FR2537131A1 - New amino acid derivatives and their therapeutic use - Google Patents

Abstract

The present invention relates to amino acid derivatives having enkephalinase-inhibitory, antalgic and hypotensive activities.

Description

 The present invention relates to again; amino acid derivatives, their preparation processes and their therapeutic application.

 The derivatives according to the present invention are inhibitors of enkephalinase which is an enzyme degrading enkephalins.

Methionine-enkephalin, [Met-E (Tyr-gly-gly-Phe
Met), and leucine enkephalin, Leu-E (Tyr-gly-gly-Phe-Leu) are peptides discovered in the brain and which are the endogenous ligands of the morphine receptor (J. Hughes et al., Nature, 258, 577 (1975).

 Their action is multiple both in the transmission of nociceptive impulses and in terms of hormonal behavior or secretion. They are considered as neuromediators with inhibitory action on the release of other neurotransmitters [J. Hughes, Nature, 278, 394 (1979); S. H. Snyder, Nature, 278, 13 (1979)]. Enkephalins have been shown to be rapidly degraded in the brain by a carboxydipeptidase which releases Tyr-gly-gly and Phe-Met residues (B. Malfroy et al, Natures 276, 523 (1978).

 Compounds capable of inhibiting enkephalinase can therefore prolong the effects of enkephalin-free agents or potentiate the action of synthetic analogs administered exogenously. These compounds are therefore likely to replace the morphine agents in all their properties without the serious drawbacks, especially in terms of addiction and dependence.

dans laquelle
A est un groupe carbonyle, méthylène, méthine ou amino; 3 est un groupe méthine; amino éventuellement substitué par un groupe alkyle en Cl à C4; carbonyle ou thio; R1 est un atome d'hy- drogène; un groupe alkyle éventuellement mono- ou polysubstitué par un atome d'halogène; un groupe phényle éventuellement mono- ou polysubstitué par un atome dthalogène, un groupe trifluorométhyle ou hydroxy; un groupe cyclohexyle ou un groupe thiényle; n-est 0 ou 1;R2 est un atome d'hy- hydrogène, un groupe alkyle en Cl à C6 linéaire ou ramifié; un groupe phényle; un groupe benzyle éventuellement monoou polysubstitué par un atome d'halogène; un groupe hydro- xyalkyle; un groupe alcoxyalkyle éventuellement substitué sur la fraction alcoxy par un groupe phényle, benzhydryle ou pyridyleN-substitué par un groupe phénylalkyle dont le noyau phényle est éventuellement substitué par un ou plusieurs atomes d'halogène; un groupe phénoxyalkyle; un groupe mercaptoalkyle éventuellement substitué sur le soufre par un groupe alkyle simple ou ramifié, phényle ou benzyle;R3 est un atome d'hydrogène; un groupe alkyle linéaire ou ramifié en Cl-C8; phényle ou phénylalkyle dont la fraction alkyle est en Ci àC8, ces deux derniers radicaux étant éventuellement mono- ou polysubstitués sur le noyau phényle par un atome d'halogène;Y est un groupe -S-; -NH- ou et X est un atome d'hydrogène; un groupe alkyle en Cl ou
C2 substitué par un groupe alcoxyearbonyle carboxy, mercapto, alkylthio éventuellement mono- ou polysubstitué par un atome d'halogène, benzylthio éventuellement mono- ou polysubstitué par un radical choisi parmi un atnie d'halogène et un groupe mono- ou polyhaloalkyle en C1 au benzhydryl thio éventuellement mono- ou polysubstitué par un atome d'halogène, acyle aliphatique (Cl ) thio éven- tuellement mono- ou polysubstitué par un atome d'halogène, benzoylthio éventuellement mono- ou polysubstitué par tun radical choisi parmi un atome d'halogène et un groupe mono- ou polyhaloalkyle en C1 à C4, benzhydryle carbonyîthia éventuellement mono- ou polysubstitué par un atome d'halogène; un groupe acyle aliphatique en C1 à C4 ou benzoyle; un groupe alcoxy-carbonyle; un groupe mercapto; acyle aliphatique (Cl 4) thio ou benzoylthio; et leurs sels d'addition avec des acides ou des bases pharmaceutiquement acceptables.The derivatives according to the invention correspond to the general formula

in which
A is a carbonyl, methylene, methine or amino group; 3 is a methine group; amino optionally substituted with C1-C4 alkyl; carbonyl or thio; R1 is a hydrogen atom; an alkyl group optionally mono- or polysubstituted with a halogen atom; a phenyl group optionally mono- or polysubstituted with a halogen atom, a trifluoromethyl group or hydroxy; a cyclohexyl group or a thienyl group; n is 0 or 1; R2 is a hydrogen atom, a linear or branched C1-C6 alkyl group; a phenyl group; a benzyl group optionally mono or polysubstituted with a halogen atom; a hydroxyalkyl group; an alkoxyalkyl group optionally substituted on the alkoxy moiety by a phenyl, benzhydryl or N-phenyl-substituted phenylalkyl group, the phenyl ring of which is optionally substituted with one or more halogen atoms; a phenoxyalkyl group; a mercaptoalkyl group optionally substituted on the sulfur by a single or branched alkyl group, phenyl or benzyl; R3 is a hydrogen atom; a linear or branched C1-C8 alkyl group; phenyl or phenylalkyl wherein the alkyl moiety is C 1 -C 8, the latter two radicals being optionally mono- or polysubstituted on the phenyl ring by a halogen atom; Y is -S-; -NH- or and X is a hydrogen atom; a C1-alkyl group or
C2 substituted by an alkoxycarbonyl carboxy, mercapto or alkylthio group optionally mono- or polysubstituted with a halogen atom, benzylthio optionally mono- or polysubstituted with a radical chosen from a halogen atom and a benzophenyl mono- or polyhaloalkyl group; thio optionally mono- or polysubstituted with a halogen atom, aliphatic acyl (Cl) thio optionally mono- or polysubstituted with a halogen atom, benzoylthio optionally mono- or polysubstituted with a radical chosen from a halogen atom and a C1 to C4 mono- or polyhaloalkyl, benzhydrylcarbonylthia group optionally mono- or polysubstituted with a halogen atom; a C1 to C4 aliphatic acyl group or benzoyl; an alkoxycarbonyl group; a mercapto group; aliphatic acyl (Cl 4) thio or benzoylthio; and their addition salts with pharmaceutically acceptable acids or bases.

Among the compounds of formula I defined above, a preferred class of compounds includes derivatives in which:
A is a carbonyl, methylene or amino group; B is an amino group optionally substituted with C1-C4 alkyl; carbonyl or thio; R1 is a hydrogen atom; a phenyl group optionally mono- or polysubstituted by a halogen atom; n is 0 or 1; R2 is a hydrogen atom; a linear or branched C1-C6 alkyl group; a benzoyl group; a benzyloxyalkyl group; R3 is a hydrogen atom; a linear or branched C1 to C8 alkyl group; Y is a group -S-; -NH- or -CH2-; and X is a hydrogen atom; a C1-alkyl group or
C2 substituted with alkoxycarbonyl, carboxy, sea-capte, aliphatic acyl (Cl 4) thio, benzoylthio; a C1 to C4 aliphatic acyl group or benzoyl; an alkoxy (Cl 4) carbonyl group; a mercapto group; aliphatic acyl (C1-4) thio or benzoyl thio.

 As a halogen atom fluorine is particularly preferred. As the aliphatic acyl group, there may be mentioned acetyl, propionyl and butyryl groups, the acetyl group being preferred.

 Preferred among the compounds of formula I are those which describe the following amino acid structures: leucine, phenylalanine, glycine and serine.

The invention envisages, as specific compounds, the following derivatives (in the formulas given, denotes by Et an ethyl radical and by a phenyl group) 1) EtO 2 C -CH 2 -L-Phe-L-Leu-OCH 3
2) HO2C-CH2-L-Phe-L-Lau OH
3) H 02C-CH 2 -L-Phe- (CH 3) -L-Leu OH
4) HO2C-CH2-L-Phe-D Leu OH
5) HO2C-CH2-D-Phe-L-Lau OH
6) HO2C-CH2-D-Phe-D-Leu OH
7) EtO 2 C-CH 2 -CH 2 -L-Phe-L-Leu OCH 3
8) HO2C-CH2-CH2-L-Phe-L-Leu OH
9) EtO 2 C-CH 2 -L-Phe-L.Ala-OCH 3 10) HO 2 C -CH 2 -L-Phe-L. Ala-OH 11) HS-CH 2 -CH 2 -L-Phe Otbu 12) # -COS-CH 2 -CH 2 -L. Phe Otbu 13) # -COS-CH2-CH2-L. Phe OH 14) # -COS-CH 2 -CH 2 -L-Phe-L-Leu OCH 3 15)) HS-CH 2 -CH 2 -L-Phe-L-Leu-OH 16) HS-CH 2 -CH 2 -L-Phe- L-Ala OH

 The compounds of formula I have one or two asymmetric carbon atoms. They therefore exist in the form of a racemic mixture or in diastereoisomeric forms. All of these compounds are within the scope of the present invention. The syntheses described below may use the racemic or one of the enantiomers as starting material.

When the racemic starting material is used in the synthesis procedure, the stereoisomers obtained in the product can be separated by conventional methods of chromatography or fractional crystallization. In general, the L isomer relative to the carbon atom of the amino acid is the preferred isomeric form.

 The compounds of formula I form salts which are also part of this invention. The salts include acid addition salts which are formed by reaction with various inorganic and organic acids providing acid addition salts, comprising for example, the hydrohalides (in particular the hydrochloride and the hydrobromide), the sulphate, the nitrate, the borate, the phosphate, the oxalate, the tartrate, the maleate, the citrate, the acetate, the ascorbate, the succinate, benzenesulfonate, methanesulfonate, cyclohexane sulfonate and toluenesulfonate.

 The base addition salts are formed by reaction with bases such as NaOH or by ion exchange reaction.

 The salts are formed in a conventional manner by reacting the free form of the product with one or more equivalents of the appropriate base or acid providing the desired anion or cation in a solvent or medium in which the salt is insoluble or in water and removing water by lyophilization. By neutralizing the salt with an insoluble acid such as a cation exchange resin in hydrogen form, for example the polystyrene-sulfonic acid-Dowex 50 resin (Mikes, Laboratory Handbook of Chromatographic Methods (Van Nostrand, 1961) page 2567, eluting with a buffervolatil (eg pyridine / acetic acid) and extracting with an organic solvent, the free formula can be obtained and, if desired, another salt can be formed.

 The compounds of the present invention are prepared by the various methods defined below.

 The compounds of formula I can be prepared by a conventional papatic condensation reaction between two suitably protected amino acid residues.

 For example, the functional group (s) (i.e., amino> carboxy, hydroxy) that are not involved in the peptide (ie, -COZXH) binding formation reaction in the The condensation reaction may be protected by one or more protecting groups before the condensation reaction.

 As the intermediate protecting groups of the amino groups, common groups such as t-butoxycarbonyl (Boc), benzyloxyearbonyl (Z), isobornyloxycarbonyl (IBOC), etc. are used.

 The carboxylic groups can also be protected, if necessary, by esterification (for example methyl, ethyl, benzyl esters, etc.).

 The condensation reaction is carried out preferably using the coupling of azides without racemization or the dicyclohexyl carbodiimide / 1-hydroxy-benzotriazole process, hereinafter referred to as DCC / HOBt or DCC / 3-hydroxy-4-oxo-3,4- 1,2,3-dihydro-benzotriazine (OOBt). Alternatively, the activated esters of the fragments may be used.

 The condensation reaction can be carried out in the presence of a solvent. The solvent may be chosen from those which are known to be useful in peptide condensation reactions. Thus, the following anhydrous or aqueous solvents may be mentioned as examples: dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, as well as suitable mixtures of such solvents.

 The reaction temperature is chosen from the known range for the reactions leading to the formation of the peptide bonds, for example normally in the range from about -200.degree. C. to about 300.degree. C. In addition, the precursor materials (protected peptides) of the desired compounds -according The present invention can also be easily prepared by solid phase synthesis methods.

After the end of the desired condensation reaction, if the product bears protecting groups, they can be removed by conventional methods. Among such conventional processes, mention may be made of: catalytic reduction in the presence of a catalyst such as palladium black, carbon on palladium, platinum, etc., solvolysis using hydrofluoric acid, trifluoric acid roacetic, etc. and reduction with metallic sodium in liquid ammonia
In particular, trifluoroacetic acid (TFA) is used to remove Boc (protecting amino) groups and saponification to remove the protective ester groups from the carboxylic groups.

 The compounds of formula I may also be prepared by condensation between a halogenated derivative moiety and the corresponding moiety of an amino or mercapto derivative.

dans laquelle R1, R2 et R3 sont tels que définis ci-dessus et A = CO ; B = NH.According to a first embodiment, the compounds of formula I in which A = CO, B = NH, Y = NH, X is a C1 or C2 alkenyl group substituted by an alkoxycarbonyl or carboxy group and R1, R2, R3 and n are as defined above, are prepared by reaction of a derivative of formula
ROOC - (CH 2) m Br III) wherein R is H or an alkyl group and m is 1 or 2, with a derivative of formula

wherein R1, R2 and R3 are as defined above and A = CO; B = NH.

avec lester

par utilisation des différentes méthodes connues précédemment indiquées telles que HOBT/DCC, anhydride mixte, ester activé. Le groupe protecteur Boc est ensuite éliminé par exemple à l'aida d'acide trifluoroacétique (TFA). The compounds of formula III are obtained by the conventional routes of peptide synthesis, that is by coupling a protected derivative.

with lester

using the various previously known known methods such as HOBT / DCC, mixed anhydride, activated ester. The Boc protecting group is then removed for example with trifluoroacetic acid (TFA).

According to another embodiment, the compounds of formula I in which A = CH 2, B = NH, Y = S, X is a hydrogen atom or a benzoyl group, R 1 is H, n = 0,
R2 and R3 are as defined above, are prepared
a) by reaction of thiirane of formula CHS with an amino acid ester of formula

un ester d'amino-acide de formule

b) optionally reaction of the product obtained in step (a) with a compound of formula
R - CO - Br in which R is a lower alkyl or phenyl group and removal of the protecting groups,
According to another embodiment of the present invention, the compounds of formula I in which A = CO,
B = NH, Y = NH or CH 2, X is a C 1 or C 2 alkyl group substituted with a mercapto group, aliphatic acyl thio or benzoylthio, R 1, R 2, R 3 and n are as defined above, are prepared a condensation reaction peptide between a compound of formula

an amino acid ester of formula

When the compound of formula IV has a group y which is a -1XH- group, it is prepared as in the previous embodiment.

(préparé selon C, Ma-nnich et K.Ritsert, 1924,:Ber 57, 1116), pour obtenir un composé de formule

When the compound of formula IV has a group
Y, which is a -CH 2 - group, is obtained by reaction of a thiocarboxylic S-aid of formula R-COSH, in which R is an alkyl or phenyl radical, on an acrylic acid derivative of formula

(prepared according to C, Ma-nnich and K.Ritsert, 1924,: Ber 57, 1116), to obtain a compound of formula

dans laquelle Hal est un atome d'halogène en particulier de brome.According to another embodiment of the present invention, the compounds of formula I in which A = CO,
B = NH, Y = S, X is a hydrogen atom, a C 1 to C 4 aliphatic acyl group or benzoyl and R 1, R 2, R 3 and n are as defined above, are prepared by reaction of a
S-thiocarboxylic acid of formula R-COSH, in which
R is an alkyl or phenyl radical, on a halogenated derivative of formula

in which Hal is a halogen atom, in particular bromine.

et un ester d'aino-acre de formule

The compounds of formula (V) are obtained by a coupling reaction analogous to peptide synthesis between a bromoacid of formula

and an amino-acre ester of formula

 According to a variant of this embodiment it is possible to first react the bromo-acid with the S-thio carboxylic acid, and then to couple the acid obtained with the amino acid ester.

ce dernier composé étant obtenu à partir d'un composé de formule I dont la synthèse a été précédemment décrite, par libération du groupe mercapto terminal.According to yet another embodiment of the present invention, the compounds of formula I in which
A = CO, B = NH, Y = S, X is a C 1 or C 2 alkyl group substituted with a mercapto group, aliphatic acyl thio or benzoyltio and R 1, R 2, R 3 and n are as defined above, are prepared by reaction of a halogenated compound of formula R-CO-S- (CH2) m-Hal, wherein Hal is a halogen atom such as bromine, R is an alkyl or phenyl group and m is 1 or 2, with a derivative of formula

the latter compound being obtained from a compound of formula I whose synthesis has been previously described, by liberation of the mercapto terminal group.

 Obtaining the mercapto-terminated derivatives of formula I is obtained by saponification with sodium hydroxide of acyl-terminated derivatives (aliphatic or aromatic) thio. This saponification leads to a preponderance of disulfide-linked duplicating compounds, this oxidation reaction being carried out mainly even by using degassed solvents and operating under a nitrogen-protective atmosphere. The mercapto-terminated compounds of formula (I) are thus obtained pure by performing a reduction in situ to the incipient hydrogen at the end of the saponification reaction.

un monoester malonique de formule

According to another embodiment, the compounds of formula I in which A = NH, B = CO, Y = NS, X is a hydrogen atom, an acyl group substituted by a carbon or mercapto group, or an alcozycarbonyl group and R 1, R2 and R3 are as defined above, are prepared by a peptide condensation reaction between a diamine
N-protected formula

a malonic monoester of formula

The diamine of formula (VI) is obtained by a Curtius reaction according to M. Chorev et al, [J. Bitter. Chem. Sac., 99, 8075 (1977)] on an N-protected amino acid according to the following reaction scheme

un ester de acide thioglycolique de formule

d'après la méthode de H. Gilman et al., J. Amer. Chem. Soc.According to another embodiment, the compounds of formula I in which A = CH 2> B = S, Y = I, X is a hydrogen atom, an alkyl group substituted by a carboxy or mercapto group, or an alkoxycarbonyl group and R1, R2, and R3 are as defined above, are prepared by a condensation reaction between an amino-N-protected tosyl derivative of formula

a thioglycolic acid ester of formula

according to the method of H. Gilman et al., J. Amer. Chem. Soc.

47, 1449 (1925).

dans lequel R est un groupe allyle, puis transformation de l'alcool obtenu en dérivé tosylé de formule VII par action de l'acide p-toluènesulfonique.The compounds of formula VII are obtained by reduction with sodium borohydride (according to the method of H. Seki et al., Chem Pharm Bull 13, 995 - VII - 1965) of an N-protected amino ester. formula acid

in which R is an allyl group and then conversion of the alcohol obtained to the tosylated derivative of formula VII by the action of p-toluenesulphonic acid.

The compounds of formula VIII are obtained by the method of E. Fischer et al. Annalen. 357, 1 (1907) or described in DOS 2,349,707 according to the following reaction scheme

d'après la technique de V.C. Sekera et al., J. Amer. Chem.According to a last embodiment, the compounds of formula I in which A = -CH2-, B = -NH-, Y = NH
X is a hydrogen atom, an alkyl group substituted with a carboxy or mercapto group, or an alkoxycarbonyl group and R1, R2 and R3 are as defined above, are prepared by a condensation reaction between a tosyl derivative N- protected amino acid of formula VII (described above) and an amino acid ester of formula

according to the technique of VC Sekera et al., J. Amer. Chem.

Soc. 55, 345 (1933).

 The following examples are given by way of illustration of the processes for preparing the compounds according to the present invention.

EXAMPLE 1
Methyl ester of N (ethoxycarbonyl-methyl) -L. phenylalanine - L. leucine
3 ml of triethylamine are slowly added under cold conditions and under nitrogen to a suspension of 10 g of dipeptide-ester trifluoroacetate: L. Phe-L. Leu OCH3 in 50 ml anhydrous benzene. At the end of the addition, the suspension becomes clear. 3.34 g of ethyl bromoacetate are then added and the solution is refluxed for 12 hours. After cooling, 0.25 N sodium hydroxide is added dropwise to an ice bath to pH = 6.

 The aqueous phase is rapidly decanted, then extracted three times (25 ml) with ether. The organic phases (ether and benzene) are combined, washed twice with 50 ml of water, then dried and evaporated to dryness. 2.6 g of N- (ethoxycarbonylmethyl) -L are obtained. Phe.-L. Leu OCH3 as a pale yellow oil. Rf = 0.77 in CHCl3 / MeOH / H2O / 9/1 / Sa t.

EXAMPLE 2 N- (carboe: y-methyl) L. Phenylalanine L. leucine
2.5 g of the methyl ester of N- (ethoxycarbonylmethyl) -L. Phe. L. Leu are dissolved in a mixture of 20 ml of methanol and 10 ml of water. 13 ml of 1N NaOH are added to OOC and the mixture is stirred for 1 hour at 0.degree. C. and then for 2 hours at 25.degree. C. The methanol is evaporated and 1N HCl is added dropwise to OOC until the pH is 2. The precipitate is filtered and recrystallized from 50/50 ether / MeOH; 1.35 g of N (carboxymethyl) -L are obtained. Phe-L.

Leu. Mp 196-2000 ° C. Rf: (BuOH / AcOH / H 2 O: 4/1/1) = 0.6.

EXAMPLE 3
N- (carboxymethyl) L. phenylalanine-N (CH3) L. leucine
By replacing the methyl ester of L. leucine with the methyl ester of N (CH3) L. leucine in the process described in Example 1, and then treating the product obtained according to Example 2, the following is obtained successively: methyl ester of N (ethoxycarbonylmethyl) -L. Phe-N (CH3)
L. Lau, then N (carboxy-methyl) -L. Phe-N (CH3)
L. Leu.

 During this synthesis, the starting compound L. Phe-N (CH 3) L. Leu OCH TFA is obtained by condensation in the presence of DCC and HOBt of Boc Phe with the methyl ester of N (CH 3) L. Lau, followed by deprotection by TFA.

EXEMPT 4
N- (carboxy-methyl) -L. D-phenylalanine. leucine
Obtained as in Example 1 by substituting D, leucine for the L-form in the starting compound.

 The methyl ester of N (ethoxycarbonyl-methyl) -L-Phe-D is obtained successively. leu, then (carboxymethyl) -L-Phe-D. Lau. Mp = 2400C.

EXAMPLE 5
N- (carboxymethyl) -D. phenylalanine-L-leucine
Obtained as in Example 1, substituting D.

L-form phenylalanine in the starting compound. Methyl ester of N- (ethoxycarbonylmethyl) -D is obtained successively. Phe-L. Lau, then N- (carboxymethyl)
D-Phe-L-Leu. Mp 2340 ° C.

EXAMPLE 6
N- (carboxymethyl) -D. D-phenylalanine. leucine
Obtained as in Example 1, replacing L.

leucine and L. phenylalanine by their isomers D. The methyl ester of N- (ethoxycarbonyl-ethyl) -D-he-D is obtained successively. Leu, then N - [(carboxy) mebhyl7
D. Phe-D-Leu. F = 2270C.

EXAMPLE 7
Methyl ester of N- (2-ethoxycarbonylethyl) L. Phe-L.

Leu
8.80 g of Phe-Leu OCH3 (obtained in base form from Phe-Leu OCH3 TFA by the action of triethylamine) are dissolved in 200 ml of anhydrous benzene under a nitrogen atmosphere. 5.8 ml of ethyl bromopropionate and 2.07 g of K 2 CO 3 are added dropwise to OOC. The mixture is heated at 800 ° C. for 4 hours. After cooling, the benzene phase is rapidly separated. 20 ml of H 2 O and 1 N HCl are then added dropwise to pH = 2.

 The benzene phase is separated, and then the acidic aqueous phase is extracted three times with 50 ml of ethyl acetate.

The combined organic phases are dried and then evaporated to dryness. 2.94 g of straw-yellow oil are obtained, which is purified by chromatography on a column of silica gel with Et 2 O / NeOH, 9/1 as eluent. 1.10 g of N (2-ethoxycarbonylethyl) methyl ester are obtained.
L-Phe-L. Leu, colorless oil, Rf = 0.81, CHCl3 / MeOH / H2O, 0/1 / Sat.

EXAMPLE 8
N- (2-carboxyethyl) L. Phe-L. Leu
720 mg of the methyl ester of N (2-ethoxycarbonylethyl) L-Phe-L-Leu are introduced into 10 ml of MeOH.

7.2 ml of 1N NaOH is added to OOC. Stirred 12 hours at room temperature. It is acidified with HCl to pH 3 and the aqueous phase is extracted with three times 20 ml of ethyl acetate. The dried and evaporated organic phases give 510 mg of N (2-carboxyethyl) L-Phe-L. Leu as a white solid. F = 161 C. Rf = 0.51. BuOH / AcOH / HSO.

EXAMPLE 9
N- (ethoxycarbonyl-methyl) L. Phe-L methyl ester

To the.

 This compound is obtained as in Example 1 by replacing the L-Phe-L-Leu OCH3 TFA with L. Phe-L TFA.

Ala OCH3 (5 g). 1.10 g of N- (ethoxy-2-carbonyl-methyl) -L-Phe-L methyl ester are obtained. Ala in the form of oil. Rf = 0.71 in CHCl3 / MeOH / H2O, 8/1 / Sat.

EXAMPLE 10 N- (carboxy-methyl) t. Phe-L. To the
The reaction is carried out as in Example 2 using the methyl ester of N- (ethoxycarbonyl-methyl) L. Phe-L. Ala (1 g). 88 mg of N- (carboxymethyl) L-Phe-L are obtained after saponification and acidification. To the.

Rf = 0.37 BuOH / MeOH / H 2 O 4/1/1.

EXAMPLE 11
T-butyl ester of N- (2-mercaptoethyl) L. Phe
0.9 ml of thiirane C2H4S, freshly distilled, are added to the ampoule to 8.7 g of oily L. pheOtBu obtained in base form from its hydrochloride. The tube is sealed and then heated for 5 hours at 1000C. After cooling, the tube is opened and the syrupy liquid is distilled.

 3.62 g of the ester t are obtained. Butyl N (2-mercaptoethyl) L. Phe. Eb0.5 = 130-134C.

EXAMPLE 12
T-butyl ester of N (benzoyl thi o-2 ethyl) -t-Phe
3.62 g of t-butyl ester of N (2-mercaptoethyl)
L-Phe are dissolved in 15 ml of H 2 O added with 628 mg of K 2 CO 3. 1.51 ml of benzoyl bromide dissolved in 15 ml of ether are slowly added. Stir 1 hour at room temperature. The solid formed is drained, washed with cold ether and then with acidic water (pH 3-4). 1.6 g of t-butyl ester of N (benzoyl'th hi-2-ethyl) -L-Phe are obtained. F = 170-1720C.

EXAMPLE 13
N- (benzoylthi-2-ethyl) -L-Phe
850 mg of t-butyl ester of N (benzoylthio-2-ethyl) -L-Phe are mixed with 3.31 ml of TFA in an ice-bath and stirred for 12 hours at room temperature. The white precipitate is filtered and washed with ether to pH 5.

620 mg of N- (benzylthio-2-ethyl) are obtained
L-Phe. Mp = 260 ° C
EXAMPLE 11
N- (benzoylthio-2-ethyl) -L-Phe-L-Leu methyl ester
0.40 ml of triethylamine is added to a solution of 620 mg of N- (benzoylthi-2-ethyl) -L-Phe, as prepared in Example 13, and 254 mg of L-Leu hydrochloride.
OCH3 in a mixture of 40 ml of CHCl3 and 10 ml of THF.

After 5 minutes, 214 mg of HOBT and 288 mg of
DCC.

 After stirring for 48 hours, the DCU is filtered off and after treatment 250 mg of N- (benzoyl-ethyl-2-yl) -L-Phe-L-Leu methyl ester is obtained.

 = = 0.42 in CHG13 / Et2O, 8/2.

EXAMPLE 15
N- (2-mercaptoethyl) -L-Phe-L-Leu
Method A
250 mg of the methyl ester of N- (benzoylthioethyl) -L-Phe-L-Leu are dissolved in 15 ml of MeOH. 1.1 ml of 1N NaOH is added, the mixture is stirred overnight at 250 ° C. under nitrogen. The methanol is evaporated, 10 ml H 2 O is added and the mixture is acidified to pH 2-3.

65 mg of white crystals of N- (2-mercaptoethyl) -L-Phe-L-Leu are obtained. Rf = 0.9 in BuOH / AcOH / H20.4 / l / l
Method B
5 g of the methyl ester of L-Phe-L-Leu, in base form are dissolved in 50 ml of anhydrous benzene in a glass ampoule. 5 ml of freshly prepared thiirane, C 2 H 4 S are added and the ampoule is sealed.

It is heated in an oven at 800C for 2 hours. After cooling and opening of the ampoule, a gelatinous precipitate is removed by filtration from the solution and the filtrate is then concentrated under vacuum until a yellow oil is obtained which, by chromatography on an alumina column, gives 2.2 g of the methyl ester of N- (2-mercaptoethyl) -L-Phe-L-Leu. The latter is saponified under a nitrogen atmosphere in a solution of MeOH and 1N NaOH.

After acidification and extraction with ethyl acetate, 1.19 g of N- (2-mercaptoethyl) -L-Phe-L-Leu identical to the compound obtained by method A are obtained.

EXAMPLE 16 N- (2-mercaptoethyl) -L-Phe-L-Ala
Obtained as in Example 15 (Method B), replacing L-Leu with L-Ala in the starting methyl ester, starting from 3 g of methyl ester, 720 mg of
N- (2-mercaptoethyl) -L-Phe-L-Ala.

Mp = 60 ° C. Rf = 0.80 BuOH / MeOH / H 2 O, 4/1/1
EXAMPLE 17
N / tR, S) -acetyl-thio-benzyl-2-propionyl-7-L-leucine methyl ester
1.4 g of acetylthio-3-benzyl-2-propionic acid obtained by addition of thioacetic acid to benzylacrylic acid obtained according to C. Mannich and K. Ritsert, 1924, are added successively to a solution cooled to 0.degree. ,
Ber 57, 1116 in 20 ml of anhydrous TTIF, a solution of 1.09 g of L-leucine methyl ester hydrochloride and 0.84 ml of triethylamine in 20 ml of dry chloroform, followed by a solution of 920 ml. mg of HOBT in 20 ml of THF and finally a solution of 1.22 g of DCC in 20 ml of CHCl 3. It is stirred for 4 hours at 0 ° C. and then for 5 hours at 200 ° C.

After filtration of the DCU formed, the mixture is evaporated to dryness and the residue is taken up in 40 ml of ethyl acetate. The mixture is washed twice with 20 ml of water, once with 20 ml of saturated NaCl solution, twice with 20 ml of a solution of 10 citric acid, three times with 20 ml of a solution. saturated with NaHCO 3, twice with 20 ml of water, once with 20 ml of saturated NaCl solution and dried over Na 2 SO 4
Evaporate to dryness. 1.9 g of a dark yellow oil are obtained. Yield = 87%; Rf: 0.86 in CHCl3 /
MeOH / H2O = 9/1 / sat.

HPLC - Vr = 28.3 ml in 10-M sulfate buffer (pH = 4.3) /
CH3CN = 55/45.

EXAMPLE 18
N [(R, S) 3-mercaptobenzyl-2-propionyl] L-leucine
1.5 g of the compound. 17 is dissolved in 20 ml of degassed methanol. At 0 ° C. under a nitrogen stream, 8.5 ml of a 1N sodium hydroxide solution are added. OOC is stirred 1 hour. The methanol is evaporated under vacuum and taken up in 20 ml of degassed water and extracted with chloroform.

 The aqueous phase is then acidified to pH = 1 with a 2N HCl solution. A white precipitate forms which dissolves after the addition of 20 ml of methanol. 500 mg of powdered Zn are added and the mixture is stirred for 1 hour at 200 ° C.

 The zinc is filtered and washed with twice 5 ml of degassed MeOH. The methanol-water phases are combined and the methanol is evaporated under vacuum. The remaining aqueous phase is extracted with chloroform.

 After evaporation to dryness, 1 g of N (R, Smercapto-3-benzyl-2-propionyl) L-leucine is obtained in the form of a white solid product. F = 500C; Yld: 79.

HPLC, Vr = 10.56 ml in 10-M acetate buffer (pH 4.3) /
CH3CN = 60/40.

Elemental analysis for C16H23NO3S
calculated: C; 62.1; H, 7.49; N, 4.53; S: 10.36
found: C, 61.98; H, 7.53; N, 4.46; S 10.51
NMR (DMSOd6). Separate signals are observed for both diastereoisomers.

HS = 2.03 and 2.15 ppm (disappear by addition of D20).

(HS) CH2 = 2 multiples centered on 2.37 and 2.56 ppm.

CH-CH2 () = Complex mass centered on 2.61 ppm.

 H (Leu): 4.06 and 4.17 ppm.

CH2ssLeu = 1.30 and 1.44 ppm.

 CH γ Leu = 1.13 and 1.58 ppm.

 CH = 0.65 and 0.80 ppm.

NH = 8.07 and 8.18 ppm.

Aromatic H = 7.13 ppm.

THE 19
Methyl ester of N / TRS) acetyl-3-thio-2-benzylpropionyl] glycine.

 To a solution of 2.34 g of 3-acetyl-3-benzylpropionic acid in 20 ml of THF cooled with OOC, a solution of 1.23 g of glycyl methyl ester hydrochloride and 1.37 g ml of triethylamine in 25 ml of chloroform, a solution of 1.50 g of HOBT in 15 ml of THF and a solution of 2.22 g of DCC in 10 ml of CHCl 3. Stir 1 hour at 00C and then 5 hours at 200C. After filtration of the DCU formed, the process described for compound 17 is treated.

 2.6 g of the NZtR (S) acetylthio-3-benzylpropionyl] glycine methyl ester are obtained in the form of a pale yellow oil.

Yield = 87 seconds; Rf = 0.76 in CHCl3 / MeOH / H2O = 9/1 / sat.

HPLC = Vr = 9.12 ml in 10-M acetate buffer (pH:
CH3CN X 55/45.

EXAMPLE 20 N- (tR, S) 3-Mercaptobenzyl-2-propionyl-glycine
960 mg of the compound of Example 19 are dissolved in 20 ml of degassed MeOH. 6 ml of a 1N sodium hydroxide solution are added at 0 ° C. under a nitrogen atmosphere and the mixture is stirred for 1 hour at 0 ° C. The methanol is evaporated under vacuum and the residue is taken up in 15 ml of water. twice with 5 ml of CHCl3.

 The aqueous phase is acidified with a 2N HCl solution to pH 1: 20 ml of degassed MeOH and then 300 mg of powdered Zn are added and the mixture is stirred for 1 hour at 20 ° C.

 The zinc is filtered and washed with twice 5 ml of MeOH. The methanol-water phases are combined and the methanol is evaporated under vacuum. The aqueous phase is then extracted with 3 times 10 ml of CHCl 3. It is evaporated to dryness and 500 mg of (R, S-mercapto-3-benzylpropionyl) glycine are obtained in the form of a white solid.

 F: 13800; Yield: 64%.

HPLC, Vr = 6.48 ml in 2M acetate buffer (pH =
CH3CN = 70/30.

For C12H15NO3S% calculated: C, 56.9; H = 5.97; N: 5, 53; S: 12.66% found: C, 56.6; H, 6.01; N, 5.62; S = 12.48
NMR (DMSOd6) a single set of signals for both enantiomers.

HS = 2.20 ppm (disappears by addition of D20).

 (HS) CH2 = 2.34 and 2.57 ppm.

CH-CH2 multiplet centered on 2.64 ppm.

 CH2 (Gly) = 3.66 ppm.

Aromatic H = 7.17 ppm.

NH = 8.28 ppm.

EXAMPLE 21
NZtR methyl ester, S) acetylthio-3-benzylpropionyl] -L alanine
To a solution cooled to 0 ° C. of 1,600 g of acetyl-3-thio-2-benzylpropionic acid in 20 ml of anhydrous THF, a solution of 940 mg of L-alanine methyl ester hydrochloride and 1 ml of triethylamine in 20 ml of CHCl3, a solution of 1.03 g of HOBT in 10 ml of THF and 1.4 g of DCC in 10 ml of
CHCl3. The mixture is stirred for 1 hour at 0 ° C. and then for 5 hours at 200 ° C. The reaction is carried out as in the previous examples and 1.75 g of N- / 1 R, S) acetylthio-3-benzylpropionyl] -Lalanine methyl ester are obtained in the form of a yellow oil.

Yield: 80 ss; Rf = 0.77 in CHCl3 / MeOH / water: 9/1 / sat.

HPLC, Vr = 10.32 in 10-M acetate buffer (pH = CH3CN = 55/45.

EXAMPLE 22 N- (RS) 3-Mercaptobenzyl-2-propionyl] -Lalanine
-1.54 g of the compound of Example 21 are dissolved in 20 ml of degassed methanol, 10 ml of a 1N sodium hydroxide solution are added at 0 ° C. and under nitrogen. The mixture is stirred for 2 hours at 0 ° C. The methanol is evaporated under vacuum and the residue is taken up in 20 ml of water. The alkaline aqueous phase is extracted twice with 5 ml of chloroform. The aqueous phase is acidified with 2N HCl to pH @ 1. 20 ml of
Degassed MeOH and 300 mg of Zn powder. The mixture is stirred for 1 hour at 20 ° C. The zinc powder is filtered and washed twice with methanol. The aqueous and methanolic phases are combined. The methanol is evaporated under vacuum and the remaining aqueous phase is extracted with chloroform. We evaporate dry. We obtain the Ô7. n - [(R, S) 3-mercapto-2-benzylpropionyl L alanine in the form of a white solid.

F <50 C; Yield: 84%.

HPLC, Vr = 5.4 ml in 10-M acetate buffer (pH =
CH3CN = 65/35.

Analysis calculated for C13H17NO3S: C, 58.40; H, 6.41; N, 5.24; S: 11.99 found: C, 58.23; H, 6.60; N, 5.31; S: 11.64
NMR (DMSOd6) 1 set of signals is observed for each diastereoisomer.

HS = 2.06 and 2.12 ppm (disappear by addition of D20).

(HS) CH2 multiplets centered on 2.34 and 2.58 ppm.

(HS) complex complex CH2 (#) centered on 2.75 ppm.

NH = 8.17 and 8.27 ppm.

H2Ala: 4.08 and 4.15 ppm.

 CH3Ala: 1.05 and 1.20 ppm.

Aromatic H: 7.17 ppm.

EXAMPLE 23
Methyl ester of O-benzyl N - [(R, S) 3-benzoylthio-2-benzylpropionyl] L-serine
To a solution cooled to 0 ° C. of 500 mg of 3-benzoylthio-2-benzylpropionic acid in 10 ml of anhydrous THF, are added successively a solution of 408 mg of benzyl-L-serine methyl ester hydrochloride and of 0.24 ml of triethylamine in 6 ml of CHC13, a solution of 250 mg of HOBT in 5 ml of
Anhydrous THF and finally a solution of 376 mg of DCC in 5 ml of CHCl3. The mixture is stirred for 1 hour at 0 ° C. and 5 hours at 20 ° C.

After filtration of the DCU formed, the mixture is treated as in the previous examples and 720 mg of O-benzyl N-ZRS (3-benzoylthio-2-benzylpropionyl) -L-serine methyl ester are obtained in the form of a white solid.

Yield: 88 ss; Mp: 62-64 ° C; Rf = 0.84 in CHCl3 / MeOH / water = 9/1 / sat.

HPLC, Vr = 9.84 ml in 10-M acetate buffer (pH:
CH3CN = 30/70.

EXAMPLE 24
O-benzyl, N- / tR, S) 3-mercaptobenzyl-2-propionyl] L-serine
660 mg of the compound of Example 23 are dissolved in 10 ml of degassed MeOH. At 0 ° C. and under nitrogen, 3 ml of a 1N sodium hydroxide solution are added. The mixture is stirred for 1 hour at 0 ° C. The methanol is evaporated under vacuum and taken up with 10 ml of water. It is extracted twice with chloroform. The aqueous phase is acidified with 2N HCl to pH 1.

10 ml of methanol and 150 mg of zn powder are added.

The mixture is stirred for 1 hour at 20 ° C. The zinc is filtered off and washed with twice 5 ml of MeOH. The aqueous and methanolic phases are combined and the methanol is evaporated under vacuum. The aqueous phase is extracted with chloroform. After evaporation, 350 mg of O-benzyl, N - [(R, S) 3-mercaptobenzyl-2-propionyl] -L-serine is obtained in the form of a very pale yellow oil. : 70,
HPLC, m.p .: 6.96 ml in 10-M acetate buffer (pH: CH3CN = 55/45.

NMR (DMSO-d6)
HS = 2.02 and 2.12 ppm (disappear by addition D20) (HS) CH2 = 2.33 and 2.56 ppm.

CHCH2 (= mass centered on 2.70 ppm.

 H &alpha; Ser = 4.37 and 4.44 ppm.

CH2ss Ser = 3.46 and 3.62 ppm.

OCH2 = 4.32 and 4.42 ppm.

Aromatic H = 7.17 ppm.

NH = 8.25 and 8.33 ppm.

Analysis for C20H23NO4S% calculated: C, 64.32; H. 6.21; N, 3.75; S: 8.58 ss found: C, 64.30; H, 6.31; N, 3.87; S: 8.66
EXAMPLE 25
N [(R, S) 3-acetylethio-3-methyl ester fluoro-2-benzylpropionyl-leucine
1.54 g of acetylthio-3-fluorobenzylpropionic acid in 20 ml of anhydrous THF are added successively to a solution cooled at 0 ° C., a solution of 1.15 g of methyl ester hydrochloride is added. L-leucine and 0.85 ml of triethylamine in 20 ml of chloroform see, then a solution of 930 mg of HOBT in 20 ml of THF and finally a solution of 1.24 g of DCC in 20 ml of CHCl3.

The mixture is stirred for 1 hour at 0 ° C. and then 5 hours at 20 ° C. After filtration of the DCU formed and treatment as in Example 17, 1.95 g of dark yellow oil are obtained. Yield: 88, Rf: 0.87
CHCl3 / MeOH / H2O = 0/1 / sat.

HPLC, Vr = 30 ml in 10-M acetate buffer (pH =
CH3CN = 55/45.

EXAMPLE 26
N - [(R, S) 2-mercapto-3-fluorobenzylpropionyl] L-leucine
1.8 g of compound 25 is dissolved in 20 ml of degassed methanol. 9.8 ml of a 1N sodium hydroxide solution are added at 0 ° C. and under a stream of nitrogen. The mixture is stirred for 1 hour at 0 C.

The methanol is evaporated under vacuum and taken up in 20 ml of degassed water and extracted with chloroform.

 The aqueous phase is then acidified to pH = 1 with a 2N HCl solution. A white precipitate forms which dissolves after the addition of 20 ml of methanol. 500 mg of powdered Zn are added and the mixture is stirred for 1 hour at 20 ° C. The zinc is filtered and washed with twice 5 ml of degassed MeOH.

The methanolic and aqueous phases are combined and the methanol is evaporated under vacuum.

The aqueous phase is then extracted with 3 times 10 ml of CHCl 3. It is evaporated to dryness and 1.08 g of the title compound is obtained as a white solid.

Yield: 68%; F <50 C.

HPLC, VR: 11 ml in 10- @ acetate buffer (pH =
CH3CN - 60/40.

NMR (DMSO-d6).

HS = 2.04 and 2.12 ppm (disappear by addition of D20) (HS) CH2 = 2 multiplets centered on 2.40 and 2.58 ppm.

Complex CHCH2 (#) massive centered on 2.63 ppm.

 Leu = 4.06 t 4.17 ppm.

 CH2 Leu = 1.31 and 1.44 ppm.

 CH &gamma; Leu = 1.13 and 1.58 ppm.

CH3 = 0.65 and 0.81 ppm.

NH = 8.08 and 8.19 ppm.

H aromatic = 2 mass centered on 7.28 and 7.95 ppm.

Analysis for C16H22NN3S s calculated: C, 58.70; H, 6.78; N, 4.28; S: 9.78fs found: C, 58.55; H, 6.60; N, 4.32; S: 9.98
EXAMPLE 27
N [(S) 2-benzoylthio-3-phenylpropionyl-7-L-leucine
To a solution of 11.2 g of R, bromo-2-phenylpropionic acid in 30 ml of THF is added 8.9 g of L-leucine methyl ester hydrochloride dissolved in a mixture of 100 ml of CHCl3 and 6.86 ml of NEt3. To the resulting solution was added 7.5 g of HOBT and 10.1 g of DCC.

After stirring 1 hour at 0 ° C., then 12 hours at 25 ° C., the DCU formed is filtered and treated by the usual process.

12.5 g of white crystals of N-methyl (3-bromophenylpropionyl) -L-leucine methyl ester are obtained by evaporation of ethyl acetate. F = 121 C.

 30 ml of 1N NaOH are added at 0 ° C. to a solution of 10.5 g of the above compound in 100 ml of MeOH. After stirring for 1 hour at 0 ° C., then 4 hours at 25 ° C., the mixture is acidified with HCl. The precipitate is collected and then crystallized in ethyl acetate. 9.07 g of N / tR) 3-bromophenylpropionyl] -L-leucine is obtained. F = 1585C.

 1.76 g of thiobenzoic acid dissolved in 40 ml of water in the presence of 2.17 g of K 2 CO 3 are added to a mixture of 7.1 g of N [(R) 2-bromo-3-phenylpropionyl] - L-leucine in 30 ml of water and 1.74 g of Na bicarbonate.

It is stirred for 12 hours at 25 ° C. It is acidified with 6N HCl, which gives a pale yellow oil which is extracted with ethyl acetate. After washing with water, the organic phase is dried and evaporated to dryness. 7.9 g of N [(S) benzoylthio-2-phenyl-3-propionyl] -L-leucine are obtained in the form of an oil which crystallizes slowly.

Mp 74 ° C-78 ° C; Rf = 0.80 in BuOH / MeOH / H 2 O, 4/1/1.

EXAMPLE 28
N [(S) 2-mercapto-3-phenylpropionyl] -L-leucine
2 g of N (S, 2-benzoylthiophenylpropionyl) -L-leucine dissolved in 10 ml of MeOH are stirred at 0 ° C. for 1 hour with 10 ml of 1N NaOH and then 4 hours at 250 ° C. under nitrogen. The methanol is evaporated, taken up in water and acidified to pH = 1 with 1N HCl. A gummy precipitate is formed which is extracted with ethyl acetate. The organic phase is dried and evaporated under vacuum. 1.45 g of N / tS) 2-mercapto-3-phenylpropionyl] -L-leucine are obtained which crystallizes slowly. Mp 67-7 ° C.

EXAMPLE 29
N [(S) 2-acetylthio-3-phenylpropionyl] -L-leucine methyl ester
3 g of S, 2-acetylthio-3-phenylpropionic acid (obtained by the action of thioacetic acid on R, 2-bromo-3-phenylpropionic acid) in solution in 30 ml of
CHCl3 are added to a solution of 2.5 g of L-leucine methyl ester hydrochloride. 5 ml of
NEt3, then 2 g of HOBT and 2.8 g of DCC. After stirring for 1 hour at 0 ° C., then 12 hours at 25 ° C., the DCU formed is filtered and treated by the usual process. 4.1 g of the methyl ester of N - [(S) -2-ethyl-2-phenylpropionyl] -L-leucine are obtained in the form of an oil.

Rf: 0.85 without CHCl3 / MeOH / H2O, 9/1 / Sat.

EXAMPLE 30
Methyl ester of N [(S) 2-mercapto-3-phenylpropionyl]
L-leucine
2 g of the compound of Example 29 are stirred for 12 hours in a methanol solution saturated with HCl under nitrogen.

It is evaporated to dryness, taken up in water and extracted with ether.

The organic phase is dried and evaporated to dryness. 750 mg of N [(S) mercapto-2-phenylpropionyl] -L-leucine methyl ester are obtained in the form of an oil.

 Rf: 0.70 in CHCl3 / Et2O, 5/5.

EXAMPLE 31
N [(S) 2-acetylthio-3-phenylpropionyl] -L-leucine
This compound is obtained as in Example 27, replacing the thiobenzoic acid with thioacetic acid.

A yellow oil is obtained which crystallizes slowly.

Mp = 50 ° C; Yield: 84 ss; Rf = 0.80 in BuOH / MeOH / H 2 O = 4/1/1.

EXAMPLE 32
N [(S) 2-mercapto-3-phenylpropionyl] -L-alanine
Obtained by following the methods successively applied in Examples 27 and 28, replacing the methyl ester of L-leucine with the methyl ester of Alanine. There is thus obtained N / tS) 2-mercapto-3-phenylpropionyl] -L-Ala, white crystals. F = 42 C.

EXAMPLE 33
N [(S) acetylthiomethylthio-2-phenyl-3-propionyl] -L-leucine
0.42 ml of acetylthio chloromethane in solution in MeOH is added to a mixture of 1.1 g of N [(S) mercapto-3-phenylpropionyl] -L-leucine obtained according to Example 28 and 552 mg. of K2C03 in water. Stirred 20 hours at room temperature, evaporated to dryness and taken up in ethyl acetate. We dry, evaporate. 252 ml of N (2-acetylthiomethylthio-3-phenylpropionyl) -L-leucine are obtained under an oil jet after purification by chromatography on an alumina column in CHCl 3 / MeOH, 7/3.

Rf = 0.77 CHCl3 / MeOH, 5/3.

EXAMPLE 34
N [(S) benzoylthio-ethylthio-2-phenyl-3-propionyl] -L-lucine
1.33 g of N [(S) 2-mercapto-3-phenylpropionyl] leucine obtained according to Example 28 are dissolved in 30 ml of water in the presence of 685 mg of K 2 CO 3. 1.32 g of 1-benzoylthio-2-bromoethane (obtained by the direct action of benzoyl bromide on the thitrane according to
B. HANSEN, Acta. Chem. Scand 11, 537 (1957) in solution in 10 ml of MeOH. Stirring is carried out for 48 hours at 25 ° C. A slight cottony precipitate is filtered off and acidified to pH 2 by
6N HCl. Extracted three times with 20 ml of ethyl acetate, dried and evaporated to dryness. 1.50 g of the expected compound is obtained as an oil which crystallizes slowly.

Mp 62-680 ° C.

EXAMPLE 35
N [(S) 2-mercapto-2-ethyl-3-phenylpropionyl] -L-leucine
800 mg of the compound of Example 34 are dissolved in 10 ml of MeOH and 3.4 ml of 1N NaOH are added under nitrogen in 30 minutes. It is left for 4 hours at 25 ° C., still under nitrogen. 20 ml of H 2 O are added, the mixture is evaporated to MeOH, filtered and acidified with 110 l of 1N until pH = 1. The mixture is extracted with three times 20 ml of ether, dried and evaporated to dryness. 544 mg of a colorless oil are obtained. Rf = 0.92 in BuOH / AcOH /
H 2 O, 4/1/1, corresponding to N [(S) mercapto-ethylthio-2-phenyl-3-propionyl] -L-leucine.

EXAMPLE 36
N [(S) 2-mercapto-2-ethyl-3-phenylpropionyl] -L-alanine
By replacing, in example 34, N / tS) 2-mercapto-3-phenylpropionyl] -L-leucine with N [(S) 2-mercapto-3-phenylpropionyl] -L-alanine obtained according to Example 32, N [(S) benzoylthio-ethylthio-2-phenyl-3-propionyl] -L-alanine is obtained. This derivative, treated as in Example 35, gives N [(S) mercapto ethylthio-2-phenyl-3-propionyl]
L-alanine. Thick oil that crystallizes slowly.

F = 34 C.

EXAMPLE 37
Preoaration of N-Boc, N '- (2-ethoxycarbonylpropionyl) 1,1-diamino-2-phenylethane
a) Nt butoxycarbonyl, N-benzyloxycarbonyl-diamino1,1-phenyl-2-ethane
1.5 g of Boc L-Phe-azide are brought to 800C for 1 hour in 10 ml of anhydrous toluene. An isocyanate is formed which is not isolated. After cooling the solution, 0.55 ml of benzyl alcohol is added, and then heated again at 80 ° C. for 1 hour. After cooling, the solution leaves to crystallize 0.9 g of
N-Boc, N'-2,2-diamino-2-phenylethane. F = 165 C.

b) N-Boc 1,1-diamino-2-phenylethane
0.4 g of N-Boc, N'-2-diamino-1,1-phenylethane are dissolved in 10 ml of methanol in the presence of 25 mg of pd / C. The ordinary pressure hydrogenation provided after filtration and evaporation to dryness 0.3 g of N-Boc-1,1-diaminophenyl-ethane. Pale yellow solid. Mp 40-50 ° C.

(c) Product set out in the title
270 mg of methyl malonic acid methyl ester (R, S) in solution in 10 ml of THF are added to a solution of 436 mg of N-Boc 1-diamino-1-phenylethane, 283 mg of HOBT and 381 mg of toluene. mg DCC in 10 ml CHCl3. After 4 hours at room temperature, the DCU formed is filtered and the solution is treated under the usual conditions of the peptide coupling. 544 mg of pale yellow solid are thus obtained. Rf = 0.80 in CHCl3 / MeOH / H2O, 9/1 / Sat, N-Boc,
N '- (2-ethoxycarbonylpropionyl) diamino-1,1-phenylethane (SR + SS).

EXAMPLE 38
N-Boc, N '(carboxy-2-propionyl) 1,1-diamino-2-phenylethane
200 mg of the compound obtained in Example 37 are dissolved in 10 ml of MeOH. 1.5 ml 1N NaOH is added, the mixture is stirred for 4 hours at room temperature, the mixture is evaporated to MeOH, the residue is taken up in H 2 O and acidified to pH 2. The mixture is extracted with ethyl acetate, dried and evaporated to dryness. 130 mg of white crystals of N-Boc, N '(carboxy-2-propionyl) 1,1-diamino-2-phenylethane (SR + SS) are obtained. F = 170-171C.

EXAMPLE 39 N- (2-Carboxypropionyl) diamino-1,1-phenyl-2-ethane
100 mg of the compound obtained in Example 38 are stirred with 0.4 ml of TFA for 1 hour at 250C. After evaporation to dryness and taken up in ethyl ether, 6-o mg of N- (2-carboxypropionyl) diamino-1,1-phenylethane (RR + RS) is obtained. Rf = 0.35 in BuOH / AcOH / H2O, 4/1/1.

EXAMPLE 40
N-carboxymethyl, N '(carboxy-2-propionyl) diamino1,1-phenyl-2-ethane
300 mg of the compound obtained in Example 37 are stirred for 1 hour at 0 ° C. with 1 ml of TFA. After evaporation to dryness and addition of ether, the white solid obtained is dried under vacuum, then suspended in anhydrous benzene and heated at reflux for 12 hours with 0.1 ml of ethyl bromoacetate.

 After filtration, a pale yellow oil of N-ethoxycarbonylmethyl, N '(2-ethoxycarbonylpropionyl) diamino-1,1-phenylethane, was saponified without purification to yield 52 mg of N-carboxymethyl, N' ( carboxy-2-propionyl) diamino-1,1-phenylethane (RR + RS).

 Rf = 0.42 in BuOH / AcOH / H2O, 4/1/1.

EXAMPLE 41
N- (2-mercaptoethyl), N '(carboxy-2-propionyl) diamino1,1-phenylethane (RR + RS)
Obtained by direct action of thimerane entube sealed on 50 mg of the compound of Example 39 following the method described in Example 15 (Method B). Mp 180-1900C.

EXAMPLE 42
Boc 2-amino-3-phenyl-1-carboxymethylthio propane
2.3 g of the p-toluenesulphonic ester of Boc-L phenylalaninol (obtained by reduction of the amount of
Boc-L Phe in alcohol, then action of tosyl chloride) dissolved in 50 ml of MeOH, to an aqueous solution of the disodium salt of thioglycolic acid. After refluxing for 12 hours, the mixture is filtered, the solution is acidified and extracted with ethyl acetate. 700 mg of a thick oil is obtained which is chromatographed on an alumina column with an Et2O / MeOH mixture, 7/3.

 550 mg of Boc-2-amino-3-phenyl-carboxy methylthio-1 propane are collected.

F = 95 ° C. Rf = 0.63 in CHCl3 / MeOH 7/3.

EXAMPLE 43
(2-amino-3-phenylcarboxymethylthio) propane trifluoroacetate
400 mg of the compound described in Example 42 are stirred for 1 hour at 25 ° C. with 1 ml of TFA. After evaporation to dryness and taken up in ether, 300 mg of (2-amino-3-phenylcarboxymethylthio-1) propane trifluoroacetate are obtained in the form of a white powder. Rf = 0.41 in BuOH / Me.OH / H 2 O 4/1/1.

EXAMPLE 44
Carboxymethyl-2-amino-3-phenyl-1-carboxymethylthio propane
250 mg of the compound described in Example 43 are suspended in 10 ml of anhydrous benzene. 0.15 ml of triethylamine and 0.21 ml of ethyl bromoacetate are added. The resulting solution is refluxed for 10 hours. Filtered, dried and evaporated to dryness. The oil obtained is chromatographed on a column of silica gel in Et2O / MeOH, 8/2.

 125 mg of carboxymethyl-2-amino-phenyl-1-carboxymethylpropane are obtained. Rf = 0.57 in CHCl3 / MeOH, 8/2.

EXAMPLE 45 (2-Mercaptoethylamino) -2-phenyl-1-carboxymethylthio propane
150 mg of the compound described in Example 43 are treated as in Example 15 (Method B) to yield 42 mg of 2- (2-mercaptoethylamino) -3-phenylcarboxymethylthio-propane. F = 201 C.

EXAMPLE 46
Methyl ester of N- (Boc-2-amino-3-phenylpropyl-1)
L-leucine
2 g of the p-toluenesulphonic ester of the
Boc-L Phe-alaninol to a solution of 1.4 g of L-leucine methyl ester (base form) in 10 ml of dioxane. Refluxed for 10 hours. The solution is evaporated to dryness and taken up with ether. A precipitate forms which is filtered and then dried. There is thus obtained 1.1 g of the methyl ester N- (Boc-2-aminophenyl-3-propyl-1) L-leucine. F = 61 C.

EXAMPLE 47
N (2-carboxymethyl-2-phenyl-3-propyl) L-leucine
500 mg of the compound described in Example 46 are stirred for 1 hour with 2 ml of TFA. After evaporation to dryness and addition of ether, a white solid is collected which is dried under vacuum. This product is placed without purification in suspension in 20 ml of anhydrous benzene and 0.2 ml of triethylamine and 0.15 ml of ethyl bromoacetate are added.

After refluxing for 15 hours, the solution is filtered and then evaporated to dryness. The oil obtained is purified by chromatography on an alumina column with a mixture of
Et2O / MeOH, 9/1. 120 mg of N methyl ester (2-carboxymethylamino-3-phenylpropyl) L-leucine are obtained.

F = 40-500C.

 100 mg of this product are dissolved in 20 ml of methanol and NaOH is added to pH = 9. After stirring for 2 hours, it is acidified with HCl (pH = 4), then extracted with ethyl acetate and evaporated at room temperature. dry.

 23 mg of N- (2-carboxymethylamino-3-phenylpropyl) -Leucin is obtained, mp = 147-151 ° C.

EXAMPLE 48
2 - [(2-Mercaptoethylamino) -3-phenyl-3-propyl] -L-leucine
Obtained as in Example 15 (method B), replacing the methyl ester of L-Phe-L-Leu with 200 mg of N-methyl ester (2-amino-3-phenylpropyl-1)
L-leucine obtained from the compound of Example 46 by the action of TFA.

 After the action of thiirane and saponification under nitrogen, 32 mg of N / tmercapto-2 ethylamino) -2-phenyl-3-propyl-1 L-leucine are obtained. Mp = 48C.

 The results of the biological and pharmacological studies reported below highlight the interesting inhibitory properties of enkephalinase, analgesic and hypotensive.

 The subject of the present invention is therefore a medicinal product having, in particular, inhibiting properties of enkephalinase, analgesic and hypotensive, characterized in that it contains as active principle a compound of formula I or an addition salt with an acid or a pharmaceutically acceptable base of this compound.

A) Biological study
Determination of enkephalinase activity (enkephalin dipeptidylcarboxypeptidase) and determination of the effect of inhibitors

 The enzymatic preparation used is a membrane fraction from the rat or mouse striatum.

 This fraction is obtained by homogenization at 40C in 20 volumes of 0.05M Tris-HCl buffer (pH 7.4) followed by two successive centrifugations (1000 g x min.

and 200,000 g x min.) at the end of which the pellet of the second centrifugation is retained. It is washed by resuspension in 10 ml of the buffer followed by centrifugation (203,000 g x min.) And the resulting pellet is itself superficially washed to complete the removal of the soluble enzymes. The membrane fraction thus obtained is taken up in the buffer at 40C so as to obtain a suspension comprising approximately 1.5 m of proteins per ml.

 A 50 μl aliquot of the membrane suspension is then incubated in a final volume of 100 to 25 ° C. in the presence of 10 μl of leucine-enkephalin (39 Ci / mmol), previously purified by chromatography on a Porapak column. Q (100-120 ms, Waters.Assoc.) And 0.1 mM puromycin, in aminopeptidase inhibitor. The duration of the incubations is generally fixed at 15 minutes. in order to measure the initial formation rate of the Tyr-Gly-Gly-3H tripeptide characteristic of enkephalin dipeptidylcarboxypeptidase (enkephalinasic) activity. The reaction is stopped by the addition of 25 l of 0.2 N HCl and the tripeptide is isolated by column chromatography on Porapak Q or on a silica thin layer according to methods described by Malfroy et al.

(B. Malfroy, JP Swerts, C. Llorens and JC Schwartz,
Neuro-Science Letters, 11, 329, 1979).

 The results obtained by each of these two methods have always been in good agreement.

 The radioactivity of the tripeptide is measured by liquid scintillation spectrometry.

 The effect of the inhibitors is established by conducting experiments in the presence of increasing concentrations of these products, which leads to the determination of the 50% inhibitory concentrations calculated by means of data analysis by the method of Parker and Waud (J. Pharmacol Exper Ther 177, 1, 1971). In some cases the competitive nature of the inhibition has been established by incubation experiments in the presence of the fixed concentration inhibitor and the increasing concentration of the substrate.

Compounds N Inhibitory concentration 50%
2 1.5 x 10-6 M
5 2 x 10-5 M
27 5 x 10-6 M
28 2 x 10-7 M
32 1 x 10-7 M
35 4 x 10-7 M
33 1 x 10-6 M
18 5 x 10-9 X
20 5 x 10-9 M
22 7 x 10-9 M
24 4 x 10-9 M
B) Pharmacological study
The pharmacological study of the products described above made it possible to demonstrate an analgesic effect and a specific hypotensive effect and a potentiating action of the effects of an enkephalin, D Ala2Met
Enkephalin (in particular analgesic and hypotensive).

The pharmacological tests carried out were as follows
I - Acute toxicity
The determination of mortality in the mouse is observed following the single intravenous administration of increasing doses of test compounds.

 The LD50 for all the compounds studied is greater than 100 mg / kg / i.v.

II - Analgesic activity
1) Hot plate test
Licking reflex of the mouse on a plate heated to 55 ° C. according to the method of Jacob et al.

(Arch Int Pharmacodyn 122, 287-300, 1959: 133, 296-300, 1961).

Potentiation of the analgesic effect of D Ala2Met Enkephalin
a) Intra-ventricular (ivt)
The following table shows that the effect of a subacute dose (0.3 &gamma;) of intraventricular D Ala2Met Enkephalin is significantly potentiated (p <0.05) by Compounds 2, 28 and 5, and this effect is antagonized by Naloxone.

b) By intravenous injection
Intravenously, No. 35 at the dose of 1.00 mg / kg g results in a 100 ss increase in licking time compared with animals treated with Ala2Met and Enkephalin (0.3 &gamma; intraventricular).

TABLE I
Hot plate
Potentiation of the analgesic effect of
D Ala2Met Enkephalin and antagonism by the
naloxone

<SEP> Dose <SEP> Increase <SEP> of <SEP> time
<tb> Product <SEP>&gamma;<SEP><SEP> Number <SEP> of <SEP> licking <SEP> ss <SEP> by
<tb><SEP> mouse <SEP> from <SEP> report <SEP> to <SEP> @@@@@@@
<tb><SEP> ivt <SEP> mouse <SEP> DAla2Met <SEP> enkepha
<SEP> line <SEP> (1)
<tb><SEP> (1) <SEP> 10 <SEP> 6 <SEP> 274 <SEP> *
<tb><SEP> + DAla2Met <SEP> Enkephalin
<tb><SEP> n <SEP> 29 <SEP> (1) <SEP> 10 <SEP> 6 <SEP> 207 <SEP><SEP> X <SEP>
<tb><SEP> (1)
<tb><SEP> + DAla2Met <SEP> Enkephalin
<tb><SEP> n <SEP> 28 <SEP> 10 <SEP> 6 <SEP> 400 <SEP> *
<tb><SEP> (1)
<tb><SEP> + DAla2Get <SEP> Enkephalin
<tb> n <SEP> 35
<tb><SEP> (1) <SEP> 10 <SEP> 10 <SEP> 240 <SEP> *
<tb> + DAla2Met <SEP> Enkephalin
<tb> n <SEP> 35 <SEP> (1) <SEP> 10 <SEP> 10 <SEP> 18
<tb><SEP> + DAla2Mt <SEP> Enkephalin
<tb><SEP> + Naloxone <SEP> (10mg / kg <SEP> ss.
<Tb>

<SEP> cut.)
<tb> - (1) the dose of D Ala2Met Enkephalin is 0.3 t mouse,
inactive dose by itself,
# P <0.05 Wilcoxon rank test.

 2) Mice withdrawal test of the tail soaked in water heated to 48 ° C according to the method of Sewell and Spencer (Neuropharmacology - 1976 - 15, pp. 683-688).

Table II shows that compounds 18 and 20 administered intravenously at the dose of 100 mg / kg
- have a clean analgesic effect,
- potentiate very significantly the
D Ala2fi and Enkephalin administered 15 minutes later intraventricularly, at subacute doses ranging from 10 to 30%.

This effect is durable and longer than 2 hours; it is also observed at a dose of 30 mg / kg for 20 TABLE II
Tail of the mouse
Own analgesic effect
Potentiation of the analgesic effect of D Ala2Met Enkephalin

<SEP> Number <SEP> Percentage <SEP> of increase
<tb> Products <SEP> and <SEP> dose <SEP><SEP><SEP> pathway <SEP><SEP> time <SEP> reaction
<tb><SEP> mice <SEP> 20mn <SEP> 45mn <SEP> 60mn <SEP> 90mn <SEP> 120mn
<tb> N <SEP> 18 <SEP> - <SEP> 100 <SEP> mg / kg <SEP> iv <SEP> 6 <SEP> 38 <SEP> 80 * <SEP> 120 * <SEP> 61 * <SEP> 90
<tb> N <SEP> 20 <SEP> - <SEP> 100 <SEP> mg / kg <SEP> iv <SEP> 6 <SEP> 59 <SEP> 196 ** <SEP> 150 * <SEP> 88 * <SEP> 85
<tb> N <SEP> 18 <SEP> - <SEP> 100 <SEP> mg / kg
<tb> + <SEP> D <SEP> Ala2Met <SEP> Enkephalin <SEP> (30 &gamma; ivt) <SEP> iv <SEP> 6 <SEP> 0 <SEP> 280 * <SEP> 166 * <SEP> 87 * <SEP> 174 *
<tb> N <SEP> 20 <SEP> - <SEP> 100 <SEP> mg / kg
<tb> + <SEP> D <SEP> Ala2Met <SEP> Enkephalin <SEP> (20 &gamma; ivt) <SEP> iv <SEP> 6 <SEP> 239 ** <SEQ> 528 *** <SEP> 321 * ** <SEP> 382 *** <SEP> 322 *
<tb> N <SEP> 20 <SEP> - <SEP> 30 <SEP> mg / kg
<tb> + <SEP> D <SEP> Ala2Met <SEP> Enkephalin <SEP> (15 &gamma; ivt) <SEP> iv <SEP> 6 <SEP> 247 ** <SEP> 353 *** <SEP> 283 * ** <SEP> 210 * <SEP> 203 *
<tb> * <0.05 ** <0.02 *** <0.01
c) Hypotensive activity
- clean
potentiator of enkephalin (LAUBIE M., SCHMITT H., VINCENT M., REMON D. Central cardiovascular effects of Morphinometic peptides in dogs, European Journal of Pharmacology, Vol 46, 67-71, 19771.

 Intravenously, compounds 18 and 24 resulted in a decrease in blood pressure at a dose of 0.3 mg / kg.

 The results of these studies demonstrate the low toxicity and the interesting inhibitory properties of the enkephalinase, analgesic and hypotensive derivatives of the invention that make them useful in human and veterinary medicine.

 The medicament of the invention is administrable to humans by the oral, parenteral or rectal route.

 Each unit dose advantageously contains from 0.5 to 100 mg of active principle, the doses that can be administered daily may vary from 0.5 to 200 mg of active principle.

Claims (6)

 1. Amino acid derivatives having the following general formula

 in which  R1 is a hydrogen atom; an alkyl group optionally mono- or polysubstituted with a halogen atom; a phenyl group optionally mono- or polysubstituted with a radical chosen from a halogen atom, a trifluoromethyl radical or hydroxy; a cyclohexyl group or a phenyl group;  n is 0 or 1;  R2 is a hydrogen atom, a linear or branched C1-C6 alkyl group; a phenyl group; a benzoyl group optionally mono- or polysubstituted with a halogen atom; a hydroxyalkyl group; an alkoxyalkyl group optionally substituted on the alkoxy moiety by a radical selected from phenyl, benzhydryl or pyridyl N-substituted by a phenylalkyl group, the phenyl ring of which is optionally substituted with one or more halogen atoms; a phenoxyalkyl group; a mercaptoalkyl group optionally substituted on the sulfur by a radical chosen from a single or branched alkyl radical, phenyl or benzyl radical;  R3 is a hydrogen atom; a linear or branched C1-C8 alkyl group; a phenyl or phenyl alkyl group whose alkyl moiety is C1 to C8, the latter two radicals being optionally mono- or polysubstituted on the phenyl nucleus by a halogen atom;  Y is -S-; NH- or -CH2-; and  X is a hydrogen atom; a C1 to C2 alkyl group substituted with a radical selected from alkoxycarbonyl radical, a carboxy radical, a sea capto radical, an alkylthio radical optionally mono- or polysubstituted with a halogen atom, an optionally mono- or polysubstituted benzylthio radical; by a group chosen from a halogen atom and a mono- or polyhalo-C 1 -C 4 alkyl group, a benzhydrylthio radical optionally mono- or polysubstituted with a halogen atom, an optionally substituted aliphatic (C1-C4) thio radical; mono or polysubstituted by a halogen atom, a benzoylthio radical optionally mono- or polysubstituted by a group selected from a halogen atom and a mono or polyhaloalkyl group C1 to C4, or a benzhydrylcarbonylthio radical optionally mono- or polysubstituted by an atom halogen; an acyl-aliphatic group C1 to C or benzoyl; an alkoxycarbonyl group; a mercapto group; an aliphatic (C1-4) acyl or benzoylthio acyl group;  and their addition salts with pharmaceutically acceptable acids or bases.  2. Derivatives according to claim 1a characterized in that  R1 is a hydrogen atom; a phenyl group optionally mono- or polysubstituted with a halogen atom;  n is 0 or 1;  R2 is a hydrogen atom; an alkyl group  C1 to C6 linear or branched; a benzyl group; a benzyloxyalkyl group;  R3 is a hydrogen atom; a linear or branched C1 to C8 alkyl group;  Y is a group -S-; -NH- or -CH2-; and  X is a hydrogen atom; a C1 to C2 alkyl group substituted with alkoxycarbonyl, carboxy, mercapto, aliphatic (C1-4) thio acyl, or benzoylthio; a C1 to C4 aliphatic acyl group or benzoyl; a C1-4 alkoxycarbonyl group; a mercapto group; an aliphatic (C1-4) acyl or benzoylthio acyl group.

 a malonic moncester of formula

 A process for preparing derivatives of formula 1a wherein Y = H, X is a hydrogen atom, an alkyl group substituted with a carboxy or mercapto group, or an alkoxycarbonyl group and R1, R2 are as defined in claim 1, characterized in that it carries out a peptidisc condensation reaction between a N-protected diamine of formula  4. The medicament having inhibitory activities of enkephalinase antalgic and hypotensive, characterized in that it contains as active principle a compound according to any one of claims 1 and 2.  5. Medicament according to claim 4, characterized in that it is presented in a form suitable for oral, parenteral or rectal administration, the action principle being associated with a suitable therapeutic vehicle.  6. Medicament according to any one of claims 4 and 5, characterized in that it is presented in unit dose form containing from 0.5 to 100 mg of active ingredient.