evaporated, taken up with water and then washed and dried to obtain 4 g of 10; 98% yield; TLC / (in the solvent described for 2), Rf: 0.30.

<Tb>

11) <SEP> Boc-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
105 g of 10 are dissolved in DES, 31 g of Boc-Phe-ONp are added to the solution, then 9.9 g of OHBT and a sufficient amount of N-ethylmorpholine are added so that the pH value is 7.

After stirring for 1 hour at ambient temperature, the mixture is evaporated under vacuum.

The oil obtained is taken up in ethyl acetate, the solid precipitate is filtered off, dried and then washed with water and with ethyl acetate and dried.

102 g of 11 is obtained, ie a yield of 89%.

<Tb>

12) <SEP> H-PHe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP> Msc <SEP> Acm
<Tb>
400 ml of TFA containing 10% of ethanedithiol and 10% of anisole are cooled in an ice bath. 101 g of solution are dissolved in this solution. After stirring for 20 minutes at room temperature, the solid formed is taken up in vacuo and the residue is taken up in ether, drained, washed three times with ether and dried.

106 g of 12 is obtained, ie a yield of 97%.

<Tb>

13) <SEP> Boc- (D) <SEP> Phe-Phe- (D) <SEP> Trp-Lys-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
10 g of 12 and 2.6 g of Boc- (D) Phe-ONp are dissolved in 60 ml of DMF, 0.8 g of OHBT and a sufficient amount of N-ethylmorpholine are added so that the pH is about 7. After stirring for 1 hour, the mixture is evaporated to dryness, diluted with ethyl acetate, the solid which precipitates is filtered off, washed three times with water and dried and then washed three times with ethyl acetate, three times with methanol and twice with acetone.

9 g of 13 is obtained, representing a yield of 83%

<tb> 14) <SEP> H- (D) <SEP> Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP>
<tb><SEP> Msc <SEP> Acm
<Tb>
40 ml of TFA containing 10% anisole and 10% ethanedithiol are cooled to 0 ° C. 8.8 g of the product 13 prepared above are added with stirring. After stirring for 20 min at room temperature, the product is evaporated for 9 sec and the residue is taken up in ether. The precipitate formed is drained, washed three times with ether and dried.

9 g is obtained, ie a yield of 100%.

<Tb>

15) <SEP> Boc-Cys (D? <SEP> Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Acm <SEP>. <SEP> Msc <SEP> Acm
<Tb>
This compound is obtained from 14 by addition of
Boc-Cys-ONp in the DES according to the process described for the preparation
Acm of 13.

<Tb>

16) <SEP> H-Cys- (D) <SEP> Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP> I <SEP> I
<tb><SEP> Acm <SEP> Msc <SEP> Acm
<Tb>
This compound is obtained from 15 according to the process described for the preparation of 14.

<tb> 17) <SEP> H.-Cys- (D) <SEP> Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe-Cys-OH
<tb><SEP> Acm <SEP> Acm
<Tb>
2.02 g of 16 are dissolved in 30 ml of DMF, after cooling to 0 ° C., 10 ml of water are added, then 15 ml of a 0.4 N solution of barite. After stirring for 30 minutes at room temperature 40 ml of water are added and neutralized with carrier gas. The pellets are centrifuged and washed with water. The aqueous phases are combined and then extracted four times with ethyl acetate and evaporated to dryness. The solid is washed with ethyl acetate, drained, washed with ether and dried.

1.4 g is obtained, ie a yield of 95%.

<Tb>

18) <SEP> H-Cys- (D) <SEP> Phe-Phe- <SEP> (D) <SEP> Trp-Lys-Thr-Phe-Phe-Cys-OII
<tb><SEP> I
<tb><SEP> Ag <SEP> Ag
<Tb>
2.02 g of 17 are dissolved in 160 ml of 50% acetic acid, the solution is degassed and then 13 ml of an aqueous solution of silver nitrate (108 mg / ml) are added.

 After one night at room temperature, protected from light, it is evaporated to dryness in the dark, the residue is taken up in ether, drained, dried and washed twice with water.

19) CM 14.479

 The silver salt obtained above is dissolved in 100 ml of DMF, the solution is degassed, sulphuretted hydrogen is bubbled for 5 min until saturation, then centrifuged. The supernatant is degassed under vacuum, then the medium is diluted by the addition of 1900 ml of degassed 0.2 N acetic acid. The following evolution is then added over 15 hours under stirring under nitrogen, the pH being maintained at 6.8 with ammonia: 1 liter of degassed water, 800 mg of potassium ferricyanide, 1.6 g ammonium acetate.

 After spinning, the solid is washed with water and then with alcohol. On the other hand, the filtrate is passed on resin AG x 3, then at pH 5 on Amberlite Gc resin 50 which fixes the product. This is then eluted with 80% acetic acid. The solution is evaporated to dryness and then taken up with ether, drained and dried.

The solid thus formed is identical to the previous one,
they are combined to be purified by chromatography on Sephadex
C 15, with 50% acetic acid as eluent.

 TLC / BPEW1, Rf 0.72.

EXAMPLE 2: CM 14.480

<tb> 1) <SEP> H-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys-Opse, <SEP> TFA
<tb><SEP> Msc <SEP> Acm
<Tb>
This compound is identical to the compound 12 described in Example 1.

<Tb>

2) <SEP> Boc-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys-OPse
<tb><SEP>'Isc<SEP> Acm
<Tb>
43 g of 1 and 10.9 g of Boc-Phe-ONp are dissolved in 200 ml of DMF, 3.4 g of OHBT and enough N-ethylmorpholine are added so that the fold is equal to 7.

 After stirring for 1 hour, the mixture is evaporated to dryness and the residue is taken up in ethyl acetate, the precipitate formed is filtered off, washed three times with water, dried and washed three times with ethyl acetate. ethyl, three times with methanol and three times with acetone.

After drying, 41 g are obtained, ie a yield of 88%.

<Tb>

3) <SEP> H-Phe-Phe- (D) -Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP> I <SEP> I
<tb><SEP> Msc <SEP> Acm
<Tb>
160 ml of TFA containing 107 ml of ethanedithiol and 10% of anisole are cooled in an ice bath and 40.5 g of 2 are added. After dissolution, the mixture is stirred for 20 minutes at room temperature and then evaporated to dryness. The residue is taken up in ether, then drained, washed three times with ether and dried.

<Tb>

4) <SEP> Boc- (D) <SEP> Ala-Phe-Phe- (D) -Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
10 g of 3 and 2.19 g of Boc- (D) Ala-OTcp are dissolved in 80 ml of DMF to which 0.730 g of OHBT and a sufficient quantity of N-ethylmorpholine are added so that the pH is equal to 7.

 After stirring for 60 min at room temperature, the mixture is evaporated to dryness, the residue is taken up in ethyl acetate, drained, washed with ethyl acetate and dried twice in succession and then washed twice with a saturated solution of sodium bicarbonate, twice with water and twice with ethanol, and then dried.

9.65 g of 4 is obtained, ie a yield of 93Z.

<tb> 5) <SEP> H- <SEP> (D) <SEP> -Ala-Phe-Pbe- <SEP> (D) <SEP> -: p-Lys-Thr-Phe-Phe- (D) ) <SEP> Cys-OPse <SEP> TFA
<tb><SEP> sc
<Tb>

40 ml of TFA containing 10% of anisole and 10% of ethanedithiol are cooled to 0 ° C., 9.6 g of 4 are then added and then the mixture is stirred for 20 minutes at room temperature. After evaporation to dryness, an oil remains which is precipitated in ether and then wrung out. Wash three times with ether and then dry to obtain 9.6 g of product; 97% yield.

<Tb>

6) <SEP> Boc-Cys- (D) <SEP> Ala-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) -Cys-OPse
<tb><SEP> Acm <SEP> Msc <SEP> Acn
<Tb>
9.5 g of 5 and 2.25 g of

<tb> Boc-Cys-ONp, <SEP> on
<tb><SEP>
<tb><SEP> Acm
<tb> adds a sufficient quantity of N-ethylmorpholine so that the pH is equal to 7. After 60 minutes of stirring, the medium is evaporated to dryness, the residue is taken up in ethyl acetate, washed twice with suction with ether, twice with ethyl acetate and dried; it is then washed three times with a saturated solution of sodium bicarbonate, twice with water and twice with methanol.

9.8 g of 6 is obtained, ie a yield of 95%.

<Tb>

7) <SEP> H-Cys- (D) <SEP> Ala-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP> Acm <SEP> Msc <SEP> Acm
<Tb>
This compound is obtained from 6 by following the procedure described for the preparation of 5.

<tb> 8) <SEP> H-Cys- (D) <SEP> Ala-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys -OH
<tb><SEP> Acm <SEP> Acm
<Tb>
2.09 g of 7 are dissolved in 30 ml of DMF, cooled to 0 ° C. and then 10 ml of water and 15 ml of 0.4 N aqueous barium solution are added at this temperature.

After stirring for 30 minutes at a temperature in the region of 15 ° C., 20 ml of water are added and the mixture is neutralized with carbon dioxide. After centrifugation, the supernatant is extracted three times with ethyl acetate, the aqueous phase is evaporated to dryness and then washed with ethyl acetate. The centrifugation pellet is taken up in 50 ml of 50% acetic acid and filtered. The filtrate is evaporated to dryness, washed with ethyl acetate, dried and washed with water.
The two products obtained are pooled from the filtrate and the supernatant to give 1.2.50 g.

<tb> 9) <SEP> H-Cys- (D) <SEP> Ala-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-Oli
<tb><SEP> Ag <SEP> Ag
<Tb>
This product is obtained by addition of silver nitrate following the procedure described for the preparation of 18 in Example 1.

10) CM 14.480

 The silver salt 9 is suspended in 400 ml of degassed DMF, the hydrogen sulfide is bubbled under stirring for 1 hour and then 100 ml of degassed water is added. It is centrifuged, the hydrogen sulfide saturation of the supernatant is controlled after further degassing, the medium is diluted with 2.5 l of a degassed solution of DMF and water (1 volume of DES for 2 volumes of water).

 This solution is added over 15 hours under nitrogen to the following solution, the pH of which is maintained at 6.8 by addition of ammonia diluted eighth: 1.5 liters of DMF and water (volume-to-volume mixture), 1 g of ferricyanide of potassium, 2 g of ammonium acetate.

 A turbid solution is obtained which is filtered, passed over resin AG x 3, brought to pH = 5 with acetic acid, then passed through Biorex resin 70. The resin is then washed with acetic acid at 2.5% and then with 80% acetic acid.

 After evaporation and drying of the product, it is purified by chromatography on Sephadex G 15, the glutant being 50% acetic acid.

TLC / BPEW1, Rf: 0.72; [&alpha;] 25 = -91.60 (c = 0.5, acid
D acetic).

EXAMPLE 3 CM 14.481

<tb> i) <SEP> H-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) -Cys-OPse><SEP> TFA
<tb><SEP> Msc <SEP> Acm
<Tb>
This product is identical to the product 3 described in Example 2.

<tb> 2) <SEP> Boc-Phe-Phe-Phe- (D) <SEP> Trp-lys-Thr-Phe-Phe <SEP> (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>

This product is obtained from 10 g of 1 by addition of 2.3 g of Boc-Phe-ONp in 80 ml of DES. 0.730 g of OHBT and enough N-ethylmorpholine are added so that the pH is equal to 7. After stirring for 60 minutes, the medium is evaporated and then taken up in ethyl acetate. After drying, the solid form is washed with ethyl acetate, dried and then washed again with ethyl acetate, water, saturated sodium bicarbonate solution, water, ethanol. After drying, 10.43 g of 2 is obtained, ie a yield of 97%.

<Tb>

3) <SEP> H-Phe-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe-CD) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP>
<tb><SEP> Msc <SEP> Acm
<Tb>
40 ml of TFA containing 10% of anisole and 10% of ethanedithiol are cooled to 0 C, 10.3 g of 2 are added and the mixture is left stirring for 25 min at room temperature. The medium is evaporated to dryness, taken up in ether, drained, washed three times with ether and dried.

10.3 g of product are obtained, ie a yield of 99%.

<tb> 4) <SEP> Boc-) ys-Phe-Phe-Phe- <SEP> (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys-OPse
<tb><SEP> Acm <SEP> Msc <SEP> Acm
<Tb>
This product is obtained from 3 by addition

<tb> of <SEP> Boc-Cys-ONp
<tb><SEP> Acm
<tb> following the procedure described for the preparation of 2.

<tb> 5) <SEP> H-Cys <SEP> -Phe-Phe-Phe- <SEP> (D) <SEP> Trp-Lys-Thr-Phe-Phe (D) -Cys-OPse, <SEP> TFA
<tb><SEP> 1 <SEP> I
<tb><SEP> Acm <SEP> Msc <SEP> Acm
<Tb>
This product is obtained from 4 following the procedure described for the preparation of 3.

<Tb>

6) <SEP> H-Cys-Phe-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OH
<tb><SEP> Acm <SEP> Acm
<Tb>
This compound is obtained by following the procedure described in Example 2 for the preparation of 8.

<tb> 7) - <SEP> H-Cys-Phe-Phe-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OH
<tb><SEP> I <SEP> I
<tb><SEP> Ag <SEP> Ag
<Tb>
This product is obtained by following the procedure described in Example 2 for the preparation of 9.

8) CM 14.481

 CM 14.481 is obtained from the silver salt 7 following the procedure described in Example 1 for the preparation of 19.

TLC / BPEW1, Rf: 0.73; [&alpha;] 25 = - 108.3
D
EXAMPLE 4: CM 14.482

<tb> 1) <SEP> Il-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP> Msc <SEP> Acm
<Tb>
This compound is the product 12 described in Example 1.

<tb> 2) <SEP> Boc-Gly-Phe- (D) <SEP> Trp-tys-Thr-Phe-Phe- <SEP> CD) <SEP> -Cys-OPse
<tb><SEP> sc <SEP> Acm
<Tb>
9.6 g of 1 are dissolved in 50 ml of DMP, 1.08 g of Boc-Cly-OH, 2.47 g of OHBT and 1.85 g of dicyclohexylcarbodiimide are added, the pH is brought to 7 by the addition of N-ethylmorpholine.

After stirring for 1 hour, 0.700 g of dicyclohexylcarbodiimide is added while controlling the pH, and then stirring is continued for 3 h.

 The dichlohexylurea is drained and the filtrate is evaporated to dryness, the residue is taken up in ethyl acetate, drained, washed three times with water, drained, washed four times with ethyl acetate, three times with methanol, three times with acetone and dried.

8.2 g is obtained, that is to say a rendement of 83%.

<Tb>

3) <SEP> H-Gly-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) -Cys-OPse, <SEP> TFA
<tb><SEP> Msc <SEP> Acm
<Tb>
40 ml of TFA containing 10% anisole and 10% ethanedithiol are cooled to 0 ° C. 9.5 g of 2 are dissolved in this mixture, the mixture is stirred for 20 minutes at room temperature, and the medium is then evaporated to dryness. The oily residue formed is taken up in ether, drained, washed three times with ether and dried.

9.3 g is obtained, a yield of 97%.

<Tb>

4) <SEP> Boc-Phe-Gly-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys-OPse
<tb><SEP> Msc <SEP> Acm
<Tb>
9.15 g of 3 and 2.2 g of Boc-Phe-ONp are dissolved in 100 ml of DES. 0.670 g of OHBT and the sufficient amount of OHBT are added to reach pH = 7. After stirring for 1 h at room temperature, the medium is evaporated to dryness, the residue is taken up in ethyl acetate, the solid formed is filtered, washed three times with ethyl acetate, three times with water, three times with methanol, three times with acetone and dried.

9.25 g is obtained, ie a yield of 94%.

<Tb>

5) <SEP> H-Phe-Gly- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse, <SEP> TFA
<tb><SEP> WIsc <SEP> Acm
<Tb>
5 is obtained from 4 following the procedure described for the preparation of 3.

<tb> 6) <SEP> Boc-Cys-Phe-G1 <SEP> y-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- <SEP> (D) <SEP> Cys-OPse
<tb><SEP> tIsc <SEP> Acm
<tb> 6 is obtained by addition of

<tb> Boc-Cys-ONp <SEP> to <SEP> 5 <SEP> in <SEP> sui
<tb><SEP> Acm
<tb> the procedure described for the preparation of 4.

<tb> 7) <SEP> H-Cys-Phe-Gly-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OPse
<tb><SEP> l <SEP> l <SEP> l
<tb><SEP> Acm <SEP> Msc <SEP> Acm
<Tb>
8.9 g of 6 are suspended in 20 ml of methylene chloride, they are then treated at 0 ° C. in TFA according to the procedure described for the preparation of 5.

<Tb>

8) <SEP> H-Cys-Phe-Gly-Phe- (D) <SEP> Trp-Lys-Thr-Phe-Phe- (D) <SEP> Cys-OH
<tb><SEP> I <SEP> 1
<tb><SEP> Acm <SEP> Msc <SEP> Acm
<Tb>
3.3 g of 7 are dissolved in 50 ml of DES. After cooling to 0 C, 20 ml of water are added, followed by 22.5 ml of 0.4 N barite.

Stirring is continued for 30 minutes at a temperature in the region of 15 ° C., then neutralized with carbon dioxide and diluted with 50 ml of water.

After centrifugation, the supernatant is extracted three times with ethyl acetate, the aqueous phase is evaporated to dryness and the residue is taken up in ethyl acetate. The solid formed is filtered off, washed with ethyl acetate and dried.

2.2 g is obtained, ie a yield of 90%.

<Tb>

9) <SEP> H-Cys-Phe-Gly-Phe- <SEP> (D) <SEP> Trp-Lys-Thr-Phe-Phe-Cys-OH
<tb><SEP> Ag <SEP> Ag
<Tb>
2.2 g of 8 are dissolved in 160 ml of 3% acetic acid, the solution is degassed and 17 ml of silver nitrate in aqueous solution at 108 mg / ml are added. After one night in the dark, evaporate to dryness, the residue is taken up in ether, drained, washed with ether and dried.

10) CM 14.482

 CN 14,482 is obtained from the silver salt 9 following the procedure described for the preparation of 19 in Example 1.

 TLC / BEPW1, Rf 0.73.

CCM/BPEW1, Rf : 0,66; [&alpha;]25 = +9,1 (c = 0,5, acide acétique).
D
EXEMPLE 6 : CM 14,498

EXAMPLE 5: CM 14.483

TLC / BPEW1, Rf 0.66; [+ alpha] 25 = +9.1 (c = 0.5, acetic acid).
D
EXAMPLE 6 CM 14.498

TLC / BPEW1, Rf 0.68.

EXAMPLE 7: 14.499

TLC / BPEW1, Rf 0.68; [&alpha;] 20 = -38.1 (c = 0.5, acetic acid).

CCM/BPEW1, Rf : 0,76; [&alpha;]20 = -2,5 (c = 0,5, acide acétique)
D
les produits des exemples 5 à 8 ont été préparés selon les modes opératoires décrits pour les exemples précédents.D
EXAMPLE 8 14.501

TLC / BPEW1, Rf: 0.76; [&alpha;] 20 = -2.5 (c = 0.5, acetic acid)
D
the products of Examples 5 to 8 were prepared according to the procedures described for the preceding examples.

 The peptides of the present invention as well as their pharmacologically acceptable salts are actively involved in the control of growth hormone secretion both in animals and in humans (therapeutics of acromegaly).

 The differential inhibitory action of these products on insulin and glucagon production and in glycemic 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 of the invention have low toxicity; in no case during the tests did the treated animals show any alarming signs.

 The dosage range of clinical use of these substances varies with the desired therapeutic effects and with each patient; 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 a phosphate buffer and always under rigorous conditions of sterility; the saline solutions contain from 1 to 5% by weight of products 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 Charles River Charles Sprague Dawley male rats of 200 9 220 g.

Dosages of insulin, glucagon and growth hormone are carried out by radioimmunological techniques.

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

 In each case, the peptide concentration inhibiting 50% of the hormonal secretion or gastric secretion is determined. In the following table the results are expressed in relation to the somatostatin used as reference product.

As the activity of somatostatin was in each case considered to be 100, the figures relating to the peptides of the invention express the ratio of the activity of the peptide under consideration to the activity of somotostatin.

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

<Tb>

<SEP> Ineuline <SEP> Clucagon <SEP> OH
<tb><SEP> Somatostatin <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> CM <SEP> 14,480 <SE> 83 <SEP> 66 <SEP> inactive
<tb> CM <SEP> 14,482 <SE> 81 <SEP> inactive <SEP> 165
<tb><SEP> CM <SEP> 14,483 <SEP> 0 <SEP> inactive <SEP> 157
<tb><SEP> CM <SEP> 14,499 <SEP> 98 <SEP> Inactive <SEP> 221
<Tb>

Claims (11)

 1 - Analogues of somatostatin, characterized in that they correspond to the general formula

 in which . R denotes an H, an amino acid, a dipeptide such as Ala-Gly by  example; X1 = (L) Phe, (D) Ala or single bond; . X2 = (L) Phe. (D) Phe or Gly;  X3 = (L) Phe, (D) Phe or single bond; . 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 somatostatin analogue 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

 5 - Products according to claim 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 analog of the so, rXatostatin has the 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 - Medicaments useful in particular for the treatment of diseases resulting from a dysregulation 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 - Drugs according to claim 10, characterized in that they are administered by intravenous, subcutaneous or intramuscular injection.