FR2577226A1 - Pepstatin analogues which are inhibitors of renine and acid proteases - Google Patents

Abstract

The subject of the invention is pepstatin analogues which are inhibitors of renine and acid proteases. These analogues are particularly appropriate for the treatment of high blood pressure.

Description

Analogues of pepstatin inhibitors of renin and acid proteases.

 The present invention relates to novel peptide derivatives which inhibit renin and proteases α-cides. It also relates to a process for obtaining them and their application in therapeutics.

In 1970, UMEZAWA isolated from a streptomyces culture a pentapeptide referred to as pepstatin, the structure of which was later established and corresponds to the formula
Isovaleryl-L-valyl-L-valyl-statyl-L-alanyl-statin, in which the term "statin" is used to designate an unusual amino acid, (3S, 4S) amino-4-hydroxy-3-methyl-heptanolic acid .

 He showed that pepstatin is an inhibitor of acid proteases and follows in particular on pepsin, cathepsin D and renin. Especially renin, an enzyme of renal origin, is involved in the angiotensinogen, angiotensin I, angiotensin II sequence in the transformation of angiotensinogen into angiotensin.

 Angiotensin II, being a powerful vasoconstrictor agent, intervenes in the regulation of arterial pressure. The use of pepstatin has been considered for the control of high blood pressure in humans.

However, since pepstatin acts on all acidic proteases and has low solubility in aqueous media and a weak affinity for renin, its use in therapy has proved difficult. Derivatives of pepstatin have been described in the scientific literature. For example, attempts have been made to solubilize pepstatin by extension of the peptide chain (J. Cardiovasc, Pharmacol., 1980, 2, 687-698).

 The present invention relates instead to peptides whose model is built on that of pepstatin. In some cases, the introduction of unnatural amino acids allows to increase the solubility of these compounds and their resistance to proteolysis.

Surprisingly, it has been found that the peptides according to the invention which carry hydrophilic residues at different sites have a high level of activity and very much higher than that of pepstatin as an inhibitor of human renin.

 The present invention relates to peptides having a high level of activity as inhibitors of renin and other acid proteases.

In the present description and in the claims, the following abbreviations will be used:
Laminated acids and protecting groups or activators.

 These abbreviations are in agreement with those indicated by the Nomenclature Commission of IUPAC-IUB Biochemistry Section. The most recent recommendations are reported in.Eur. J. Biochem.

1984, 138, 5-7 and 9 to 37.

Amino acids :
Ala: Alanine
Asn: Asparagine
Asp: Aspartic acid
Gln: Glutamine
Gly: Glycine
Ris: Histidine
Island .: Isoleucine
Leu: Leucine
Met: Methionine
Nle: Norleucine
Nva: Norvaline
Phe: Phenylalanine
Ser: Serine
Sta: Statine
When the Statin is in the central position in the peptide, it always has the configuration (3S, 4S); when the Statin is in the terminal position, it can have either configuration (3S, 4S) or (3R, 4S).

(tauMe) His : (télé-méthyl)histidine

Amino acid derivatives
Met (02): Methionine dioxide
Phg: Phenylglycine
Hph: Homophenylalanine (Pyr-3) Ala: (Pyridyl-3) Alanine (piMe) His: (pro-Methyl) histidine

(tauMe) His: (tele-methyl) histidine

(tauEt) His: (tele-ethyl) histidine ((CH3) 3CO) Ser: Serine t-butyl ether
(PhCH20) Ser: Serine benzyl ether
(CH3 CH20) Ser: serine ethyl ether
(Pentyl) Ala: 2-Octanoic Acid
(Ph) Gln: N (phenyl) glutamine
(Ph) Asn: N (phenyl) asparagine
These amino acids are, unless otherwise indicated, L configuration.

Protective groups and activators:
Ac: Acetyl
Boc: t @ butyloxycarbonyl
(Boc) 20: bis-terbutyloxycarbonic anhydride HON Su: N-hydroxysuccinimide
OEt: Ethyl ester
OMe: Methyl ester
ONp: p-nitrophenyl ester ONSu: Ester of N-hydroxysuccinimide oTcp: 2,4,5-trichlorophenyl ester Va: Isovaleryl
Z: Benzoyloxycarbonyl
In addition, the following abbreviations will be used:
AcOEt: Acetate of ethyl
AcOH:: Acetic acid
Bop: Benzyloxy tris hexafluorophosphate
dimethylamino phosphonium C CM: Thin layer chromatography
DCCI: Dicyclohexylcarbodiimide
DCHA: Dicyclohexylamine DCI) Dicyclohexylurea
DIPEA: Diisopropylethylamine
DMF: Dimethylformamide
DMSO: Dimethyl sulfoxide
Ether: Ether
HOBt: 1-hydroxybenzotriazole KHSO4-K2 504: Aqueous solution containing 16.6 g of
potassium bisulfate and 33.3 g of
potassium sulphate for 1 liter
MeOH: Methanol
NEM: N-ethyl morpholine
NMM: N-methyl morpholine
TA: Room temperature
TFA: Trifluoroacetic acid.

dans laquelle - R1 représente l'un des groupements suivants

The compounds according to the invention correspond to the following general formula

in which - R1 represents one of the following groupings

NH2CH2CH2 - SO2
BocNHCH2CH2 - SO2
Z '- NHCH2CH2 - 502
Alk-SO2
C6H5 (CH2) a - SO2 -, where a P is 0.1 or 2, - Alk being an alkyl group of C1 to C4; R2 represents the grouping (CH2) b R5
in which :. b IQ, 1, 2, 3 or 4 and
R5 is a hydro atom
gene or a phenyl radical
or naphthyl or cyclohexyl
or pyridyl; - R3 represents one of the following groupings
an alkyl (C2 to C6)
alkenyl (C2 to C6)
. a group A - R6
in which
1) A is either - (CH2) d, where d = 1,2,3,4
either - CH-Alk
and R6 is one of the following residues - NH2, - N (Alk) 2, NHW where W = group
protector such as benzyloxycarbonyl;
COOH, COO-Alk, COOCH2C6H5;
'- CONR7R8 where R7 and R8 are different
or identical and represent H or Alk
or C6H5; OH, O-Alk, OCH2C6H5, OCH2-Pyr,
where Pyr represents the radical 2-, 3- or 4
pyridyl ;; - -S (O) eAlk, where e = 0, 1, 2
Alk is an alkyl group of C1 to C4;
2) A is either a direct link
either (CH2) d, where d = 1,2,3,4
be -CH-Alk
R6 is one of the following remains
phenyl, naphthyl or cycloalkyl
carrying 3 to 6 carbon atoms,
monocyclic heterocycle such as: imi
dazole, oxazole, thiazole or tetrazole,
thiophene, pyrrole, triazole, pyridine ,.

this heterocycle being possibly
substituted by Alk or CF3,
Alk being as defined above; - R4 represents one of the following groups: -OH, -OAlk ',
NH 2, NALK '2, OM, where M is a metal and Alk' is a C1 to C6 alkyl group; - R2 is other than benzoyl, when R3 represents either an alkyl or a benzyl, or the group (CH2) 4NH2 or (CH2) 4NHZ.

 The present invention also includes the salts of inorganic or organic acids of the pharmaceutically acceptable peptides of formula (I).

 The products according to the invention can be prepared according to the usual methods of peptide chemistry. More particularly, from a lower alkyl ester of the statin, the various suitably protected amino acids are coupled step by step, the product obtained at each step being deprotected, according to known methods, before being subjected to a new coupling, each of the coupling operations being carried out using either an activated ester of the amino acid to be coupled, or the N-protected amino acid in the presence of dicyclohexylcarbodiimide. The starting material is a lower alkyl ester of the C-terminal amino acid on which the following amino acid of the sequence is condensed. After the release of the amine function of the dipeptide, the peptide chain is elongated. by coupling the next amino acid, suitably protected. Each coupling phase is followed by a selective amine release operation that will come into action during cremation of the bond.

next peptide. The various coupling operations are carried out either by using an activated ester of the amino acid to be coupled, or by using the amino acid
N-protected in the presence of dicyclohexyl carbodiimide.

The phases of selective deprotection of the amine are carried out according to the nature of the protecting group used either by hydrogenolysis or by acidolysis in a strong acid medium such as trifluoroacetic acid. When the amino acid to be introduced into the sequence has a reactive function in its side chain, it should be blocked by a suitable protecting group which is subsequently removed.

avec l'aminoacide suivant
Lorsque le composé (I) porte un substituant R3 tel que le résidu correspondant est un aminoacide non naturel, celui-ci est préparé selon des méthodes connues. I1 est ensuite couplé avec 1' aminoacide suivant selon la méthode habituelle. The protection of the amino acid initiated by the group R1 is carried out according to known methods, before the coupling of the residue

with the next amino acid
When the compound (I) bears a substituent R3 such that the corresponding residue is a non-natural amino acid, it is prepared according to known methods. It is then coupled with the next amino acid according to the usual method.

Peptides (I) in acid form (R4 =
OH) can be obtained from the corresponding esters by saponification in a dilute alkaline medium. Their salts can also be prepared (R4 I OM). The peptides (I) in amide form (R4-NH2 or R4 = N (Alk) 2) are obtained directly by taking as starting material the amino acids in amide form which exist commercially.

 According to the general scheme, it is possible either to prepare the entire desired sequence, or to prepare 2 fragments of this sequence that one couples..finally to obtain the desired peptide.

 The compounds of the present invention have an inhibitory action on human plasma renin activity very important and generally much higher than that of the natural product, pepstatin; as such they can be used in the treatment of high blood pressure.

 They also possess a marked inhibitory action on acid proteases, in particular pepsin. It is therefore possible to envisage the use of the products according to the invention in therapeutic areas where the inhibition of such enzymatic systems is justified, in particular, in addition to arterial hypertension, peptic ulcer and inflammatory diseases.

 The present invention also relates to antihypertensive drugs containing the peptides of formula (I) or their pharmaceutically acceptable salts as active principle.

 The peptides of the present invention can be used therapeutically by intravenous, intramuscular or subcutaneous injection. They are used in a solvent such as saline (isotonic saline) or in a buffer, such as phosphate buffer; they can also be suspended in a diluent, aqueous or otherwise.

aqueous, pharmaceutically acceptable. Each dosage unit may contain from 1 to 1000 mg of active ingredient.

 The amount of active ingredient to be used varies according to the desired therapeutic effects, the severity of the condition to be treated and the route of administration chosen. It must be determined for each patient and is most often between 0.100 g and 2 g of active ingredient.

 The following nonlimiting examples are given by way of illustration of the present invention.

 In all of these examples, the pH of solutions in an organic solvent is measured or monitored using wet pH indicator paper.

 The indicated melting points (Fc) are taken from triturated solids; they are measured in the capillary tube.

 Finally, the nuclear magnetic resonance spectra were recorded at 250 MHz in solution in DMSO, the internal standard being hexamethyldisiloxane.

The following abbreviations are used.
s: singlet
d: doublet
m: multiplet or massive.

 The chemical shifts (delta) are measured in ppm.

EXAMPLE 1
Boc-Phe-Met-Sta-Ala-Sta-Ome (SR 42572) 1. Z-Ala-Sta-OMe.

1.44 g of H-Sta-OMe, TFA, 595 mg of NEM, 2.06 g of Z-Ala-OTcp and 1.29 g of HOBt are dissolved at room temperature in 35 ml of DMF. The pH is adjusted to 6-7 on HP paper, if necessary by NEM, and stirred at
TA for 48 h. DMF was evaporated in a water bath at 400 under 0.01 mm. The residual oil is taken up in 50 ml of AcOEt and washed successively with 5% KHSO4-K2SO4.
NaCl / H 2 O, NaHCO 3, N aCl 2 / H 2 O, dried over MgSO 4 and the solvent evaporated. It is taken up in ether, a solid appears.

After an hour in the refrigerator, squeeze and dry.

 Yield: 1.46 g (86%); Fc: 117-120C.

2. H-Ala-Sta-Oe
1.46 g of Z-Ala-Sta-OMe are dissolved in 20 ml of MeOH and, at RT, 1.03 g of ammonium formate is added, the mixture is stirred until dissolution and then 400 mg of Pd / C are added. 10% and stirred 5 min at RT. After 5 min, a TLC control shows the disappearance of the starting material. The pH is 8 to pH paper. The whole is filtered through celite and the solvent evaporated. The methanol is filtered through an Amberlite IR 45 (OH) resin column and the methanol is evaporated. It is taken up in ether and evaporated to dryness and then dissolved in CH 2 Cl 2, the insoluble material is filtered and evaporated to dryness to obtain a white powder.

 Yield: 810 mg (75%); Fc: 123-1340C.

3. Boc-Sta-Ala-Sta-OMe
5 ml of H-Ala-Sta-OMe, 1 g of Boc-Sta-OH, 420 mg of HONSu, 750 mg of DCCI are dissolved successively in 50 ml of CH 2 Cl 2. Very quickly a white precipitate appears. After stirring at RT, the DCU formed is filtered off and washed with CH 2 Cl 2. The organic solution is washed successively with KHSO4-K2SO4 (5% H2O), NaHCO3 (5Z H2O). It is dried over MgSO 4, filtered and the solvent evaporated.

The product is solubilized in the minimum of chloroform-MeOH 97.5 / 2.5 and deposited at the top of a column of silica gel, eluted with the same mixture and fractured
Ur lot of pure product and two batches of product to be recycled under the same conditions are collected, hence an overall yield: Yield: 67%; Fc: 95-980C.

4. H-Sta-Ala-Sta-OMe, TFA
At 0 ° C., 3.5 ml of TFA are treated with 200 mg of
Boc-Sta-Ala-Sta-OMe. The mixture is stirred for 20 minutes at RT. Then, after concentration in vacuo, the oily residue is depleted by successive additions and concentrations of anhydrous ether. A white solid is obtained.

 Yield: 170 mg; Fc: 148-1510C.

5. Boc-Met-Sta-Aya-Sta-OMe.

 In 5 ml of dioxane, a solution of 149 mg of DCCI, 88 mg of HOBt, 100 mg of DIPEA and 119 mg of DCCI is prepared. After 30 minutes, the TPA salt obtained in step 4 is poured into this solution and the pH is adjusted to 7 by adding DIPEA. After 24 h, the solid is filtered, evaporated to dryness, water is added, extracted with 3 volumes of AcOEt, washed with a K2CO3 solution, with water, with saturated saline solution and then dried over MgSO4. After evaporation. it is purified by filtration on silica gel, eluting with AcOEt.

 Yield: 127 mg (51%).

6. H-Met-Sta-Ala-Sta-OMe, TFA.

 120 mg of the preceding peptide are treated at 0 ° C. with 3 ml of TFA.

7. SR 42572.

In 5 ml of CH 2 Cl 2, a solution of 85 mg of Boc-Phe-ONp and 34 mg of HOBt in the presence of 20 mg of NEM is prepared. After 15 minutes, this solution is poured onto the salt of
Dry TFA obtained in the previous step and neutralized by NEM. After 24 h, evaporate to dryness, add water and AcOEt, wash with KHSO4 solution, water, brine, and dry over MgSO4. The residue obtained is purified by chromatography on silica gel with
AcOEt as eluent. After trituration in hexane, 90 mg of white powder is obtained.

 Yield: 61Z.

NMR spectrum

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 8.05-6.90 <SEP> m <SEP> 10 <SEP> H <SEP> 4 <SEP> NH-CO
<tb><SEP> 1 <SEP> NH-CO2
<tb><SEP> 5 <SEP> H <SEP> Aromatic
<tb> 4.95-4.78 <SEP> m <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb> 4.41-4.00 <SEP> m <SEP> 3 <SEP> H <SEP> CH <SEP> (alpha)
<tb> 3.86-3.67 <SEP> m <SEP> 4 <SEP> H <SEP> CHOH <SEP> and <SEP> CHNR <SEP> (.Sta) <SEP>
<tb><SEP> 3.48 <SEP> s <SEP> 3 <SEP> H <SEP> O-CH3
<tb><SEP> 1.96 <SEP> s <SEP> 3 <SEP> H <SEP> S-CH3
<tb> 1,62-0,67 <SEP> m <SEP> 30 <SEP> H <SEP> H <SEP> aliphatic
<tb><SEP> (CH3) 3C <SEP>
<tb><SEP> (CH3) 2CH <SEP>
<tb><SEP> (CH3) 2-CH-CH2
<tb><SEP> CH3 (Ala)
<Tb>
EXAMPLE 2 Boc-Phe-Met (02) -Sta-Ala-Sta-OMe (SR 42845) 1. H-Sta-Ala-Sta-OMe. TFA.

 Prepared according to Example 1 from 103.5 mg of Boc-Sta-Ala-Sta-OMe.

2. Boc-Met (02) -Sta-Ala-Sta-OMe.

 On the white solid of TFA salt obtained, 10 ml of DMF is added and the pH is brought to 7 by NMM. At -50C, 56.3 mg of Boc-Met (O 2) -OH, 45.9 mg of HOBT and 45.4 mg of DCCI are introduced. After pH control, the mixture is stirred for 6 hours at RT. 1 drop of AcOH is then added to the reaction mixture and the mixture is stirred for 30 min and then the DCU is filtered off; the filtrate is taken up in an AcOH-butanol mixture 1 to 75/25 by volume; the organic phase is washed successively with a saturated solution of NaCl, with a 1% solution of KHSO 4, with water, with a 1% solution of NaHCO 3 and finally with water. After drying and concentration, 120 mg of a white solid residue are recovered, which is chromatographed on silica p.ar AcOEt then with a 9/1 by volume AcOEt-MeOH mixture. 60 mg of an amorphous white solid precipitated in hexane are obtained.

3. SR 42845
The 60 mg of the product obtained in the preceding step is added to 2 ml of TFA at 0 ° C. and the mixture is stirred for 20 minutes at RT. After concentration in vacuo, the oily residue is extracted by adding and successive concentrations of anhydrous ether. On the white solid of TFA salt thus obtained, 10 ml of CH 2 Cl 2 are added and the pH is brought to 7 by NMM. 33.4 mg of Boc-Phe-ONp and 14 mg of HOBt are then introduced; after pH control, stirring is continued for 72 hours at RT. The solution is diluted by adding
CH 2 Cl 2 and the organic phase is washed successively with a saturated solution of ClNa, with a 1% solution of KHSO 4, with water, with a 1% solution of NaHCO 3 and then with water. After drying and concentration 58 mg of a solid residue are recovered which is chromatographed on silica with AcOEt then with a 9/1 by volume AcOEt-MeOH mixture. 37 mg of an amorphous white solid precipitated in hexane are obtained.

NMR spectrum.

<Tb>

<SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.9 <SEP> - <SEP> 7.35 <SEP> 5 <SEP> d,
<tb> 7.6 <SEP> - <SEP> 7.85 <SEP> j = 8 <SEP> Hz <SEP> 5H <SEP> 5NH
<tb> 8.05
<tb> 7.1 <SEP> - <SEP> 7.25 <SEP><SEP> 5H <SEP> H <SEP> Aromatic <SEP> (Phe)
<tb> 4.8 <SEP> - <SEP> 5 <SEP><SEP> 2H <SEP> OH <SEP> (Sta)
<tb><SEP> 2.9 <SEP> s <SEP> 2H <SEP> CH3-SO2
<tb><SEP> 3.45 <SEP> s <SEP> 3H <SEP> OCH3
<tb><SEP> 1.25 <SEP> s <SEP> 9H <SEP> C (CH3) 3
<Tb>
EXAMPLE 3
(R, S) Boc-Phe-Hph-Sta-Ala-Sta-OMe (SR 42727).

1. Boc-Ala-Sta-OMe.

3
4.1 g of H-Sta-OMe, TFA are introduced in 250 cm
3 of CH2Cl2; 1.7 cm 3 of NEM, 3.86 g of Boc-Ala-ONSu and 2 g of HOBt are successively added, then adjusted to pH 7 by addition of NEM and stirred overnight at RT. The mixture is washed twice with 500 cm 3 of a saturated solution of NaHCO 3,
2 times per 500 cm of a solution of KHSO4-K2SO4 and 500 cm3 of a saturated solution of ClNa and then dried over
MgSO 4 and evaporated to dryness under reduced pressure.

 Yield: 3.86 g (74.7).

2. (R, S) Hph, HCl.

 Homophenylalanine hydrochloride is prepared according to the method described in J. Med. Chem., 1968, 11, 1258-1262.

3. (R, S) Boc-Hph, HCl.

 In 50 ml of water, 2.8 g of the above hydrochloride and triethylamine are mixed to neutralize to pH 7-8.

25 ml of dioxane are then slowly added at approximately 30-400 ° C., in which 3.12 g of terbutyloxycarbonyl (Boc) 2 O anhydride is dissolved before being stirred overnight at RT. The solution obtained is diluted with water, washed twice with ether; AcOEt is then added and the pH of the aqueous phase is gradually brought to 2.5-3 by addition of H 2 SO 4, 6N. After 3 extractions with AcOEt and washing the organic phase with water saturated with NaCl, dried over NaSO 4 and then concentrated. 1 g of an amorphous white solid is recovered which precipitates in hexane.

4. (R, S) Roc-Hoh-Sta-OMe.

 To 5 ml of TFA at 0 ° C., 434 mg of Boc-Sta-OMe are added and the mixture is stirred for 20 min at 0 ° C. After concentration in vacuo at RT, the oily residue is extracted by adding and successive concentrations of anhydrous ether. To the white solid residue thus obtained, 15 μl of CH 2 Cl 2 are added and the pH is brought to 7 by NMM and then 309.5 mg of DCCI, 229.5 mg of HOBt and 419 mg of Boc-Hph, HCl are added. previously obtained. The pH (pH 7) is checked and the mixture is stirred for 72 hours at RT.

The insoluble material is filtered, and the chloromethylenic filtrate is washed successively with a saturated solution of ClNa, a solution of KHSO4, water, a 1% solution of NaHCO 3, water. After drying and concentration,
3, 900 mg of an oily residue are obtained which is chromatographed on fine silica using AcOEt. 230 mg of an amorphous white solid precipitated in hexane are obtained.

5. (R, S) Boc-Hph-Sta-Ala-Sta-OMe.

 182 mg of Boc-Ala-Sta-OMe obtained in step 1 are added to 4 ml of TFA at 0 ° C., the mixture is stirred for 30 minutes with OOC and the excess of TFA is then concentrated; the oily residue is then exhausted by successive additions and concentrations of anhydrous ether. There remains a white residue to which 10 ml of CH 2 C 12 is added, the pH is brought to 7 by NMM, then 76.5 mg of HOBt, 103.2 mg of DCCI and 220 mg of the product obtained are added. step 4. The pH (7) is checked and the mixture is stirred for 72 hours at RT. The insoluble material is filtered and the filtrate is washed with saturated NaCl solution, 1% KHSO 4 solution, water, 1% NaHCO 3 solution, water.

After drying and concentration, 364 mg of a residue is recovered as fine silica chromatography with AcOEt.

190 mg of a white amorphous solid is obtained which is a mixture of isomers.

6. SR 42727.

 170 mg of the product obtained in the preceding step are added to 3 ml of TFA with OOC; After 30 minutes stirring at 0 ° C., the excess of TFA is concentrated and the oily residue is exhausted by adding and successive concentrations of anhydrous ether. To the white residue obtained, 10 ml of CH 2 Cl 2 and NMM are added to bring the pH to 7, then 96.6 mg of Boc-Phe-ONp and 38.3 mg of HOBt; adjusted to pH 7 and stirred for 120 h at RT.

The solution is diluted with CH 2 Cl 2 and the organic phase is washed successively with a solution of
NaCl, a 1% solution of KHSO4, water, a 1% solution of NaHCO3, water. with drying and concentration, 300 mg of a residue is recovered which is chromatographed on fine silica with AcOEt then with a 9/1 by volume AcOEt-MeOH mixture. 170 mg of an amorphous b-lance solid are obtained. This is the expected product (SR 42727) in the form of a mixture of isomers at the level of Hph.

NMR spectrum.

<Tb>

<SEP> Delta <SEP> | <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 7 <SEP> - <SEP> 8.1 <SEP> m <SEP> 15 <SEP> H <SEP> 5NH
aromatic <tb><SEP> 10 <SEP> R <SEP>
<tb><SEP> (Phe, <SEP> Hph)
<tb> 4.75 <SEP> and <SEP> 4.9 <SEP> d <SEP> J = 4 <SEP> Hz <SEP> 2 <SEP> H <SEP> 2 <SEP> OH <SEP> (Sta)
<tb><SEP> 5 = 4 <SEP> Hz
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3 <SEP>
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (cH3) 3 <SEP>
<Tb>
EXAMPLE 4
Boc-Phe- (piMe) His-Sta-Ala-Sta-OMe (SR 43061).

1. Bis(Boc)His-Sta-OMe. This product is prepared from listiistidine protected by 2 Boc groups, one of which is on the amine function and the other on the telly nitrogen of histidine:

1. Bis (Boc) His-Sta-OMe.

3.7 g of Boc-Sta-OMe are reacted with 15 ml of
TFA between 0 C and + 50C for 20 min. Concentrate, take 3 times with anhydrous ether and concentrate to a maximum.

The TFA salt obtained is placed in 150 ml of CH 2 Cl 2, brought to pH 7 by the DIPEA under cooling. 6.6 g of Bis-Boc-His are then added in the form of DCHA salt, brought to pH 7 by DIPEA, 6.3 g of Bop are added and the mixture is stirred for 3 days at RT at pH 7. Washing with water salted, with bicarbonate water and then with water, dried and concentrated. By chromatography on silica with AcOEt as eluent, 7.8 g of white powder are collected.

2. Boc-Phe- (iMe) His-Sta-OH.

 The mixture of 1 g of the product obtained in the preceding step, 10 ml of anhydrous DMF and 5 ml of methyl iodide is refluxed gently; after 48 hours of rest, concentrated under vacuum pump. By taking up again with anhydrous ether, a yellow gum is obtained which hardens in AcOEt and is concretized in the anhydrous ether. We squeeze and spray dry. The concreted portion and the concentrated mother liquors are added to 20 ml of TFA at 0 ° C. for 20 min, concentrated, treated with anhydrous ether 3 times and concentrated to a maximum.

The TFA salt obtained is dissolved in 25 μl of CH 2 Cl 2 and brought to pH 7 with NMM. It adds 724.5 mg of
Boc-Phe-ONp and 299 mg of HOBt and stirred for 4 days at RT.

Wash with salt water, bicarbonate water and then with water, dry and concentrate. Chromatography on silica and elution with AcOEt-MeOH 8/2 by volume, 2 polar fractions are collected concretized in anhydrous ether.

639 mg of product are obtained. Saponification is then carried out from 300 mg, to which 13 ml of MeOH, 2 ml of water and 167 mg of hydrated barium are added, and then, after 1 hour, the ester has disappeared on TLC. Small pieces of dry ice are added to a pH of less than 7, the mixture is filtered off with celite, rinsed with methanol and the filtrate is concentrated; the residue is taken up with the minimum of warm MeOH, filtered and concentrated, thereby obtaining 248 mg of white powder.

3. SR 43061.

 The compound obtained in the preceding step is coupled with Boc-Ala-Sta-OMe by operating according to the methods previously described.

NMR spectrum.

<Tb>

<SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.9 <SEP>;<SEP> 7.4
<tb> 7.6 <SEP>;<SEP> 7.8 <SEP> 5 <SEP> d,
<tb> 8.05 <SEP> J = 8 <SEP> Hz <SEP> 5H <SEP> NH
<tb> 7.1 <SEP> - <SEP> 7.25 <SEP> m <SEP> 5d <SEP><SEP> 5 <SEP> H <SEP> Aromatic
<tb><SEP> (Phe)
<tb> 6.7 <SEP> and <SEP> 7.4 <SEP> 2 <SEP> s <SEP> 2H <SEP> 2 <SEP> H <SEP> Imidazole
<tb><SEP> 3.5 <SEP> s <SEP> 3H <SEP> OCH3 <SEP>
<tb><SEP> 3.55 <SEP> s <SEP> 3H <SEP> (piCH3) His <SEP>
<tb><SEP> 1.25 <SEP> s <SEP> 9H <SEP> C (CR3) 3 <SEP>
<Tb>
EXAMPLE 5
Boc-Phe- (tauMe) His-Sta-Ala-Sta-OMe (SR 43062).

1. The (tauMe) His is prepared according to the method described in JR Neth. Chem. Soc., 1978, 97, 293-295.

 2. (tauMe) His-OMe, 2HCl.

 4.5 g of the dihydrochloride of the preceding product are dissolved in 50 ml of MeOH; saturated with gaseous HCl at 10-15 ° C and then stirred at RT for 72 h.

It is concentrated to dryness, taken up in ether 2 times, then the residue is taken up in acetone and crystallized. After spinning, 4 g are obtained.

3. Boc-Phe- (tauMe) His-OMe.

 508 mg of the above product are placed in 25 ml of CH 2 Cl 2 and brought to pH 7 by the addition of DIPEA. 772 mg of Boc-ne-ONp and 306 mg of HORk are added. After stirring for 2 days at RT, it is washed with iced water and then dried and concentrated. By chromatography on silica and solution with a 9/1 by volume AcOET-MeOH mixture, 849 mg of the expected product are collected.

4. Boc-Phe (tauMe) His.

768 mg of the above product are dissolved in 33 ml of MeOH and 5 ml of water to which 561 mg of hydrated barium are added. After 1 h, 280 mg of barite are added and the mixture is stirred for a further 1 hour. We then add small pieces of dry ice until the pH is less than 7, then we spin on celite, rinsed by
MeOH and concentrated. It is taken up in MeOH, the light insoluble filter and concentrated. It is taken up in ether and then filtered to obtain 634 mg of white product.

5. H-Sta-Ala-Sta-OMe, TFA.

 It is prepared according to the method described in Example 1 from 130 mg of Boc-Sta-Ala-Sta-OMe.

6. Boc-Phe- (tauMe) His-Sta-Ala-Sta-OMe.

To the previously obtained TFA salt placed in 10 ml of CH 2 Cl 2, DIPEA is added to bring the pH to 7; then 124 mg of Bop and 104 mg of the product obtained in step 4 are added, 3 ml of anhydrous DMF are added and the mixture is stirred for 72 hours at pH 7. The mixture is washed with salt water and then with dilute bicarbonate and then with water, dry and concentrate. Chromatography on silica eluting with a mixture
AcOEt-MeOH 9/1 by volume. A fraction of 80 mg of white product is isolated.

NMR spectrum. t

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 7.0 <SEP>;<SEP> 7.3 <SEP> 5d, <SEP> 5 <SEP> H <SEP> 5 <SEP> NR
<tb>7.4;<SEP> 7.95 <SEP> J = 8 <SEP> Hz
<tb> 8.1 <SEP> Jt10 <SEP> Hz
<tb><SEP> J = 6 <SEP> Hz
<tb><SEP> J = 6 <SEP> Hz
<tb><SEP> J = 8 <SEP> Hz
<tb> 6.8 <SEP> and <SEP> 7.4 <SEP> 2 <SEP> s <SEP> 2 <SEP> H <SEP> 2 <SEP> H <SEP> imidazolic <SEP>
<tb> centered <SEP> to
<tb><SEP> 4.95 <SEP> m <SEP> 2 <SEP> H <SEP> 2 <SEP> OH <SEP> (Sta)
<tb> 7.1 <SEP> - <SEP> 7.3 <SEP> m <SEP> 5 <SEP> H <SEP> 5 <SEP> H <SEP> Aromatic
<tb><SEP> (Phe) <SEP>
<tb><SEP> 3.5 <SEP> d <SEP> 6 <SEP> H <SEP> 0CR3 <SEP> (tauCH3) His <SEP>
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3? 3
<Tb>
EXAMPLE 6
Boc-Phe- (cyclohexyl) Gly-Sta-Ala-Sta-OMe
(SR 43091).

 N-Boc (2-alpha cyclohexyl) glycine is prepared from alpha phenyl glycine by catalytic hydrogenation over Adams catalyst (Pt 02). The procedure is then carried out according to the methods previously described for preparing SR 43091.

NMR spectrum.

<Tb>

<SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 6.9-7.85 <SEP> m <SEP> 5 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> centered <SEP> to
<tb><SEP> 7.2 <SEP> m <SEP> 5 <SEP> H <SEP> 5 <SEP> H <SEP> Aromatic
<tb> 4.8 <SEP> and <SEP> 2 <SEP> d, <SEP> J = 5 <SEP> Hz <SEP> 2 <SEP> H <SEP> 2 <SEP> OH <SEP> (Sta )
<tb> 4.95 <SEP> J = 4 <SEP> Hz
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3) 3 <SEP>
<Tb>
EXAMPLE 7
Boc-Phe (Pyr-3) Ala-Sta-Ala-Sta-OMe
& 43429.

1. 2-Acetamido 2-ethoxycarbonyl-3-pyridylpropionate
ethyl.

 11 g of a sodium solution in 300 ml of EtOH are added, 48 g of diethyl acetamidomalonate are added, and 36 g of 3-chloromethyl-pyridine hydrochloride are added under reflux, and the mixture is kept under reflux for 5 hours. EtOH is evaporated, taken up in water, extracted, dried, concentrated and chromatographed on silica. The CH 2 Cl 2 -AcOEt 60/40 mixture by volume elutes 23 g of the expected compound.

2. (Pyr-3) Ala, 2HCl
15 g of the preceding diester are refluxed in 200 ml of 5N HCl for 5 hours. Concentrate dry, adds
EtOH, evaporate. The process is repeated until recrystallization of the dihydrochloride: 15 g.

3. Boc (Pyr-3) Ala.

 1 g of the above dihydrochloride is dissolved in a mixture of equal volume water-dioxane. 350 mg of sodium hydroxide and 1 g (Boc) are added, followed by stirring overnight at RT. The dioxane is evaporated, water is added, neutralized with 8 g of amberlite CG 120 1 (acid form), stirred 1 h at RT and then the medium is placed on a column containing 8 g of the same resin. It is eluted with distilled water until pH 5 is obtained and then eluted with a solution of 4X ammonia in distilled water. The waters are concentrated. 0.69 g of the expected product is collected.

4. Boc- (Pyr-3) Ala-Sta-OMe.

The mixture is stirred for 3 days at RT in 10 ml of acetonitrile and a mixture of 465 mg Sta-OMe, TFA, 500 mg of
Boc- (Pyr-3) -Ala in the presence of 780 mg of Bop, the medium being maintained at pH 7 by addition of DIPEA. It is then evaporated to dryness, water is added, the mixture is extracted with AcOEt, the solution is washed with Na2CO3 solution, with water, with brine, and then dried over MgSO4 and evaporated to dryness.

The residue is chromatographed on silica gel with a CH2Cl2-AcOEt eluent mixture at 1/9 by volume, which elutes successively
the less polar isomer A: 150 mg;
the most polar isomer B: 180 mg.

5. SR 43429.

 The expected compound is obtained as a pure isomer from isomer B, using the usual coupling methods.

RMX spectrum.

<Tb>

<SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 8.41 <SEP> s <SEP> 1 <SEP> H <SEP> H2 (PYr)
<tb><SEP> 8.32 <SEP> d, <SEP> J = 4 <SEP> Hz <SEP> 1 <SEP> H <SEP> H6 (pyr)
<tb><SEP> 8.02-7.33 <SEP> m <SEP> 6 <SEP> H <SEP> NHCO, <SEP> H <SEP> (pyr)
<tb><SEP> 7,27-7,04 <SEP> m <SEP> 5 <SEP> H <SEP> C6H5
<tb><SEP> 6.87 <SEP> d <SEP> J = 8 <SEP> Hz <SEP> 1 <SEP> H <SEP> NH-C02
<tb><SEP> 4.97 <SEP> d <SEP> J = 4 <SEP> Hz <SEP> 1 <SEP> H <SEP> OH
<tb> 1 <SEP> 4.85 <SEP> d <SEP> J = 4 <SEP> Hz <SEP> 1 <SEP> H <SEP> OH
<tb><SEP> 4.64 <SEP> - <SEP> 3.90 <SEP> m <SEP> 3 <SEP> H <SEP> CH <SEP> alpha <SEP> ((Ala,
<tb><SEP> Phe, <SEP> (pyr <SEP> - <SEP> 3) Ala)
<tb><SEP> 3.90 <SEP> - <SEP> 3.67 <SEP> m <SEP> 4 <SEP> H <SEP> CHNH, <SEP> CHOH <SEP> (Sta)
<tb><SEP> 3.50 <SEP> s <SEP> 3 <SEP> H <SE> O <SEP> CH3
<tb><SEP> 3,10-2,47 <SEP> m <SEP> 4 <SEP> H <SEP> CH2C5H4N <SEP>
<tb><SEP> CH2C6H5
<tb><SEP> 2.96-1.90 <SEP> m <SEP> 4 <SEP> H <SEP> CH2CO <SEP> (Sta)
<tb><SEP> 1.57-1.00 <SEP> m <SEP> 18 <SEP> K <SEP> (CH3) 3C, <SEP> CH3 <SEP> (Ala)
<tb><SEP> CH, <SE> CH <SEP> (Sta)
<tb><SEP> 0.87-0.66 <SEP> m <SEP> 12 <SEP> H <SEP> CH3 <SEP> (Sta) <SEP>
<Tb>
Using the same coupling methods, the following compounds were prepared:

<tb>. <SEP> SR <SEP> Formula
<tb> 42823 <SEP> Boc-Phe-Phg-Sta-Ala-Sta-OMe
<tb> 42928 <SEP> Boc-Phe-Asn-Sta-Ala-Sta-OMe
<tb> 42939 <SEP> Boc-Phe. (PheCH20) Ser-Sta-Ala-Sta-OMe <SEP>
<tb> 42941 <SEP> (R, S) <SEP> Boc-Phe- (Pentyl) Ala-Sta-Ala-Sta-OMe
<tb> 42980 <SEP> Boc-Phe-Gln-Sta-Ala-Sta-OMe
<tb> 43075 <SEP> (R, S) <SEP> Boc-Phe- (thienyl-2) Gly-Sta-Ala-Sta-OMe
<tb> 43266 <SEP> Boc-Phe- (Ph) Gln-Sta-Ala-Sta-OMe
<tb> 43281 <SEP> Boc-Phe- (Ph) Asn-Sta-Ala-Sta-OMe
<tb> 43297 <SEP> Boc-Phe- (CH3) 3CO) Ser-Sta-Ala-Sta-OMe
<tb> 43365 <SEP> Boc-Phe- (CH3CH2O) Ser-Sta-Ala-Sta-OMe
<tb> 43391 <SEP> Boc-Phe- (tauEt-His) -Sta-Ala-Sta-OMe
<Tb>
These products are characterized by their spectrum of
NMR.

SR 42823

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 7-8.3 <SEP> m <SEP> 15 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 10 <SEP> H <SEP> Aromatic
<tb> 4.8 <SEP> and <SEP> 5 <SEP> 2 <SEP> d,
<tb><SEP> J = 4 <SEP> Hz <SEP> 2 <SEP> H <SEP> 2 <SEP> OH <SEP> (Sta)
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3 <SEP>
<tb><SEP> 1.2 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3) 3
<Tb>
SR 42928

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.85-8.25 <SEP> m <SEP> -12 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 5 <SEP> H <SEP> Aromatic
<tb><SEP> CONR2
<tb> 5.8 <SEP> and <SEP> 5.9 <SEP> 2 <SEP> d, <SEP> 2 <SEP> H <SE> OH <SEP> (Sta)
<tb><SEP> J = 6 <SEP> Hz
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3) 3
<Tb>
SR 42939

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.9-8.1 <SEP> m <SEP> 15 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 10 <SEP> H <SEP> Aromatic
<tb> 4.85 <SEP> and <SEP> 2 <SEP> d <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb><SEP> 4.95 <SEP> 3 = 5 <SEP> Hz
<tb><SEP> 4.45 <SEP> s <SEP> 2 <SEP> H <SEP> OCH2C6H5 <SEP>
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3 <SEP>
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> - <SEP><SEP> C (CH3) 3
<Tb>
SR 42941

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.9-7.9 <SEP> m <SEP> 10 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 5 <SEP> H <SEP> Aromatic
<tb> 4.75-4.9 <SEP> m <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> 0CR3 <SEP>
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3) 3
<Tb>
SR 42980

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.75-8 <SEP> m <SEP> 12 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 5 <SEP> H <SEP> Aromatic
<tb><SEP> NH2 <SEP> (Gln)
<tb><SEP> 4.85 <SEP> and <SEP> 2 <SEP> d <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb><SEP> 4.95 <SEP> J = 5 <SEP> Hz
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3) 3
<Tb>
SR 43075

<tb><SEP> Delta <SEP> Aspect <SEP> - <SEP> Integration <SEP> Attribution
<tb><SEP> 6.9-8.5 <SEP> m <SEP> - <SEP> 13 <SEP> H <SEP> 5 <SEP> NH
<tb> 5 <SEP> H <SEP> aromatics
<tb><SEP> 3 <SEP> @ <SEP> H <SEP> Thiophenic
<tb><SEP> 4.8-4.9 <SEP> m <SEP> 2 <SEP> H <SEP>. <SEP>. <SEP><SEP> OH <SEP>(Sta>
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> C (CH3) 3
<Tb>
SR 43266

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 9.8 <SEP> s <SEP> 1 <SEP> H <SEP> NH <SEP> (NH <SEP> C6H5)
<tb> 6.9-9.1 <SEP> m <SEP> 15 <SEP> H <SEP> 5 <SEP> NH <SEP>
<tb><SEP> 10 <SEP> H <SEP> Aromatic
<tb> 4.85-5 <SEP> m <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> 0CR3 <SEP>
<tb><SEP> 1.25 <SEP> -s <SEP> 9 <SEP> H <SEP> C (CH3) 3
<Tb>
SR 43281

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 10.1 <SEP> s <SEP><SEP> H <SEP> NH <SEP> (NH-C6H5),
<tb> 6.95-8.4 <SEP> m <SEP> complex <SEP> 15 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 10 <SEP> H <SEP> Aromatic
<tb> 4.8 <SEP> and <SEP> 2 <SEP> d <SEP><SEP> 2 <SEP> H <SEP> 2 <SEP> OH <SEP> (Sta)
<tb><SEP> 4.95 <SEP> J = 6 <SEP> Hz
<tb><SEP> 3.8 <SEP> s <SEP> Wide <SEP> 2 <SEP> H <SEP> 2 <SEP> H <SEP> Alpha <SEP> (Sta)
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> CH <SEP> ester
<tb><SEP> 3
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> Boc
<Tb>
SR 43297

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 7 <SEP> to <SEP> 7.85 <SEP> m <SEP> complex <SEP> 10 <SEP> H <SEP> 5 <SEP> NH
<tb><SEP> 5 <SEP> aromatic <SEP>
<tb> 4.6 <SEP> to <SEP> 5 <SEP> m <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb><SEP> 3.8 <SEP> s <SEP> Wide <SEP> 2 <SEP> H <SEP> 2 <SEP> H <SEP> Alpha <SEP> (Sta)
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> CH3 <SEP> ester
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> Boc
<tb> 1.05and <SEP> 1 <SEP> 2 <SEP> peaks <SEP> 9 <SEP> H <SEP> t.bu <SEP> 0
<tb><SEP> isomers
<Tb>
SR 43365

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb><SEP> 7.2 <SEP> m <SEP> 5 <SEP> H <SEP> Aromatic
<tb>(<SEP> 6.95
<tb>(<SEP> 7.30 <SEP> 5 <SEP> d <SEP> 5 <SEP> H <SEP> NH
<tb>(<SEP> 7.45 <SEP> J = 8 <SEP> Hz
<tb>(<SEP> 7.80
<tb>(<SEP> 8.0
<tb> 4.8-4.95 <SEP> 2 <SEP> d <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta).
<Tb>

<SEP> J = 4 <SEP> Hz
<tb>(<SEP> 3.75 <SEP> 4 <SEP> H <SEP> alpha
<tb>(<SEP> 4.2 <SEP> 3m <SEP> 4 <SEP> H <SEP> (Phe, <SE> Ser, <SE> Sta)
<tb>(<SEP> 4,4
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3
<tb><SEP> 3.35 <SEP> quadruplet <SEP> OCH2CH3
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> Boc
<tb><SEP> 1.0 <SEP> t <SEP> 3 <SEP> H <SEP> OCH2CH3
<tb> SR 43391

<tb><SEP> Delta <SEP> Aspect <SEP> Integration <SEP> Attribution
<tb> 6.8 <SEP> to <SEP> 8.2 <SEP> m <SEP> complex <SEP> 12 <SEP> H <SEP> 5 <SEP> NH,
<tb><SEP> 5 <SEP> aromatics,
<tb><SEP> 2 <SEP> His
<tb><SEP> 4.95 <SEP> m <SEP> Complex <SEP> 2 <SEP> H <SEP> OH <SEP> (Sta)
<tb><SEP> 3.5 <SEP> s <SEP> 3 <SEP> H <SEP> OCH3 <SEP> ester
<tb><SEP> 1.25 <SEP> s <SEP> 9 <SEP> H <SEP> Boc
<Tb>
The products according to the invention have been studied with regard to their therapeutic properties and their enzymatic inhibitory action. More particularly, the compounds were evaluated "in vitro" on the inhibition of Human Plasmatic Renin (ARP!
I - METHOD.

 Our method is inspired by GUYENE (J. Clin.

Endocrinol. Metab., 1976, 43, 1301, to the extent. where the inhibition of A.R.P. is evaluated from a pool of human plasmas, rich in renin (15 to 20 mg of angiotensin I released per milliliter per hour) incubated at 370C in the presence of increasing concentrations of the product to be studied.

 The angiotensin I released during the reaction (human plasma contains and the substrate angiotensin, and the enzyme: renin) is measured by radioimmunoassay using a kit.

 Of course, an inhibitor of the conversion enzyme of PMSF (phenylmethylsulfonyl) fluoride is added to the incubation medium.

 The experiments were carried out at a pH of 7.4, which is physiological (phosphate buffer, 60 min incubation).

The results are expressed by the dose of. compound evaluated in moles, which inhibits by 50Z (IC50) the Activity
Human Plasmatic Renin presents in the absence of inhibitor.

 A solution of acetic acid is prepared in. methanol (19/1 by volume) and a soda solution in methanol (2/1 by volume). In an equivolumic mixture of these 2 solutions, a stock solution of the peptide at 0.01M is prepared. The subsequent dilutions of the peptide are then carried out in the appropriate buffer according to the protocol described above.

 The amount of solvent present in a solution of the peptide at a concentration less than 0.0001M does not interfere with the results.

 II - RESULTS.

The results obtained with various products of the invention are shown in the following table which shows the IC50's of each molecule with respect to their inhibition of Human Plasmatic Renin Activity at pH 7.4. From 5 to 10 doses were needed to determine these IC50s. Pepstatin, as a reference substance, is always tested in parallel in each experiment. The results are expressed by their logarithmic value (-Log IC50).

<Tb>

<SEP> Inhibition <SEP> ARP
<Tb>

<SEP> Human
<tb> No. <SEP> SR <SEP><SEP> -Log <SEP> .1C50 <SEP> M <SEP>
<tb><SEP> pH <SEP> 7.4
<tb><SEP> Pepstatin <SEP> 4.92
<tb><SEP> 42 <SEP> 572 <SEP> 6.50
<tb><SEP> 42 <SEP> .727 <SEP> 4.69
<tb><SEP> 42 <SEP> 823 <SEP> 7.03
<tb><SEP> 42 <SEP> 845 <SEP> 5
<tb><SEP> 42 <SEP> 928 <SEP> 5.92
<tb><SEP> 42 <SEP> -939 <SEP> 6.76
<tb><SEP> 42 <SEP> 941 <SEP> 6.26
<tb><SEP> 42 <SEP> 980 <SEP> 6.50
<tb><SEP> 43 <SEP> 061 <SEP> 5.52
<tb><SEP> 43 <SEP> 062 <SEP> 7,11
<tb><SEP> 43 <SEP> 075 <SEP> .6,60 <SEP>
<tb><SEP> 43 <SEP> 091 <SEP> 6.82
<tb><SEP> 43 <SEP> 266 <SEP> 4.85
<Tb>
The toxicity of the products according to the invention is compatible with their use in therapeutics.

Claims (4)

1. Peptide derivatives of formula

 in which - R1 represents one of the following groupings

 pharmaceutically acceptable substances.  - (CH2) 4NHZ, or one of its salts of mineral acids or orga  alkyl, either benzyl or the - (CH2) 4NH2 group or  - R2 is other than benzyl, when R3 represents either a  an C1 to C6 alkyl group;  -OAlk ', -NH2, -NAlk'2, -, where M is a metal and Alk' represents  - R4 represents one of the following groups: -OH,  Alk being as defined above;  optionally substituted with Alk or CF3,  pyrrole, triazole, pyridine, this heterocycle being  oxazole, thiazole or tetrazole, thiophene,   monocyclic heterocycle such as: imidazole,  6 carbon atoms  phenyl, naphthyl or cycloalkyl having 3 to  R6 is one of the following remains  be -CH-Alk  or (CH2) d, where d - 1, 2, 3, 4  2) A is either a direct link  Alk is a C1 to C4 alkyl group;  - 4-pyridyl; - S (O) Alk, where e 8 O, 1, 2  where Pyr represents the radical 2-, 3- or  or C6H5; OH, O-Alk, OCH2C6H5, OCH2-Pyr,  or identical and represent H or Alk  '- CONR7R8 where R7 and R8 are different  protector such as benzyloxycarbonyl; - COOH, - C00-Alk, - COOCH2C6H5;  and R6 is one of the following residues - NH2 - (N (Alk) 2, NHW where W = group  either - CH Alk  1) A is either - (CH2) d, where d 1 1,2,3,4  . a group A - R6 in which  alkenyl (C2 to C6)  an alkyl (C2 to C6)  pyridyl; - R3 represents one of the following groupings  phenyl or naphthyl or cyclohexyl or  R5 is a hydrogen atom or a radical  C6H5 - (CH2) a - SO2 -, where a = 0,1 or 2, -Alk being a C1 to C4 alkyl group; R2 represents the group (CH2) b R5 in which: b = 0, 1, 2, 3 or 4 and  Alk-SO2  Z-NHCH2CH2 - SO2  BocNHCH2CH2 - SO2  NH2CH2CH2 - SO2  2. Process for preparing a peptide according to  claim 1, characterized in that from an ester  lower alkylate of the statin on couple, step by  stage, the various amino acids suitably protected,  the product obtained at each stage being deprotected, according to  known methods, before being subjected to a new coupling, each of the coupling operations being effec  killed using either an activated ester of the amino acid  to be coupled, ie the N-protected amino acid in the presence of  dicyclohexylcarbodiimide.  3. Pharmaceutical compositions characterized in that  they contain a product according to claim 1  as an active ingredient. 4. Pharmaceutical compositions according to claim 3, usable in particular for the treatment of arterial blood pressure, characterized in that they contain from 1 to 1000 mg of product according to claim 1 per unit.  dosing mixture with a pharmaceutical excipient.