FR2597865A1 - New derivatives of a benzylalkylcarboxylic acid substituted with a 4-pyridylaminocarbonyl radical, process for their preparation, the new intermediates obtained, their application as medicaments and the pharmaceutical compositions which contain them - Google Patents

Abstract

Compounds of formula (I): in which X represents one of the following groups: -COOH, -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms, , Y represents one of the following groups: R1 being a hydrogen atom, an alkyl radical containing from 1 to 5 carbon atoms, an alkoxy radical containing from 1 to 5 carbon atoms, a hydroxyl radical, a halogen atom or a trifluoromethyl radical, as well as their addition salts with acids and bases. Another subject of the invention is the processes for the preparation of the compounds (I) and the new intermediates thus obtained, their application as medicaments and the pharmaceutical compositions which contain them.

Description

 The present invention relates to novel benzylalkylcarboxylic acid derivatives substituted with a (4-pyridinylaminocarbonyl) radical, process for their preparation, novel intermediates obtained, their use as medicaments and pharmaceutical compositions.

dans laquelle X représente l'un des groupements suivants : -COOH, -COOR, R étant un radical alcoyle renfermant de 1 à 5 atomes de carbone,

The subject of the invention is the compounds of formula (I)

in which X represents one of the following groups: -COOH, -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms,

Y represents one of the following groupings

R 1 being a hydrogen atom, an alkyl radical contains from 1 to 5 carbon atoms, an alkoxy radical containing from 1 to 5 carbonyl atoms; a hydroxy radical, a halogen atom or a trifluoromethyl radical as well as their addition salts with acids and bases.

 When X represents a -COOR group, R is preferably a methyl, ethyl, isopropyl, n-propyl, n-butyl, isobutyl or tert-butyl radical. R 1 is preferably a hydrogen atom or a methyl or ethyl radical. methoxy or ethoxy radical, a chlorine atom, a hydroxy or trifluoromethyl radical.

 Among the acid addition salts, mention may be made of those formed with mineral acids such as hydrochloric, hydrobromic, sulfuric or phosphoric acids as well as those formed with sulphonic acids, such as alkyl or arylsulphonic acids, for example methanesulfonic acid or para-toluenesulfonic acid.

 Among the base addition salts are those formed with alkali metals such as sodium and potassium and amines, for example, trimethylamine or dimethylamine.

The preferred products are the products in which R 1 represents a hydrogen atom.

The invention relates in particular to the compounds of formula (I) in which X represents a group

ainsi que leurs sels d'addition avec les acides et les bases et tout particulièrement le N'-hydroxy 2-(phenylméthyl) N-(4-pyridinyl) 1,4-butane diamide et son chlorhydrate.

as well as their addition salts with acids and bases and especially N'-hydroxy 2- (phenylmethyl) N- (4-pyridinyl) 1,4-butane diamide and its hydrochloride.

The invention also relates to a process for preparing the products of formula (I '). in which X represents one of the following groups: - C00H, -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms,

-CONHNHCHO et Y' représente

-CONHNHCHO and Y 'represents

dans laquelle R est un radical alcoyle renfermant de 1 à 5 atomes carbone et R1 a la signification indiquée précédemment, à un ...ent de chloruration et en ce que l'on condence le chlorure de l'acide de formule II' ainsi obtenu avec la 4-aminopyridine pour obtenir le produit de formule (IA.) :

que l'on saponifie, le cas échéant, pour obtenir le produit de formule (I'B)

ou soumet, le cas échéant, la fonction carboxyle de ce produit (I'B) soit à une chloruration pour obtenir le chlorure de l'acide alpha-[(4 pyridinylamino)carbonyl] benzène propanoïque que l'on traite - avec l'ammoniac pour obtenir le produit de formule fi 'C)

- ou bien avec la bis triméthylsilylhydroxylamine, pour obtenir le prpduit de

soit à une activation et traite le produit de formule (I'B) ainsi activé - avec la benzylhydroxylamine pour obtenir le produit de formule (I'E)

que l'on soumet, le cas échéant, à une hydrogénation catalytique pour obtenir le produit de formule (I'D) telle que décrite cidessus - ou bien à la formylhydrazine pour obtenir un produit de formule (I F)

ou bien avec un produit de formule R2-NHNH2, R2 étant un groupement bloqueur de l'hydrazine, pour obtenir le produit intermédiaire de formule III' suivant

produit intermédiaire dont on élimine le groupement bloqueur, pour obtenir le Droduit de formule (I'G)

produit intermédiaire (I'G) que l'on soumet éventuellement à l'action du mélange acide formique-anhydride acétique pour obtenir le produit de formule (I'F) telle que décrite ci-dessus produits de formule (I') que l'on soumet éventuellement à l'action d'un acide ou d'une base pour en former le sel correspondant.R 1 being a hydrogen atom or an alkyl radical containing from 1 to 5 carbon atoms, an alkoxy radical containing from 1 to 5 carbon atoms, a hydroxyl radical, a halogen atom or a trifluoromethyl radical, characterized in that the product of formula (II ') is subjected

in which R is an alkyl radical containing from 1 to 5 carbon atoms and R 1 has the meaning indicated above, at a chlorination stage and in that the chloride of the acid of formula II 'thus obtained is with 4-aminopyridine to obtain the product of formula (IA):

which is saponified, if necessary, to obtain the product of formula (I'B)

or subject, where appropriate, the carboxyl function of this product (I'B) to chlorination to obtain alpha - [(4 pyridinylamino) carbonyl] benzene propanoic acid chloride which is treated with the ammonia to obtain the product of formula fi 'C)

- or with bis-trimethylsilylhydroxylamine, to obtain the product of

either to an activation and treats the product of formula (I'B) thus activated with benzylhydroxylamine to obtain the product of formula (I'E)

that, if appropriate, subjected to a catalytic hydrogenation to obtain the product of formula (I'D) as described above - or to formylhydrazine to obtain a product of formula (IF)

or with a product of formula R2-NHNH2, R2 being a blocking group of hydrazine, to obtain the intermediate product of formula III 'next

intermediate product from which the blocking group is eliminated, to obtain the product of formula (I'G)

intermediate product (I'G) which is optionally subjected to the action of the formic acid-acetic anhydride mixture to obtain the product of formula (I'F) as described above products of formula (I ') that the it is optionally subjected to the action of an acid or a base to form the corresponding salt.

The subject of the invention is also a process for preparing the products of formula (I ") in which X represents one of the following groups: -COOH, -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms,

et Y" représente

and Y "represents

R1 being a hydrogen atom, an alkyl radical containing
1 to 5 carbon atoms, an alkoxy radical containing 1 to 5 ~ carbon atoms, a hydroxy radical, a halogen atom or a
trifluoromethyl radical characterized in that 1 is subjected to
product of formula (II ") ::
Wherein R 2 is an alkyl radical containing from 1 to 5 carbon atoms

R 1 having the above meaning to a chlorinating agent and in that the chloride of the acid of formula IV thus obtained is condensed with 4-aminopyridine to obtain a mixture of the two products of formula (I ")

R and Y "having the above meaning, products of formula (II) which are separated and then saponified, where appropriate, to obtain the 2 products of formula (II) ::

Y "having the meaning given above, products of formula (I" B) which is activated, where appropriate, the acid function and then treated with either ammonia to obtain a mixture of 2 products of formula (I '' c) following

Y "having the meaning given above as well as the cyclization product of formula V following

et du produit de cyclisation de formule (V) précédemment décrit, produits de formule (I"E) que l'on isole du mélange et que l'on soumet, le cas échéant, à une hydrogénation catalytique pour obtenir les produits de formule (I"D) :

R1 having the previously indicated values, products of formula (I-C) which is isolated from the mixture, or with benzylhydroxylamine to obtain a mixture containing the following two products of formula (I "E)

and the cyclization product of formula (V) previously described, products of formula (I "E) which is isolated from the mixture and which is subjected, if appropriate, to a catalytic hydrogenation to obtain the products of formula ( I "D):

Y "having the meaning given above.

Z ayant la signification donné précédemment et du produit de cyclisation de formule (V) précédemment décrit, produits de formule (I"F) que l'on isole du melange, soit avec un produit de formule R2NHNH2, R2 étant un groupement bloqueur de l'hydrazine, pour obtenir les produits intermédiaires suivants

with formylhydrazine to obtain a mixture containing the following two products of formula (I "F)

Z having the meaning given above and the cyclization product of formula (V) previously described, products of formula (I-F) which is isolated from the mixture, or with a product of formula R2NHNH2, R2 being a blocking group of hydrazine, to obtain the following intermediate products

Y" ayant la signification donnée précédemment que l'on soumet, le cas échéant, à l'action du mélange anhydride acétique-acide formique, pour le produit de formule (I"F) telle que décrite cidessus produits de formule (I") que l'on soumet, le cas échéant, a l'action d'un acide ou d'une base pour en former le sel correspondant.Y "having the meaning given above, in the form of a mixture of products (III"); these mixtures also containing the cyclization product of formula (V,) intermediate products which are isolated from the mixture obtained and which the blocking group is removed to obtain the products of formula

Y "having the meaning given above which is subjected, if appropriate, to the action of acetic anhydride-formic acid mixture, for the product of formula (I-F) as described above products of formula (I") if appropriate, it is subjected to the action of an acid or a base to form the corresponding salt.

 in the preferred conditions for carrying out the process which is the subject of the invention, in the starting products of formula (II ') or (II "), R is an ethyl radical.

The chlorinating agents of the acids of formula (II '), (II ") or (I'8) are customary: thionyl chloride phosgene, oxalyl chloride e.

The condensation of the acid chloride of formula (II ') or of the acid chloride of formula (II ") is carried out in tetrahydrofuran between -10 and -20 ° C.

The activation of the acid functions is carried out by the action of 1-ethyl-3 (3-dimethylamino propyl) carbodiimide (EDAC). Hydroxybenzotriasole can also be used in the presence of dicyclohexyl carbodiimide.

On fait, par exemple, agir à -20 C E l'ammoniac sur le chlorure de l'acide alpha-E4-pyridinylaminocarboxyl] benzène propanoique dans l'éther.- To obtain the product of formula (I'c) in which

For example, ammonia is reacted with ammonia on alpha-E4-pyridinylaminocarboxylbenzenepropanoic acid chloride in ether.

The action of trimethylsilyhyldroxylamine is carried out in the presence of a tertiary base such as triethylamine.

In the products of formula (III ') and (III "), the R2 blocking group of hydrazine is a benzyloxycarbonyl (Z) or tert-butyloxycarbonyl (-Boc) group.The elimination of the Z blocking group from the amino function is In particular, it is carried out by catalytic hydrogenation with palladium on activated charcoal.The Boc blocker group is eliminated in an acidic medium, in particular by trifluoroacetic acid in a solvent such as ethyl acetate or methylene chloride.

 - The products of formula (IA) (I-C), (IE), (I "1, F> (here") obtained in the form of a mixture are separated by chromatography on silica The compounds of formula (I) as defined above have interesting pharmacological properties.

They have, in particular, very interesting properties of inhibitors of enkephalinase and aminopeptidase sensitive to the bestatin, enzymes of degradation of -enkephalins. They are, therefore, endowed with analgesic and psychotropic properties.

Enkephalinase is a dipeptidylcarboxypeptidase that specifically hydrolyzes methionine and leucine enkephalin between the 3rd and 4th amino acids, thereby releasing a tripeptide
Tyr-Gly-Gly (JP Swerts, Perdrisot R., Patey G., Baume S.

and Schwartz J.C. Europ. J. Pharmacol., 1979, 57, 279).

Bestatin-sensitive aminopeptidase releases tyrosine from the peptide.

 Enkephalinase and aminopeptidase contribute to the physiological degradation of enkephalins, endogenous natural ligands of opiate receptors. The compounds of the invention, which delay the degradation of enkephalins, thus stimulate the body's defense reactions against pain.

These properties justify their application in therapy and the subject of the invention is also, as medicaments, the compounds of formula (I) as defined above. The subject of the invention is more particularly, as medicaments, the compounds of formula (I) in which X represents a group

et Y représente

and Y represents

as well as their addition salts with acids and bases
pharmaceutically acceptable.

The invention relates particularly as a medicinal
N'-hydroxy 2- (phenylmethyl) N- (4-pyridinyl) 1,4-butane
diamide and its hydrochloride.

The medicaments, object of the invention, can be used
in the treatment of muscle, joint or
nerves, rheumatic conditions, dental pain, zonas and migraines, as well as in the treatment of
inflammatory diseases, including arthrosis, lumbago, and
also as a complementary treatment in the states
infectious and feverish.

The invention extends to pharmaceutical compositions
containing as active ingredient the medicinal products defined in
above.

These pharmaceutical compositions can be administered
by the oral, rectal, parenteral or
local, topically applied to the skin and mucous membranes.

These compositions may be solid or liquid and
present in the pharmaceutical forms commonly used in
human medicine such as, for example, single tablets or
sugar coated tablets, capsules, granules, suppositories,
injection preparations, ointments, creams, gels and
aerosol preparations; they are prepared according to
usual methods. The active ingredient can be incorporated into
excipients usually used in these compositions
pharmaceutical products, such as talc, gum arabic, lactose,
starch, magnesium stearate, cocoa butter,
aqueous vehicles or not, fatty substances of animal origin or
plants, paraffinic derivatives, glycols, various
wetting agents, dispersants or emulsifiers, preservatives.

The dosage varies in particular depending on the route
administration, the condition being treated and the subject.

For example, in adults, it can vary between 20 mg and
2g of active ingredient, per day, orally.

The products of formula (III '), (V), (III ") and the chloride of
alpha-Ef4-pyridinylamino) carbonyl] benzenepropanoic acid
are new industrial products.

 The subject of the invention is therefore the products of formula (III ") and the chloride of alpha-E (4-pyridinylamino) carbonyl] benzenepropanol, as new industrial products and in particular intermediate products necessary for implementation of the methods of the invention.

 The invention also relates, as a new chemical, to the product of formula (V).

 The compounds of formula (II ") used as starting materials of one of the processes of the invention are prepared as described in Journal of the American Chemical Society / 90 13 / June 19, 1963.

The examples given below illustrate the invention without
however, limit it
Example 1: alpha - ((4-pyridinyl) amino carbonyl] benzene propa
noate of ethyl.

22.3 g of 2-phenylmethyl are heated at 700 ° C. for 1 hour.
monoethyl propanedioate in 100 ml of thionyl chloride.

There is a significant evolution of gaseous hydrochloric acid,
then cool the solution to room temperature, evaporate the
solvent under reduced pressure, take up the residue twice with 100 ml
of benzene and removes solvents again under pressure
scaled down. The acid chloride thus obtained is taken up in 100 ml.
methylene chloride and added dropwise at -10 ° C to a
solution containing 18.8 g of 4-amino pyridine in 150 ml of
methylene chloride and in the presence of 13.9 ml of triethylamine.

It is allowed to come back to room temperature and maintains
stirring for 12 hours. The suspension thus obtained is filtered off, the filtrate is washed with 0.05 N hydrochloric acid and
the water, dry and evaporate the solvent under reduced pressure. The
desired compound is purified by chromatography on silica under a
pressure of 8 bar with the following eluate: methylene chloride
methanol (98-2). 21 g of the expected product F = 100 ° C. are obtained.

Example 2: alpha- (4-pyridinylamino) hydrochloride
"carbonyl benzene propanol.

8 ml of 1N sodium hydroxide dissolved in 30 ml of ethanol in 2 g of the compound of Example 1 in 20 ml are added dropwise to OC.
'Methanol. The mixture is warmed to room temperature, for
the reaction for 12 hours, then evaporate the solvent
reduced pressure. The residue is taken up in 50 ml of water, washed with methylene chloride, the aqueous phase is acidified to pH = 6.
recover the precipitate obtained by filtration, wash with ether
ethyl and dry. 1.85 g of the expected product under
base form which is dissolved in 18.5 ml of N. hydrochloric acid

 The expected hydrochloride is obtained after lyophilization - F 1960C.

Example 3: hydrazide of alpha - ((4-pyridinyl amino) acid
carbonyl) benzene propanol.

Stage A: Hydrazide of alpha-E (4-pyridinyl amino) acid
carbonyl) benzene propanoic.

1.235 ml are added dropwise at room temperature
triethylamine in 2 g of the compound of Example 2 in suspension.
otherwise in 50 ml of chloroform. 1.70 g of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDAC) are then added in small portions. The solution obtained is stirred for 15 minutes and then 1.5 g of benzyloxycarbonyl hydrazine dissolved in 10 ml of chloroform are added.

 The reaction is continued for 12 hours at room temperature and the resulting solution is washed with 0.05 N hydrochloric acid and with water.

 The organic phase is dried, the solvents are eliminated under reduced pressure, the residue is taken up in methylene chloride, the precipitate is filtered off and dried to obtain 1 g of the expected product.

 The filtrate is concentrated under reduced pressure, the residue is chromatographed on silica under a pressure of 4 bars of ethyl acetate, and 460 mg of expected product is obtained.

 Thus, a total of 1.46 g of expected product F m 176 ° C is obtained.

Stage B: hydrazide of alpha-Ef4-pyridinyl amino acid)
carbonyl) benzene propanoic.

 100 mg of palladium on charcoal are added to 1 g of the product obtained in stage A in solution in 20 ml of a water-acetic acid mixture (9/1). It is hydrogenated under a pressure of 2 atmospheres, at room temperature for 1 h 30. The suspension and the filtrate thus obtained are filtered off, the solvent is evaporated under reduced pressure and the residue is taken up with 10 ml of a water-methanol mixture (1 / 1). Purify by chromatography on silica (eluent: methanol-water (1/1).

 620 mg of expected product F - 193 ° C is obtained.

Example 4: hydrochloride of N1-hydroxy 2- (phenylmethyl) N3 (4-pyridinyl) 1,3-propanediamide.

 500 mg of product of Example 2 in base form dissolved in 10 ml of thionyl chloride are heated at 70 ° C. for 1 hour.

 It is then cooled to room temperature, the thionyl chloride is eliminated under reduced pressure, the residue is taken up in 20 ml of benzene which is then evaporated under reduced pressure.

We repeat the operation twice.

The acid chloride thus obtained is dissolved in 20 ml of methylene chloride. 258 μl of triethylamine are added dropwise to the solution and then a mixture comprising 435 μl of bis-trimethylhydroxylamine and 258 μl of triethylamine dissolved in 10 ml of methylene chloride. Stirring is maintained for 1 hour at room temperature, then the solvent is evaporated off under reduced pressure, the residue is taken up in 30 ml of methanol and the mixture is stirred for 30 minutes at ambient temperature, the precipitate is filtered off and dried under reduced pressure to obtain 498 mg of expected product. in the form of a base which is dissolved in 1 ml of water containing 1 equivalent of hydrochloric acid. The solution obtained is lyophilized and the desired product is obtained in the hydrochloride form.
F 12100C.

Example 5: ethyl beta-Ef4-pyridinylamino) carbonyl] benzene butanoate
50 ml of 4-ethyl 2-phenylmethyl butane dioate are cooled to 50 ° C. (Journal of the American Chemical Society 90: 13, June 19, 1968) in 100 ml of methylene chloride and then 10 ml of sodium chloride are added dropwise. oxalyl.

 Stirring is continued, allowing the mixture to return to ambient temperature for 2 hours.

 200 ml of tetrahydrofuran are then added, the mixture is cooled again to -15 ° C. and then 329 of 4-aminopyridine in solution in 300 ml of tetrahydrofuran are added.

 After the mixture is at room temperature, the solvents are filtered off and removed under reduced pressure. A brown oil is obtained which is chromatographed on silica (eluent: methylene chloride) and collects 24 g of a first yellow oil, 0.5 g of a yellow solid corresponding to a low-polar impurity and 2.49 of a second yellow oil.

 29 of the first yellow oil previously obtained is chromatographed again on silica (eluent methylene chloride: 100-methanol: 4.5) and 1.8 g of expected product is obtained in base form. The product is solubilized in 50 ml of ethyl acetate, 63 ml of 0.1 N hydrochloric acid are added, the solvent is evaporated off under reduced pressure at 20 ° C. and the remaining aqueous phase is lyophilized to obtain the expected product in the hydrochloride form after drying at 60 ° C (F ~ 78 ° C).

 2.4 g of the second yellow oil obtained above (eluant: methylene chloride 100-methanol 5) are chromatographed again on silica and 2.3 g of a colorless oil whose NMR spectrum confirms that it corresponds to to the position isomer of the product of Example 5, i.e. ethyl (alpha- [2-oxo 2- (4-pyridinylamino) ethyl] benzene propanoate).

Example 6: beta - [[(4-pyridinyl) amino] carbonyl]
butanoic benzene.

 17 g of product of Example 5 dissolved in 200 ml of methanol are cooled to 10 ° C., then 55 ml of sodium hydroxide N are added dropwise and the mixture is allowed to return to ambient temperature in the course of 1 hour.

 The reaction mixture is evaporated by half under reduced pressure and then the remaining solution is cooled to -20 ° C. and the pH is adjusted to 4.5 with 2N hydrochloric acid. 200 ml of cold ethyl acetate is added to the suspension obtained, the mixture is stirred for 10 minutes, the precipitate is filtered off, rinsed with ether and dried to obtain 9.49 of crude product.

 It is removed lg and recrystallized in ethanol.

 0.79 of expected product is obtained.

 Example 7: N '- (phenylmethoxy) 2- (phenylmethyl) N- (4-pyridyl) 1,4-butane diamide.

 With 0.1 N hydrochloric acid, the pH is adjusted to 3 of a solution containing 1.959 of the product obtained in Example 6 in 399 ml of water and 100 ml of tetrahydrofuran.

 3.28 g of benzylhydroxylamine are added at ambient temperature while maintaining the pH at 3 + 0.2 with a saturated sodium bicarbonate solution and then, during 15 minutes, 2.63 g of 1-ethyl-3- ( 3-dimethylaminopropyl) carbodiimide (EDAC) in solution in 15 ml of water and stirred for 1 hour 30 minutes. The filtrate is filtered and the filtrate is poured into 500 ml of a 5% aqueous sodium bicarbonate solution, extracted with water. ethyl acetate, combines the organic phases and eliminates the solvents under reduced pressure. The residue obtained is chromatographed on silica (eluent methylene chloride methanol 100-3.5).

We obtain
0.709 of a cyclization product: 3- (phenylmethyl)
1- (4-pyridinyl) 2,5-pyrrolidine dione (F-112 ° C)
0.809 of the expected product (FX 80 "C) and
0.229 of the position isomer of the expected product or the
N '- (phenylmethoxy) 3- (phenylmethyl) N- (4-pyridinyl)
1,4-butanediamide (mp 103 ° C).

Example 8: hydrochloride of 2-hydroxy-2- (phenylmethyl)
N-4 (4-pyridinyl) 1,4-butanediamide.

3.26 g of the product of Example 7 dissolved in 1 liter of methylene chloride are cooled to -20 ° C. 3.29 ml of a mixture of ether-hydrochloric acid 2.24N in solution in 100 ml of methylene chloride are added over 30 minutes at -20 ° C. Finally
of addition, argon is bubbled into the mixture
reaction, the solvent is removed under reduced pressure at 20 ° C. and then
solubilizes the product obtained in cold water. 400 mg is added
of palladium on charcoal and hydrogen for 3 hours under a
pressure of 1.4 bar.

 The solution obtained is filtered and lyophilized to give 1.91 g of expected product, F = 137 ° C.

Example 9: N'-hydroxy-3- (phenylmethyl) hydrochloride
N- (4-pyridinyl) 1,4-butane diamide.

The procedure is as in Example 8, starting from 710 mg of
N '- (phenylmethoxy) 3- (phenyl methyl) N- (4-pyridinyl) 1,4-butane
diamide obtained in Example 7 of 0.84 ml of the ether-acid mixture
hydrochloric acid and 200 mg of palladium on charcoal.

 0.31 g of the expected product (mp> 125 ° C.) is obtained.

It has also been prepared by one of the methods
the invention the next product
read 2 alpha-2-E4-pyridinyl amino formylhydrazide)
carbonyl) benzene propanol F = 207 C.

Example 10
Tablets having the formula
- Compound of Example 8 ................................ 8 50 mg
- Excipient qs for a tablet finished at ............. at 350 mg
(details of the excipient: lactose, starch, magnesium stearate, talc).

biological study of the product of Example 8 1.- Determination of the inhibitory effect of enkephalinase and the aminopeptidase
a) Inhibitory effect of enkephalinase
The activity of enkephalinase is determined from a renal membrane fraction of rat. The kidneys are collected on ice and homogenized in 0.05M HEPES pH 7.5 buffer containing magnesium chloride (50 times the volume). After a first centrifugation at 15009, the particulate fraction is obtained after centrifugation at 15 0009 for 20 minutes. The pellet is then washed and suspended in HEPES buffer and stored at -20C.

An aliquot of this membrane preparation is placed in the presence of octyl beta-D glucopyranoside (50 mM in final concentration) for 15 minutes at 4 ° C. After centrifugation at 100,000 g for one hour, the supernatant is removed and frozen in aliquots at C. The protein concentration is determined by the Comassie blue method.

 An aliquot of the protein fraction thus prepared is preincubated at 25 ° C. for 10 minutes in 0.05M HEPES buffer pH 7.5 or in the presence of the product to be studied.The substrate added is a non-natural enkephalin of the formula Succinyl-Ala. Ala-Phe-aminomethylcoumarin (reference 1) (40 μM in final concentration), incubation continued at 37 ° C for 30 minutes. The hydrolysis reaction is stopped by heating at 95 ° C. for 5 minutes and each incubate is centrifuged A solution of aminopeptidase M (4 M in final concentration) and thiorphan (reference 2) (10 M) is added to the supernatant and the whole is brought to 37 ° C for one hour. The reaction is stopped as previously described and. the fluorescence of 7-aminomethylcoumarin thus produced determined. The effect of the products to be tested is determined by the calculation of the IC 50 concentration which inhibits the hydrolysis of the substrate.

Reference 1: A. A. Munford, PA Pierzchala, A. Strauss and M.

Zimmerman
Purification of a membrane-bound metalloendo
peptidase from porcine kidney that degrades peptides
hormones.

 Proc. Natl. Acad. Sci. 78, No. 11, 6623.

Reference 2: Enkephalinase inhibitor described in Example 20
BF - 2480747. The IC50 found is 10M.

b) Inhibitory effect of aminopeptidase
The Phe-aminomethylcoumarin substrate (40 μM) solubilized in 50 mM HEPES buffer pH 7.4 is concentrated in the presence of aminopeptidase (0.2 μg / ml) and the inhibitor for thirty minutes at 37 ° C. Hydrolysis reaction is stopped by heating to 950C and the fluorescence of 7-aminomethylcoumarin thus formed is measured.

The effect of the test product is determined by the calculation of the
IC50 or concentration that inhibits by 50% the hydrolysis of the substrate.

The IC50 found is 9 x 10 7M.

2.- Study of analgesic activity in mice
a) Stretching caused by acetic acid in the mouse.

 The test employed is based on the fact reported by R. KOSTER and Cool., (Fed Proc., 1959, 1B 412) that intraperitoneal injection of acetic acid causes, in the mouse, repeated stretching movements, torsion may persist for more than 6 hours. Analgesics prevent or diminish this syndrome which can be considered as the exteriorization of diffuse abdominal pain. A solution of 1% acetic acid in water administered in a volume of 10 ml / kg is used.

 The studied product is administered orally half an hour before the injection of acetic acid, the mice being fasted for six hours at least.

 Stretches are observed and counted for each mouse during a 15 minute observation period.

 The results are expressed by means of the DA50, ie the dose which makes it possible to obtain a 50% decrease in the number of stretches compared with the control animals. The DA50 found is 40 mg / kg.

b) Hot plate test in mice
Female mice of average body weight 229, in the water diet for 6 hours, are placed, one by one, on a copper plate maintained at 560C: the reaction to pain is manifested by the licking of the anterior legs or the leap of the animal; the time of this reaction is noted and only the mice that react in less than 8 seconds are retained.

 The mice are divided into groups and treated per os, a group receiving only the vehicle. The pain reaction time is again measured 30 - 60 and 90 minutes after the treatment. The reaction time variations are expressed as a percentage of the starting time, taking into account variations in the control group. The DA100, or dose which increases the reaction time by 100%, is determined graphically 60 minutes after the treatment; The DA100 found is 100 mg / kg.

Claims (11)

 in which X represents one of the following groups: -COOH, -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms,

 1) The compounds of formula (I)

 being an aromatic atom, an alkyl radical having 1 to 5 carbon atoms, an alkoxy radical containing 1 to 5 carbon atoms, a hydroxy radical, a halogen atom or a trifluoromethyl radical and also their addition salts with acids and bases.

Y represents one of the following groupings 2) The compounds of formula fi) as defined in claim 1 in which X represents a group

 as well as their addition salts with acids and bases.

 and Y represents

3) Nitroxy 2- (phenylmethyl) N- (4-pyridinyl) 1,4-butane amide and its hydrochloride. 4) process for the preparation of the products of formula (I ') in which X represents one of the following groups

 -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms,

 and Y 'represents

 if necessary, subject to a catalytic hydrogenation to obtain the product of formula (I D) as described above.

 (IE)  either to an activation and treats the product of formula (zig) thus activated - with benzylhydroxylamine to obtain the product of formula

 - or with bis-trimethylsilylhydroxylamine, to obtain the product of

 where appropriate, the carbonyl function of this product (I'B) is subjected to chlorination to obtain the alpha - ((4 pyridinylamino) carbonyl] benzene propanoic acid chloride which is treated with the ammonia to obtain the product of formula (I ')

 which is saponified, if necessary, to obtain the product of formula (I'B)

 in which R is an alkyl radical containing from 1 to 5 carbon atoms and R 1 has the meaning indicated above, with a chlorinating agent and in that the chloride of the acid of formula II 'thus obtained is condensed with 4-aminopyridine to get the pro

R 1 being a hydrogen atom, or an alkyl radical containing from 1 to 5 carbon atoms, an alkoxy radical containing from 1 to 5 carbon atoms, a hydroxyl radical, a halogen atom or a trifluoromethyl radical, characterized in that that the product of formula (II ') is subjected  optionally subjected to the action of the formic acid-acetic anhydride mixture to obtain the product of formula (I'F) as described above, products of formula (I ') which are optionally subjected to action of an acid or a base to form the corresponding salt.

 intermediate product from which the blocking group is eliminated in order to obtain the product of formula (I'G)

 or with a product of formula R2-NHNH2, R2 being a blocking group of hydrazine, to obtain the intermediate product of formula III 'next

or else with formylhydrazine to obtain a product of formula (I F) 5) Process for the Preparation of the Products of Formula (11) in which X represents one of the following groups: -COOH, -COOR, R being an alkyl radical containing from 1 to 5 carbon atoms,

 and Y "represents

 Wherein R is an alkyl radical having from 1 to 5 carbon atoms and R 1 being a hydrogen atom, an alkyl radical containing from 1 to 5 carbonyl atoms, an alkoxy radical containing from 1 to 5 carbonyl atoms, a hydroxyl radical, a halogen atom or a trifluoromethyl radical, characterized in that a product of formula (II ") is subjected

R 1 having the above meaning to a chlorinating agent and in that the chloride of the acid of formula IV thus obtained is condensed with 4-aminopyridine to obtain a mixture of the two products of formula (I ")

 yH having the meaning given above, products of formula whose, if appropriate, the acid function is activated and which is then treated with either ammonia to obtain a mixture of the following products of formula (I-C)

R and Y "having the above meaning, products of formula (I" A) which are separated and then saponified, where appropriate, to obtain the 2 products of formula (I "B)

Y "having the meaning given above as well as the cyclization product of formula V following  Y. having the meaning given above.

 and the cyclization product of formula V previously described, products of formula (I "E) which is isolated from the mixture and which is subjected, if appropriate, to a catalytic hydrogenation to obtain the products of formula (I" not) :

R1 having the values previously indicated, products of formula (I "c) which is isolated from the mixture, or with benzylhydroxylamine to obtain a mixture containing the following two products of formula (I" E ")

with formylhydrazine to obtain a mixture containing the following 2 products of formula (IIIF)

Y "having the meaning given above and the cyclization product of formula V previously described, products of formula (I * F) which is isolated from the mixture, or with a product of formula R2NHNH2, R2 being a blocking group of the hydrazine, to obtain the following intermediate products  Y "having the meaning given above which is subjected, if appropriate, to the action of acetic anhydride-formic acid mixture, for the product of formula (I-F) as described above.

Y "having the meaning given above, in the form of a 'mixture of products III'; ; these mixtures also comprising the cyclization product of formula V, intermediates which are isolated from the mixture obtained and from which the blocking group is removed to obtain the products of formula products of formula (I) which, if appropriate, are subjected to the action of an acid or a base in order to form the corresponding salt. 6) As medicaments, the compounds of formula (I) as defined in claim 1 and their addition salts with pharmaceutically acceptable acids and bases. 7) As medicaments, the compounds of formula (I) as defined in claim 2, and their addition salts with pharmaceutically acceptable acids and bases. 8) As a medicament, the compound of claim 3. 9) Pharmaceutical compositions containing as active ingredient * at least one of the drugs as defined in claims 6 to 8. 10) As new industrial compounds, the products of formula III 'and alpha - [(4-pyridinylamino) carbonyl] benzenepropanoic acid chloride as defined in claim 4. 11) As new industrial compounds the products of formula V and III "as defined in claim 5.