FR2469395A1 - N-dodecyl piperidyl carbonyl cyclo-peptide - as antitubercular agent and antibacterial prepd. from cyclo:peptide and piperidine carboxylic acid - Google Patents

Abstract

N-Dodecyl -L-piperidyl carbonyl-L-transmethyl prolyl cyclopeptide of formula (I) and its acid addn. salts are new. (I) is prepd. by reaction of the cyclopeptide (II) with N-dodecyl piperidine -2- carboxylic acid (III) or a reactive deriv. of this acid. This reaction may be effected in the presence of a carbodiimide such as dicylohexyl carbodiimide and 1-hydroxy benzotriazole in an organic solvent at 0-30 deg.C. Reactive derivs. of (III) that may be used include the azide, esters, and mixed anhydrides. Cpds. (I) are anti-tubercular agents and Gram positive and negative bactericides.

Description

ses sels addition avec les acides, son procédé de préparation et les médicaments qui le contiennent.The present invention relates to a novel cyclopeptide, cyclo-L-leucyl-L trans-4-methyl-prolyl-leucyl N-methylvalyl
Prolyl-N N -methylleucyl-glycyl-N- (N-dodecyl-L piperidyl-2-carbonyl)
Transmethyl-4-prolyl) 1. N-methylthreonyl] of the formula

its addition salts with acids, its method of preparation and the drugs which contain it.

et R représente un radical alcanoyle, alcénoyle, alcadiénoyle, alcoyl ozcycarbonyle, aroyle, aralcanovie, arylsulfényle, arylsulfinyle, arylsulfonyle, cycloalcoylcarbonyle, hétérocyclylcarbonyle, hétérocyclylalcanoyle ou un reste de radical peptidique linéaire rattaché par un groupe carbonyle à l'atome d'azote de la L transméthyl-4 proline de la chaîne latérale du cyclopeptide A. In formula (I):
MePro means: L transmethyl-4 proline
MeThr means: N-methylthreonine
MeVal means: L N-methylvaline
MeLeu means: D N-methylleucine
Pro means: proline
Gly means: glycocolle
Leu means: L leucine
Thr means: threonine
In Japanese Patent 743,248 has been described the preparation of cyclopeptides of the general formula
R - cyclopeptide A (II) in which cyclopeptide A denotes a nonapeptide whose structure is

and R represents an alkanoyl, alkenoyl, alkadienoyl, alkyloylcarbonyl, aroyl, aralkanoyl, arylsulfenyl, arylsulfinyl, arylsulfonyl, cycloalkylcarbonyl, heterocyclylcarbonyl, heterocyclylalkanoyl radical or a linear peptide radical residue attached by a carbonyl group to the nitrogen atom of the Transmethyl-4 proline of the side chain of cyclopeptide A.

 In the foregoing - the alkyl, alkenyl or alkadienyl portions of the alkanoyl, alkenoyl, alkadienoyl, aralkanoyl or heterocyclylalkanoyl radicals are straight or branched chains containing from 1 to 50 carbon atoms optionally substituted with one or more halogen atoms or amino or alkoylthio containing 1 to 5 carbon atoms, - the alkyl portions of the alkyloxycarbonyl radicals contain 1 to 15 carbon atoms and may be substituted by a dialkylamino radical whose alkyl portions each contain 1 to 5 carbon atoms, - the aromatic portions of the radicals aroyls, aralkanoyls, arylsulfenyls, arylsulfinyls or arylsulfonyls represent a benzene or naphthalenic ring optionally substituted by one or more alkyl, hydroxyl, benzoyl, amino, dialkylamino, alkanoylamino or nitro radicals (the alkyl radicals and the alkyl portions of the other radicals containing 1 to 12 atom the heterocyclic portions of the heterocyclylcarbonyl or heterocyclylalkanoyl radicals represent 5- or 6-membered mononuclear heterocycles containing one or more nitrogen atoms or a phenothiazinyl radical optionally substituted with one or more alkyl or nitro radicals, the cycloalkyl portions; cycloalkylcarbonyl radicals represent 5- or 6-membered cycloalkyl radicals, optionally substituted with one or more amino radicals, the radicals of the linear peptide radicals contain 2 to 15 amino acids, and the amino radicals which substitute the alkyl, alkenyl or cycloalkyl portions of the radicals. defined above and the amine functions of the linear peptide radicals may be optionally substituted by one or more alkyl, alkanoyl, aralkyl, alkyloxyberryl, aralkyloxycarbonyl or alkyloxycaralkyloxycarbonyl radicals, alkyl radicals and polyamines. Alkyl radicals of other radicals containing 1 to 30 carbon atoms.

 These products have antituberculous properties and they show good activity on Gram-positive and negative germs.

 These products are, for the most part active in vitro when studied according to the dilution method in Dubos medium.

Their in vivo activity can be determined, in the mouse, using in particular the following test
B6D2F1 mice are inoculated intravenously with the human strain Du. They receive 0.2 cm 3 of an inoculum prepared by grinding in fraction V of bovine albumin at 0.5% of the colonies taken from Lowenstein-Jensen's medium, ie approximately 0> 0015 mg (dry weight) of bacilli per ml. mouse. The treatments are started the day after infection and are continued for 4 weeks at a rate of 5 treatments per week. The products, in the form of a base or salt, are dissolved in water and are administered by oesophageal probe at the rate of 0.5 cm 3 per mouse.

Control mice receive only water. Animals are not sacrificed. Survival times are noted and average survival times (T50) are calculated using the Litchfield method.

 Under these conditions, only (N-benzyl N-methyl L valyl) cyclopeptide A, (N-benzyl N-methyl DL valyl) cyclopeptide A methanesulphonate, N-benzylsarcosyl-cyclohexyl methanesulphonate and the hydrochloride of N-benzyl N-methyl-L-methionyl-cyclopeptide A and (N-hexyl-N-methyl-L-valyl) -cyclopeptide A hydrochloride lead to a percentage increase in survival time greater than 200%, at doses between 100 and 300 mg / kg in. These products, however, do not exhibit sufficient activity to permit convenient use in human therapy.

 Among the products which are included in Japanese Patent 743,248, N-lauryl N-methyl D valy-cyclopeptide A, which is obtained by the action of N-lauryl N-methyl D valine on cyclopeptide A in the presence of dicyclohexylcarbodiimide has been shown to be active in mice at much lower doses than the products already described, thus at the dose of 5 mg / kg pO 0 the increase in survival time is 15% and it reaches 60% at the 7.5 mg / kg po

This product, although having a much higher activity, however, has adverse side effects that compromise its potential for use in human therapy. Thus in the mouse, at doses of 225, 75 and 25 mg / kg po / day for 24 days, there is, from the 9th day of treatment, tetany phenomena which disappear only 7 to 10 days after the end of the treatment. . In the dog, at doses of 75 and 25 mg / kg p.o. per day for 3 months, (N-lauryl N-methyl-D-valyl) -cyclopeptide A produces neuromuscular disorders. In rats, it leads in addition to functional disorders of the liver.

 It has now been found that the product of formula (I), which exhibits a higher in vitro and in vivo activity than the products already described in this series, does not, surprisingly and unexpectedly, have the harmful side effects which have been mentioned above.

 Thus in vitro the minimum inhibitory concentration on the 1137Rv strain of the product of formula (I) is 0.05 μg / cm3. In vivo, at the dose of 7.5 mg / kg p.o., the percentage increase in survival is close to 200%.

 In rats at 225, 75 and 25 mg / kg po daily for 3 months and in dogs at 150, 75 and 25 mg / kg po, the product is well tolerated and particularly neuromuscular disorders is not recorded.

 As a result, the product of formula (I) is a particularly useful antituberculous agent.

 According to the present invention, the product of formula (I) can be obtained by condensation of N-dodecylpiperidine-2-carboxylic acid or an activated derivative of this acid on cyclopeptide A, that is to say on the product of general formula (II) in which R represents a hydrogen atom, by all the known methods used in peptide chemistry.

 As a condensation method, it is worth mentioning, for example, the auxcarbodiimide method, the azide method, the activated ester method and the mixed anhydride method.

 More particularly, when N-dodecylpiperidine-2-carboxylic acid is used, the reaction is carried out in an organic solvent such as ethyl acetate, dimethylformamide, acetonitrile or methylene chloride, at a temperature of temperature between 0 and 300C and in the presence of a carbodiimide such as dicyclohexylcarbodiimide and hydroxy-1 benzotriazole.

More particularly, if the azide of the acid is used
N-dodecyl piperidine-2 carboxylic acid> the reaction is carried out in an organic solvent such as ethyl acetate optionally in the presence of an organic base such as triethylamine and at a temperature between -15 and 250C.

 More particularly, if an activated ester of N-dodecylpiperidine-2-carboxylic acid such as a phenol ester (p.nitrophenol, 2,4,5-trichlorophenol) or N-hydroxylated nitrogen heterocycle (N hydroxysuccinimide, hydroxy-1-piperidine) the reaction is carried out in an organic solvent such as ethyl acetate or dimethylformamide, in the presence of a carbodiimide such as dicyclohexylcarbodiimide and at a temperature between - 15 and 250C, optionally in the presence of an organic base such as triethylamine.

 More particularly, if a mixed anhydride of N-dodecylpiperidine-2-carboxylic acid, obtained for example by the action of an alkyl halogenoformate on N-dodecylpiperidine-2-carboxylic acid, is used, the reaction is carried out in an organic solvent. , such as methylene chloride, in the presence of an organic base such as triethylamine and at a temperature of -100C.

 The process according to the invention can be carried out either on pure cyclopeptide A or on the crude product of hydrolysis of antibiotic 11072 RP, the product of formula (I) being, in either case obtained in the purified state after chromatography on a suitable support.

 Cyclopeptide A can be obtained by hydrolysis of antibiotic 11072 RP according to the method which is described in Japanese Patent 638,380.

 N-dodecylpiperidine-2-carboxylic acid can be obtained by the action of lauric aldehyde on L-piperidine-2-carboxylic acid in the presence of hydrogen and a catalyst such as palladium on support.

 L-piperidine-2-carboxylic acid can be obtained according to the process described by H. BEYERMAN, Rec. Trav. Chim. Netherlands 78, 134 (1959).

 The new product of formula (I) can be converted into an acid addition salt. An addition salt can be obtained by the action of the new cyclopeptide on an acid in a suitable solvent. Generally, the base is solubilized in water by adding the theoretical amount of acid and then the solution obtained is freeze-dried.

 For medicinal use, the new product can be used either in the base state or as an addition salt with a pharmaceutically acceptable acid.

 As pharmaceutically acceptable addition salts may be mentioned salts of mineral acids (such as hydrochloride, sulfate, nitrate, phosphate) or organic (such as acetate, propionate, succinate, benzoate, fumarate maleate, tartrate, methanesulfonate v theophylineacétate salicylate, phenolphthalinate, methylene bis-s-oxynaphthoate) or substitution derivatives of these acids.

 The following examples, given in a non-limiting manner, show how the invention can be put into practice.

EXAMPLE 1
To a solution maintained at 0 ° C. of 210 g of N-dodecyl L-piperidine-2-carboxylic acid and of 116 g of 1-hydroxybenzotriazole in 2.8 liters of anhydrous tetrahydrofuran, a solution of 162 g of dicyclohexylcarbodiimide is rapidly added. in 700 cm3 of anhydrous tetrahydrofuran. The mixture is stirred for 1 hour at 0 ° C. and then for 1 hour at 20.degree. C., the cyclopeptide A solution (described below) is added thereto and the mixture is stirred for 48 hours at 20.degree. The insoluble matter is separated by filtration and then washed 3 times with a total of 1050 cm 3 of tetrahydrofuran. The combined filtrates are concentrated to a volume of 2 liters under reduced pressure (20 mm of mercury) at 45 ° C., taken up in 4 liters. of ethyl acetate, concentrated again to a volume of 2 liters under the same conditions, taken up in 2 liters of ethyl acetate and filtered to remove an insoluble material The filtrate is washed 3 times with 4.2 liters in total of a 5% solution of sodium bicarbonate and 1 time per 700 cm3 of water, dried over sodium sulfate and concentrated to dryness under reduced pressure (20 mm Hg) at 45 ° C. 1350 g of a brown-brown thick oil are thus obtained. 965 g of this oil are dissolved in 3 liters of ethyl acetate and then 1.64 liters of cyclohexane are added. The solution thus obtained is poured into 10 kg of neutral silica gel (0.05-0.2 mm) contained in a column of 15 cm in diameter. 33 liters of an ethyl acetate-cyclohexane mixture (1-1 in volumes) are eluted successively with 38 liters of ethyl acetate and 21 liters of a mixture of ethyl acetate-methanol (9-1 by volume). and 12 liters of an ethyl acetate-methanol mixture (3-1 by volume), collecting 3-liter fractions. Fractions 19 to 26 are combined and concentrated to dryness under reduced pressure (20 mm Hg) at 400 ° C. and dried under reduced pressure (5 mm Hg) at 350 ° C.

262 g of cyclo [L-leucyl-4-transmethyl-prolyl-L-leucyl-L-N-methylvalyl-L-prolyl-N-methyl-1-glycyl-N- (N-dodecyl) are thus obtained.
L -Piperidyl-2-carbonyl-4-transmethyl-prolyl) -N-methylthreonyl].

Calc Analysis. % C 65.02 H 9.45 N 11.32
Tr. 64.9 9.5 11.2
Rf = 0.75 (silica gel, 1,2-dichloroethane-methanol,
65-35 in volumes).

N-dodecyl L piperidine-2 carboxylic acid can be prepared as follows
314 g of L-piperidine-2-carboxylic acid are dissolved in 1.23 liters of water by heating at 5 ° C. and a solution of 560 g of lauryl aldehyde in 6.1 liters of ethanol is added, followed by 310 g of palladium on black (3% palladium). The mixture is heated to 50.degree.-55.degree. C., the apparatus is purged with nitrogen and then fed with hydrogen at atmospheric pressure.After 4 hours of stirring, the volume of hydrogen absorbed ( 60 liters) no longer varies, the apparatus is purged with nitrogen and the reaction medium is filtered in the presence of a filtering agent The insoluble material is washed successively with 3 times 0.9 ml of ethanol and 2 times with 500 cm3 total of water The combined filtrates are concentrated to dryness under reduced pressure (30 mm of mercury) at 450 ° C. The pulp thus obtained is dissolved in 3 liters of water and extracted successively with 2 liters and 3 liters of water. per 3 liters in total of methylene chloride.

and
The organic extracts are combined / concentrated to dryness under reduced pressure (30 mm of mercury) at 40 ° C. The solid obtained is recrystallized after dissolution in 500 cm 3 of ethanol, by addition of 2.5 liters of ethyl acetate. The white suspension is filtered, washed 3 times with ethyl acetate (900 cc in total) and dried under reduced pressure (30 mm Hg) at 20 ° C. 570 g of diacid are thus obtained
N-dodecyl L-2-piperidine carboxylic acid melting at 184 ° C.

Calc Analysis. % C 72.68 H 11.86 N 4.71
Tr. 72.5 11.2 4.7
L-piperidine-2-carboxylic acid can be prepared according to the method of H. BEYERMAN Rec. Trav. Chim. Netherlands 78, 134 (1959).

The cyclopeptide A solution is prepared as follows
924 g of antibiotic 11072 RP, prepared according to the process described in Japanese Patent 446,686, are dissolved in 12.425 liters of 3N hydrochloric methanol. It is stirred for 2 hours at 200 ° C. and then concentrated to dryness under reduced pressure (20 mm of mercury) at 50 ° C. The residue obtained is taken up twice with 350 cm 3 of ethyl acetate and each time concentrated to dryness under pressure. The meringue finally obtained is dissolved in 1.75 liters of anhydrous tetrahydrofuran.The solution obtained is brought to pH 5 by addition of 750 cm 3 of triethylamine.The precipitate formed is separated by filtration. and washed 3 times with 1.05 liter total of tetrahydrofuran.The combined filtrates are brought to pH 7 by addition of 4 cm3 of N-ethyl morpholine This solution is used as it is.

EXAMPLE 2
Cyctilus leucyl-t-transmethyl-4-propyl-L-leucyl
N-methylvalyl-L-prolyl-N-methyleucyl-glycyl-N- (N-dodecyl L -piperidyl-2-carbonyl-4-transmethyl-prolyl) -N-methylthreonyl] can be prepared under the same conditions as in the example 1 from pure cyclopeptide A in dimethylformamide
From 1.19 g of N-dodecyl L-piperidine-2-carboxylic acid and 4.22 g of cyclopeptide A with 648 mg of 1-hydroxy benzotriazole, 908 mg of dicyclohexylcarbodiimide and 80 cm 3 of dimethylformamide, 2 is obtained, 28 g of product of formula (I).

 Cyclopeptide A may be prepared according to the process described in Japanese Patent 638,380.

 Drugs which contain the product of formula (I) in the form of base or of addition salt, in the pure state or in the form of medicinal compositions (in the presence of a diluent or a coating), constitute another object of 1 invention. These drugs can be used orally, parenterally or rectally.

 As solid compositions for oral administration, tablets, pills, powders (especially in gelatin capsules) or granules may be used. In these compositions, the active product is mixed with one or more inert diluents such as sucrose, lactose or starch. These compositions may also include substances other than diluents, for example a lubricant, such as magnesium stearate.

 As liquid compositions for oral administration, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents such as water or paraffin oil may be used. These compositions may also comprise substances other than diluents, for example wetting, sweetening or flavoring products.

 The compositions according to the invention for parenteral administration may be aqueous or non-aqueous sterile solutions of suspensions or emulsions. As the solvent or vehicles propylene glycol, a polyethylene glycol, vegetable oils, in particular olive oil and injectable organic esters, for example ethyl oleate, may be used. These compositions may also contain adjuvants, in particular wetting agents, emulsifiers and dispersants. The sterilization can be done in several ways, for example by means of a bacteriological filter, by incorporating sterilizing agents into the composition, by irradiation or by heating. They can also be prepared in the form of sterile solid compositions which will be dissolved. at the time of use in sterile water or other sterile injectable medium.

 The compositions for rectal administration are suppositories which contain, in addition to the active product, excipients such as cocoa butter or suppo-wax.

 The doses depend on the desired therapeutic effect, the route of administration and the duration of treatment. In human therapy, it is possible to use daily doses of between 0.5 and 1 g of active product for an adult.

The following example illustrates a composition according to the invention
EXAMPLE
Tablets having the following composition - product of formula (I) are prepared according to the usual technique ............................... 0.500 g - wheat starch ........................................ 0.150 g - colloidal silica ..................................... 0.040 g - magnesium stearate ..... ............................ 0.010 g
In the treatment of tuberculosis the drug is preferably administered orally.

Claims (3)

 1 - CycloLL leucyl-L-4-transmethyl-prolyl-L-leucyl N-methylvalyl-L-N-methyl-prolyl-N-methyl-glycyl-N- (N-dodecyl-L -piperidyl-2-carbonyl-L -transyl-4-prolyl) -L-N-methylthreonyl Formula

 and its addition salts with acids.  2 - Process for the preparation of the product according to claim 1 characterized in that N-dodecylpiperidine-2-carboxylic acid or an activated derivative of this acid is reacted with the cyclopeptide of formula

 and isolates the product obtained, optionally in the form of an addition salt with an acid.  3 - Drug characterized in that it contains the product of claim 1 optionally in salt form, pure or in combination with one or more compatible and pharmaceutically acceptable diluents or adjuvants.