EP0054025A1

EP0054025A1 - New derivatives of l'alpha, alpha'-trehalose and drugs containing them

Info

Publication number: EP0054025A1
Application number: EP19810900515
Authority: EP
Inventors: Pierre Lefrancier; Michel Level; Françoise Audibert; Monique Parant; Jean Choay; Louis Chedid; Edgar Lederer
Original assignee: Agence National de Valorisation de la Recherche ANVAR
Current assignee: Bpifrance Financement SA
Priority date: 1980-03-10
Filing date: 1981-03-09
Publication date: 1982-06-23
Also published as: WO1981002576A1; FR2477551B1; FR2477551A1

Abstract

Composes ayant des proprietes immunostimulantes constitues par des derives de l'(Alpha)-D-glucopyranosyl-(Alpha)-D-glucopyranoside ((Alpha), (Alpha)'-trehalose), substitue asymetriquement sur ses deux fonctions alcools primaires, en position C-6 de chacun des monosaccharides constituants, selon la formule generaleCompounds having immunostimulatory properties constituted by derivatives of (Alpha) -D-glucopyranosyl- (Alpha) -D-glucopyranoside ((Alpha), (Alpha) '- trehalose), substituted asymmetrically on its two primary alcohol functions, position C-6 of each of the constituent monosaccharides, according to the general formula

Description

NOVEL DERIVATIVES OF O, α'-TREHALOSE AND MEDICINAL PRODUCTS

CONTAINING

The invention relates to new compounds endowed with biological and pharmacological properties of great value, and in particular among these products to those which have properties regulating the immune mechanisms.

The invention also relates to the applications of these new products, as well as the compositions specially suitable for their use.

The compounds according to the invention are derivatives of α-D-glucopyranosyl-α-D-glucopyranoside (α, α'-trehalose), substituted asymmetrically on its two primary alcohol functions, in position C-6 of each of the monosaceharides constituents, according to the general formula

in which :

- R ₁ is a lipophilic group comprising at least 30 carbon atoms, preferably from 60 to 90 atoms, in particular a mycolic acid comprising from 80 to 90 carbon atoms;

- R ₂ is either OH, or an OR ester in which R is a radical comprising from 1 to 10 carbons, or NH ₂ , the hydrogens of the amino group possibly being substituted by alkyl residues of 1 to 4 carbons, an aminoacyl residue substituted or not;

- X ₁ . is either a hydrogen or a muramyl or nor-muramyl or homo-muramyl residue which may or may not be substituted on the amino function at C-2 by acetyl or glycolyl groups, on the hydroxyl function at C-4 by an acyl group comprising at most -4 carbons and on the C-6 hydroxyl function by an acyl group comprising up to 90 carbons;

- X ₂ is an aminoacyl residue from the group comprising: L-alanyl, L-arginyl, L-asparaginyl, L-aspartyle, L-cysteinyle, L-glutaminyle, L-glutamyle, glycyle, L-histidy L-hydroxyprolyle, L- isoleucyle, L-leucyle, L-lysyle, L-méthionyle, L-ornithyle, L-phenylalanyl, L-prolyl, L-seryle, L-threonyle, L-tryptophanyle, L-tyrosyle et L-valyle, atom d nitrogen of the characteristic chain element -NH -C0- of said aminoacyl residue being the case optionally N- substituted by a hydrocarbon group, in particular alkyl, comprising from 1 to 4 carbon atoms;

- X ₃ may either not exist, or be an aminoacyl residue of the group indicated above or else a hydrocarbon residue comprising up to 10 carbons _. in the formula of which there are two functions, such as for example alcohol, amino or carboxyl, able to be coupled, on the one hand with the γ-carboxylic function of the D-glutamyl residue and, on the other hand, with the alcohol function in C-6 of the glucopyranosyl residue.

The group X ₁ can more particularly be represented by the formula:

in which :

-R ₃ . is a hydrogen atom or a CH ₂ OH or CH _{3 group} ;

- R ₄ e: - '. T of hydrogen or an acyl group comprising at most 4 carbon atoms;

- R ₆ is hydrogen or an acyl group comprising up to 90 carbon atoms;

- R ₇ is a hydrogen atom or a methyl or ethyl radical.

The preferred compounds according to the invention are those in which X ₂ is an L-alanyl, -N-methyl-L-alanyl or L-seryl residue.

In these compounds, the group R ₃ , is more particularly a methyl group.

In the compounds according to the invention, the group R ₇ is preferably a hydrogen atom or a methyl group. More preferably, and in the different classes of compounds envisaged above, R ₂ . is an amino or ester group, in particular an n-butyl ester.

In general, the invention also also relates, as preferred compounds, to those in which the groups R ₄ and R ₆ are both hydrogen.

X ₃ is advantageously an L-alanyl group. The products according to the invention are advantageously produced by reaction between a compound of formula

in which Ts is a tosyl group capable of reacting with the alkali salt, for example potassium salt of a carboxylic function, and a compound of formula

in which

-X ₁ , X ₂ and X ₃ have the meanings indicated above the other functions of the product of formula IV, in particular the amino functions, being protected in a manner known per se. The trehalose derivatives of formula III can be prepared according to the method described in POLONSKY et al.

Carbohydr. Res. 65 (1978), 295-300. The derivatives of formula IV can also, for their part, be produced by any known general method, for example that described in published European patent application No. 79400183.4 filed on March 20, 1979. Additional characteristics of the invention will become apparent during the description of examples of the manufacture of preferred compounds according to the invention, their biological properties and the conditions under which they can be used, so that manifest the different biolngic activities which characterize them. 6- (Boc-L-alanyl) 6 '- (Mycoloyl) -α, α-trehalose (I)

Λ 3 * 1 ^m S of the potassium salt of Boc-I.-alanine (150 ymoles) and at ^ 0 mg of an ether of the type known as "18-CROWN-6" dissolved in 2 ml of anhydrous benzene, 170 mg of 6, (tosyl) 6 '(mycoloyl) -α, α'-trehalθae (100 uπ.oles) are added in solution in 2 ml of benzene. The reaction mixture is brought to reflux under rigorously anhydrous conditions, then, after 6 hours, concentrated to dryness. The. syrup obtained is chromatographed on a column of silica gel eluted with a chloroform-methanol mixture (7/1): 132 mg. 6, - (L-alanyl) 6 '(mycoloyl) -α, α-trehalose, hydrochloride (II) 132 mg of (I) are treated for 45 minutes for 1 Til of HCl N in glacial acetic acid. The reaction mixture is concentrated to dryness. 109 mg. 6, -Jjoc-L-alanyl-D-isoqlutaminyl-L-alanyl) 6 '(mycoloyl) - α, α-trehalose (III) (or DP-L-Ala-trehalo_se mycolate)

Α 25 mg of Boc-L-Αla-D-isoGln _ (PLEFRAJCIER and E. BRICAS, Bull. Soc. Chim. Biol. (1967, 49, 135) dissolved in 2 ml of anhydrous THF are added 10 μl of cl. lorofor ethyl miate and 6 μl of N-ethyimorpnoline, at -15 ° C. After 5 minutes, are mixed with the reaction mixture 109 mg of (II) dissolved in 1 ml of anhydrous THF containing 5 μl of N-methylmorpholine . After k hours at room temperature, the reaction mixture is expanded with 50 ml of a mélance élhyle acetate-ether (1-1) and was washed successively with NaHCO ₃ M, water, 10% citric acid, water. the organic phase is dried over Na ₂ So ₄ and concentrated in a syrup (129 mg), which is purified by chromatography on a column of silica gel washed with chloroform-methanol (4-1), then by preparative chromatography on a thin layer of silica gel, developed in the cliloroform-ethanol mixture (1-1): 32.5 mg (17%). 6, - (L-alanyl-D-isoglutaminyl-L-alanyl) 6 '(mycoloyl) α, α- trehalose, hydrochloride (IV)

32.5 mg of (III) are treated for 45 minutes with 1 ml of HCl N in glacial acetic acid. The reaction mixture is concentrated to dryness: 29 mg. Rf.

(chloroform-methanol 4-1). Elemental analysis: caloulé pour c ₁₀₇ H ₂₀₃ 0 ₁₇ N ₄ Cl, 2, 5CH COOH, 1CHC1: C% 64, 0,1-H% 10,1-N% 2,6 - Found: C% 64,11- H% 10.0-N% 2.4. 6, - (N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl) -6 '(mycoloyl) α, α-trehalose (V) (or MDP-L-Ala-trehalose-mycolate A 66, 5 mg of N-acetyl-muramyl-L-alanyl-D-isoglutamine (3) dissolved in 5 ml of a THF-DMF mixture (1-1) containing 15 μl of N-methylmorpholine, 18 μl of chloroformate are added isobutyl, at -15 ° C. After 5 minutes, 148 mg of (II) dissolved in 5 ml of THF, containing 10 μl of N-methylmorpholine, are added. After 2 hours, 200 μl of KHC0 ₃ 2 is added, 5 M, then 60 ml of water, at 0 ° C. The emulsion is extracted with an ether-ethyl acetate mixture (1-1), then with chloroform. The two organic phases are combined and concentrated to dryness 140 mg The product is obtained by chromatography on a column of silica gel eluted with a chloroform-methanol mixture (7-1): 82.3 mg. (0.8-CHCl.,) Elementary analysis: calculated for ₁₂₁ H 3CH ₃ C00H: C% 65.79 - H% 10.47-N% 3.02 - Found: C% 65, 32-H% 10, 68-N% 3.25. Biological properties

The compounds according to the invention have non-specific stimulating properties, in particular adjuvants of immunity and anti-infectives. They also have anti-tumor properties with regard to certain types of tumors, in particular those which are capable of being treated by intra-lesional injections.

The adjuvant properties are found, more particularly in the compounds of the invention in which the group X .. is a derivative of the muramic type. They can be demonstrated in particular by the administration of these substances within a water-in-oil emulsion in the guinea pig vis-à-vis ovalbumin. The results are shown in Table I by comparison with the adjuvant properties of N-acetyl-muramyl-L-alanyl- D-isoglutamine (MDP). The technique used is that described in the published European patent application mentioned above.

Demonstration of anti-infectious properties is also done by using the technique described in the above-mentioned European patent application. The results obtained with the compounds used by way of example appear in Lableau II below.

* Percentage of survivor in the batch of animals treated minus that of the controls.

The antitumor properties can be demonstrated by using the technique described by ELIYAHU YARKONI and collaborators; "International Journal of Cancer": 22,564-569 (1978), by injection of an emulsion of compounds according to the invention in grafts of syngeneic murine fibro sarcoma in guinea pigs or mice. To form the emulsion, any pharmaceutically acceptable oil can be used, in particular that known under the name of squalene or squalane. The anti-tumor effect is manifested by a significant regression of the tumors.

The invention relates to bio.1oqic.ues reagents which can be constituted using the compounds according to the invention, in particular with a view to studying any standard adjuvant properties or, on the contrary, as eg ceptible of s' oppose certain effects linked to the administration of immunosuppressive substances.

More particularly, the invention relates to medicaments? reifering as active principle at least one of the compounds according to the invention, this medicament being applicable as a regulator of the immune response of the subject to which it is administered.

These drugs are especially applicable for the treatment of infectious diseases of bacterial or parasitic origin, or the inhibition of tumoral diseases. In particular, the medicaments containing the compounds according to the invention can be used for the treatment of infants caused by agents resistant to antibiotics.

The application of these drugs is not only curative, it can also be done as a preventive measure. The medicaments according to the invention can be administered to a host - animal or human being in any way suitable for obtaining the desired effect.

The invention naturally also relates to the various pharmaceutical compositions into which the compounds according to the invention can be incorporated, where appropriate in combination with other active substances.

Advantageous pharmaceutical compositions consist of injectable solutions or suspensions tables containing an effective dose of at least one product according to the invention. Preferably, these solutions or suspensions are produced in an isotonic sterilized aqueous phase, preferably saline or glucose. The invention relates more particularly to such suspensions or solutions which are capable of being administered by intradermal, intramuscular or subcutaneous injections, or even by scarification.

Other advantageous pharmaceutical compositions consist of the liposomal forms of the compounds according to the invention. As is known, the liposomes, due to the lipid (and in particular phospholipid) nature of the elements entering into their composition, constitute, for certain cases, a particularly suitable presentation.

The invention also relates to pharmaceutical compositions which can be administered by other routes, in particular by oral or rectal route, or also in forms intended to come into contact with mucous membranes, in particular the ocular, nasal, pulmonary or vaginal mucous membranes. Consequently, it relates to pharmaceutical compositions in which at least one of the compounds according to the invention is found associated with pharmaceutically acceptable excipients, solid or liquid, suitable for constituting forms of oral, ocular or nasal administration or with excipients suitable for constituting forms of rectal administration, or alternatively with excipients suitable for vaginal administration, for example gelatinous. Finally, it relates to compositions intended for the pulmonary route, in particular solutions prepared for administration by means of a conventional aerosol device.

The invention also consists of a method for strengthening the immune defenses of the host, comprising administering to it an effective dose of at least one of the products according to the invention, in one of the administration forms which has was mentioned above. By way of example of doses capable of inducing an action, doses of 10 to 1000 μg per kg will be mentioned. of body for example 50 μg when the administration is carried out parenterally, or else a dose of 200 to 20,000 ug per kg. body, for example 1000 μq for other modes of administration, such as for example the oral route.

The invention also relates to the compositions containing at least one of the compounds according to the invention and being in the form of water-in-oil emulsions. By way of example, these emulsions contain from 1 to 10% by volume of oil, in particular squalene or squalane.

These compositions can be used for performing intra-lesional injections in tumors of the mammary tumor, melanoma and other solid tumors type.

The invention finally relates to compositions of the above-mentioned type containing, in addition to the compounds according to the invention, in particular at the unit doses which have been indicated above, active principles of vaccine: the use of these compositions then being recommended when we want to take advantage of the adjuvant effect, immunologically non-specific of the compounds according to the invention in order to enhance the effect of the above vaccinating principles. The invention therefore also relates to vaccine compositions containing at least one of the above compounds. As an example of a vaccine antigen forming the active principle of such compositions, mention may be made of that of influenza. The invention is obviously not limited to the embodiments described above by way of example and the man of the art r> had to make modifications without departing from the scope of. claims below.

Claims

CLAIMS 1 - Compounds derived from 1 'α -D-glucopyranosyl- α-D- glucopyranoside (α, α' -trehalose), asymmetrically substituted on its two primary alcohol functions, in position C-6 of each of the constituent monosaccharides, according to general formula

in which :

- X ₁ is either a hydrogen, or a muramyl or nor-muramyl or homo-muramyl residue which may or may not be substituted on the amino function at C-2 by acetyl or glycclyl groups, on the hydroxyl function at C-4 with a acyl group comprising at most 4 carbons and on the C-6 hydroxyl function by an acyl group comprising up to 90 carbons;

- X ₂ is an aminoacyl residue of the group compre- nant: L-alanyl, L-arginyl, L-asparaginyl, L-aspartyle, L-cysteinyle, L-glutaminyle, L-glu ± amyl, glycyle, L-histidyle, L-hydroxyprolyle, L-isoleucyle, L-leucyle, L-lysyl, L-methioethyl, L-ornithyle, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tryptophanyl, L-tyrosyl and L-valyle, the nitrogen atom of the element of characteristic chain -NH-CH-CO- of said aminoacyl residue being optionally N- substituted by a hydrocarbon group, in particular alkyl, comprising from 1 to 4 carbon atoms; X-, may either not exist; either be an aminoacyl residue of the group indicated above or else a hydrocarbon residue comprising up to 10 carbons in the formula of which there are two functions, such as for example alcohol, amino or carboxyl, capable of being coupled, on the one hand with the Y-carboxylic function of the D-glutamyl residue and, on the other hand, with the C-6 alcohol function of the glucopyranosyl residue-

2 - Compound according to claim 1 characterized in ccee qquuee ddaannss llaa formula (I), the radical X., is characterized by the formula

in which :

- R ₃ is a hydrogen atom or a CH ₂ OH OR CH _{3 group} ;

- R ₄ is hydrogen or an acyl group comprising at most 4 carbon atoms; - R ₆ is hydrogen or an acyl group comprising up to 90 carbon atoms;

- R ₇ is a hydrogen atom or a methyl or ethyl radical 3 - Compound according to claim 2 characterized in what in the group represented by the formula (I) x ₂ . is an L-alanyl, N-metbyl -L-alanyl or L-seryl residue.

4 - Compound according to claim 2 or claim 3, characterized in that in the group represented by the formula (II) R ₃ is a methyl group.

5 - Compound according to any one of claims 2 to 4 characterized in that in the group represented by formula (II) R ₇ is a hydrogen atom or a methyl.

6 - Compound according to any one of Claims 2 to 5, characterized in that in the group represented by formula (I) R ₂ is an amino or ester group, in particular butyl.

7 - Composition according to any one of claims 2 to 6 characterized in that in the group represented by the formula (II) R ₄ and R ₆ are both 1 hydrogen.

8 - Le 6 - (Boc-L-alanyl-D-isoglutaminyl-L-alanyl) 6 '(mycoloyl) - α, α-trehalose.

9 - Pharmaceutical composition having in particular immunostimulatory and antitumor properties, characterized in that it contains an effective dose of at least one compound according to any one of claims 1 to 8 in association with a pharmaceutically acceptable vehicle or excipient. 10 - Pharmaceutical composition according to claim 9 consisting of a solution or suspension of said compound in an injectable medium, in particular in the form of an aqueous solution or a water-in-oil emulsion. 11 - Composition according to claim 9 or claim 10, characterized in that it also contains a vaccinating principle.