FR2839310A1 - NOVEL PROCESS FOR THE PREPARATION OF ALPHA-GLYCOSYLCERAMIDES, NOVEL ALPHA-GLYCOSYLCERAMIDE DERIVATIVES AND THEIR APPLICATIONS - Google Patents

Abstract

The invention relates to new α-glycosylceramide derivatives corresponding to the general formula (S-I), a process for their preparation as well as the pharmaceutical compositions comprising them and their uses.

Description

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 The invention relates to new a-glycosylceramide compounds and in particular to new a-galactosylceramide compounds, to a new process for the preparation of a-glycosylceramide derivatives, to pharmaceutical compositions and to biological reagents comprising them, as well as the use of these derivatives as regulators of the activity of NKT cells ("Natural Killer T cells").

 NKT cells have been shown to be important effectors in the mechanisms of metastasis control of liver and lung tumors in mice (Hashimoto, W. et al., J Immunol., 154, 4333 (1995)); Anzai R. et al., Immunol., 88.82 (1996)). These data suggest that NKT cells are likely to play an important role in the eradication of cancers. In addition to this activity, it is also believed that they are likely to play a role in the eradication of parasites, protozoa and intracellular bacterial infections such as Listeria monocytogenes and Mycobacterium tuberculosis (Seki S. et al., Clin. Immunol., 28.1069 (1996)).

 It is also known that NKT cells are closely associated with rejection in bone marrow transplants (Yankelevich, B. et al., J Immunol., 142, 3423 (1989)) and with the control of the production of IgE antibodies by the control the differentiation of T cells into Thl and Th2 (Yoshimoto, T. et al., J.

Exp. Med., 179, 1285 (1994)).

 NKT Va 14+ cells are a subcategory of NKT cells. They have been shown to be closely associated with the development of autoimmune diseases in mice (Mieza, MA et al.; J. Immunol., 156, 4035 (1996); Makino, Y. et al., Clin Immunol., 28.1487 (1996)). In humans, Va24JaQa cells, homologs of mouse NKTVal4 + cells, have been shown to be involved in various autoimmune diseases such as multiple sclerosis, lupus erythematosus (Sumida, T. et al., J Exp. Med., 182, 1163 (1995) or autoimmune diabetes (SHARIF S. et al., Nature Medecine, 2001,7, 1057-1062). These data confirm the pivotal role played by these cells in the immune system homeostasis.

 Compounds extracted from sponges (family of agélasphines) were isolated in 1993 by a Japanese team then tested. Some of these derivatives have been

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selected for their antitumor and immunoregulatory activities (T. Natori, Y.
Koezuka; T. Higa, Tetrahedron Lett., 1993, 34, 5591; EP-0 609 437). Analogs were then synthesized and tested by these authors (M. Morita, K. Motoki, K.

 Akimoto, T. Natori, T. Sakai, E. Sawa, K. Yamaji, Y. Koezuka. E. Kobayashi, H.

 Kukushima, J Med. Chem., 1995, 38, 2176; H. IIjima, K. Kimura, T. Sakai, A.

Ichimura, T. Shimizu, H. Ueno, T. Natori, Y. Koezuka, Bioorg. Foot. Chem., 1998,6,
1905; US-5,849,716; US-5,780,441; EP-0 666 268; US-5,936,076; EP-0 694 558).

Anti-tumor and immunostimulatory properties have been described for these compounds.

Some have been tested for their radiation-protective properties, and for the treatment of diseases due to the destruction or weakening of bone marrow cells. And for the treatment of thrombocytopenia (EP-650-0 650 732; US -6,017,892; US-6,071,884).

 These molecules of the α-galactosylceramide type interact with a major histocompatibility complex of the CDld type allowing the recruitment and proliferation of NKT lymphocytes (Z.H. Zeng, A.R. Castano, B.W. Segelke, E.A.

Stura, P.A. Peterson, LA. Wilson, Science, 1997, 277, 339). They are remarkable for their quality of potential biological tools making it possible to decipher and control the immune responses.

 A-galactosylceramide derivatives have been synthesized and described, in particular in, EP-0 988 860, and it has been shown that they stimulate the cytotoxic anti-cancer activity of NKT cells, that they prevent the appearance of autoimmune encephalomyelitis in mice and diabetes in mice Nod.

Certain derivatives of the α-galactosylceramide type have a therapeutic effect on viral diseases, in particular AIDS, hepatitis B, cytomegalovirus infections (see in particular patent application EP-A-0 957 161).

 However, the compounds described in the prior art have been obtained by long and difficult synthetic routes. Obtaining optically active molecules comprising several asymmetric carbons and several reactive functionalities on the same molecule makes the synthesis of these compounds complicated and difficult to rationalize.

 The authors of the present invention have set themselves the objective of designing and perfecting a new pathway for the synthesis of derivatives.

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 a-glycosylceramide, in particular a-galactosylceramide derivatives, this synthetic route making it possible, using the same intermediate, to lead to molecules with various functionalities, in particular molecules capable of serving as markers in activity tests organic. This intermediate has the basic structure characteristic of α-glycosylceramides, in particular α-galactosylceramides and it is furthermore endowed with an amine residue onto which various functional fragments can be grafted. The proposed synthetic route therefore makes it possible to prepare, by a single process, an amino α-glycosylceramide intermediate, in particular amino agalactosylceramide, and then to decline it into multiple molecules at the end of one or two stages of additional synthesis. . This synthetic route therefore has the advantage of being extremely rational, and therefore more easily extrapolable on a large, economical scale. In addition, it provides access to new molecules, which can be used for their immunoregulatory, antiinfectious, anti-tumor and anti-diabetic properties. This synthetic route makes it possible to use any sugar in the glycosidic part of the molecule, such as for example agalactose, a-glucose, a-mannose and their derivatives. In addition, this synthetic route makes it possible to produce mono- or di-a-glycosylceramides, the sugars used for a di-glycosylceramide being identical or different.

(S-I) dans laquelle : The present invention relates to new α-glycosylceramide derivatives corresponding to the general formula (SI):

(SI) in which:

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- Su represents a glycoside residue which can, for example, be chosen from glucose, mannose, galactose and certain glycoside derivatives; - X represents a C1-C30 alkane di-yl radical, linear or branched, or an aryl group; x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents a hydrogen atom; a linear or branched C1-C20 alkyl group, or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or a glycoside residue which can, for example, be chosen from glucose, mannose, galactose and their derivatives; R may be the same or different from the radical Su; - B represents a group chosen from: a hydrogen atom, and then x = y = 0;
B can also represent a single bond, a linear, branched or cyclic C1, C30 alkyl, alkenyl or alkynyl radical, optionally substituted by one or more groups — OH, -NH2, halogen, -COOH, -CONH2, by a group optionally substituted aryl or a heteroaryl group, by a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; may be an optionally substituted aryl group or a heteroaryl group, a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; can also represent a peptide or pseudopeptide chain comprising for example from 2 to 20 amino acids, preferably from 2 to 10 amino acids; - K represents a pharmacophore group, such as a fluorescent group, a photoactivable group, a radiolabelled group or any other group allowing the detection and the quantitative evaluation of the product corresponding to formula (I) in a biological sample without degradation of the biological sample.

 Among the glycoside derivatives represented by the symbol Su, there is denoted in particular glycosides, such as for example galactose, glucose, mannose, the hydroxyl functions of which are protected, in particular by groups protecting the sugars traditionally used such as residues

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 acetyl, benzyl etc ... The di-glycoside and polyglycoside compounds are also designated, protected or not on their hydroxyl functions. Preferably, the glycosides and glycoside derivatives used in the present invention are molecules of configuration a.

(I) dans laquelle : - Y1, Y2, Y3 et Y4 représentent un atome d'hydrogène, un groupement benzyle ou un monosaccharide qui peut par exemple être choisi parmi le glucose, le mannose, le galactose, le fucose, la glucosamine et la galactosamine ; - X représente un radical alcane di-yle en C1-C30, linéaire ou ramifié, ou un groupement aryle ; - x est un entier égal à 0 ou à 1 ; - y est un entier égal à 0 ou à 1 ; - A représente un atome d'hydrogène ; un groupement alkyle en C1-C20, linéaire ou ramifié, ou un groupement aryle ; - Z représente -CH2-CH2- ou -CH=CH- ; - R représente un atome d'hydrogène ou un radical a-galactosyle répondant à la formule (II) ci-dessous : In a preferred implementation, the present invention relates to new α-galactosylceramide derivatives corresponding to the general formula (I):

(I) in which: - Y1, Y2, Y3 and Y4 represent a hydrogen atom, a benzyl group or a monosaccharide which can for example be chosen from glucose, mannose, galactose, fucose, glucosamine and galactosamine; - X represents a C1-C30 alkane di-yl radical, linear or branched, or an aryl group; - x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents a hydrogen atom; a linear or branched C1-C20 alkyl group, or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical corresponding to formula (II) below:

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dans laquelle Y1, Y2, Y3, Y4 ont les mêmes définitions que celles indiquées ci- dessus dans la formule (I) ; - B représente un groupement choisi parmi : un atome d'hydrogène, et alors x=y=0 ;
B peut également représenter une simple liaison, un radical alkyle, alcényle ou alcynyle en C1-C30, linéaire, ramifié ou cyclique, éventuellement substitué par un ou plusieurs groupements-OH, -NHz, halogène, -COOH, -CONH2, par un groupement aryle éventuellement substitué ou un groupement hétéroaryle, par un hétérocycle comprenant de 5 à 10 chaînons et un ou plusieurs hétéro atomes choisis parmi 0, N, S ; peut être un groupement aryle éventuellement substitué ou un groupement hétéroaryle, un hétérocycle comprenant de 5 à 10 chaînons et un ou plusieurs hétéro atomes choisis parmi 0, N, S ; peut aussi représenter une chaîne peptidique ou pseudopeptidique comprenant, par exemple, de 2 à 20 acides aminés, de préférence de 2 à 10 acides aminés ;
K représente un groupement pharmacophore, tel qu'un groupement fluorescent, un groupement photoactivable, un groupement radiomarqué ou tout autre groupement permettant la détection et l'évaluation quantitative du produit répondant à la formule (I) dans un échantillon biologique sans dégradation de l'échantillon biologique.

in which Y1, Y2, Y3, Y4 have the same definitions as those indicated above in formula (I); - B represents a group chosen from: a hydrogen atom, and then x = y = 0;
B may also represent a single bond, a C1-C30 alkyl, alkenyl or alkynyl radical, linear, branched or cyclic, optionally substituted by one or more groups — OH, -NHz, halogen, -COOH, -CONH2, by a group optionally substituted aryl or a heteroaryl group, by a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; may be an optionally substituted aryl group or a heteroaryl group, a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; may also represent a peptide or pseudopeptide chain comprising, for example, from 2 to 20 amino acids, preferably from 2 to 10 amino acids;
K represents a pharmacophore group, such as a fluorescent group, a photoactivatable group, a radiolabelled group or any other group allowing the detection and the quantitative evaluation of the product corresponding to formula (I) in a biological sample without degradation of the biological sample.

 Among the pharmacophoric groups which can be used within the meaning of the present invention, there may be mentioned in particular: the fluorescent compound known under the trade name of BODIPY # (Boron Dipyrromethene difluoride or dipyrromethene boron difluoride, sold by the company Molecular Probes, Eugene, Oregon, USA ), nitrobenzoxadiazole (NBD, azirine functional groups, iodinated heterocyclic groups, radioactive groups as well as dansyl groups.

 By halogen is meant within the meaning of the present invention an atom preferably chosen from fluorine, chlorine, bromine, iodine.

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 By aryl is meant within the meaning of the present invention an unsaturated cyclic hydrogen carbon derivative, such as a phenyl radical for example, this derivative being optionally substituted by one or more alkyl, alkenyl, C 1 -C 6 alkynyl, C1 haloalkyl radicals -C6, halogen, -OH. -NH2, -COOH or-CONH2.

 Among the heteroaryl groups which can be used within the meaning of the present invention, mention may, for example, be made of pyridine, pyrimidine, imidazole, indole, thiazole, thiophene, oxazole, carboline and quinoline groups.

 Among the heterocycles comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S, which can be used in the present invention, mention may be made, for example, of the piperazine, morpholine, oxazolidine, thiazolidine and quinolizidine groups.

 According to a first variant of the invention, the molecule corresponding to formula (S-I) or to formula (I) is characterized in that: x = y = 0 and B = H.

The molecule thus obtained, and in which the other parameters have the same definition as above, can be represented by the formula (S-Ia) or respectively by the formula (la) below:

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(la)
Conformément à l'invention, les molécules répondant à la formule (S-I) ou plus particulièrement (I) dans laquelle x=l ou y=1, et BH sont obtenues par un procédé caractérisé en ce que l'on fait réagir le composé (S-Ia), respectivement (la), avec un composé répondant à la formule (Ib) ci-dessous :

(Ib) suivant le schéma S-E, respectivement E, ci-dessous :

(the)
According to the invention, the molecules corresponding to the formula (SI) or more particularly (I) in which x = l or y = 1, and BH are obtained by a process characterized in that the compound ( S-Ia), respectively (la), with a compound corresponding to formula (Ib) below:

(Ib) according to the diagram SE, respectively E, below:

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Diagram E
In the formula (Ib), x, y, B and K have the same definition as above and T is chosen to carry out the alkylation (x = 0) or the acylation (x = l) of the free amine function of ( S-Ia), respectively (la).

 Such reactions are well known to those skilled in the art. In the case where x = l, one can in particular refer to J. March, Advanced Organic Chemistry, Third Edition, 1985, 370-377. The acylation of the amine function of (la) can in a known manner be carried out by reaction with an activated acid in the form of acid chloride, amide, activated ester or anhydride. Examples of embodiments will be given later in the description. In the case where x = 0, reference may be made to J. March, cited above, pages 798-800 (reductive alkylation of amines).

 Preferably, in formula (SI), respectively (I), and in formulas (S-Ia) and (la) above one or more of the conditions below are met: - Y1, Y2, Y3 and Y4 represent the hydrogen atom - X represents a linear or branched C6-CI5 alkane di-yl radical or an aryl group; - x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents: a linear or branched C4-CI5 alkyl group or an aryl group; - Z represents -CH2 -CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical:

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B represents a group chosen from: the hydrogen atom, and then x = y = 0, or
B represents a linear or branched C 1 -C 2 alkyl or alkenyl group,
K represents a pharmacophore group, such as a fluorescent group, a photoactivatable group, a radiolabelled group chosen from: BODIPY, NBD, groups with azirine function, iodinated heterocyclic groups, radioactive groups and also dansyl groups.

In a particularly preferred manner in the formula (SI), respectively (I), or the formula (S-Ia), respectively (la) one or more of the conditions below are met: - Y1, Y2, Y3 and Y4 represent the hydrogen atom - X represents a C6-C15 alkane di-yl radical, linear or branched; - x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents: a linear or branched C4-CI5 alkyl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical:

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B represents a group chosen from: a hydrogen atom and then x = y = 0, or B represents a C1-CI2 alkyl group, linear or branched; when B H, K represents a BODIPY group.

 Even more preferably, within the meaning of the present invention, the products of formula (SI), respectively (I), or (S-Ia) or (la), are characterized in that one or more of the conditions below are met: - Y1, Y2, Y3 and Y4 represent the hydrogen atom, - X represents a linear C6-C15 alkane diyl radical; - x is equal to 1; - y is an integer equal to 0 or 1; - A represents a linear C10-C15 alkyl group; - Z represents -CH2-CH2-; - R represents a hydrogen atom; - B represents a group chosen from: a hydrogen atom, and then x = y = 0, a linear C1-C6 alkyl group; - when B # H, K represents a BODIPY group.

 In the case where K represents a BODIPY group, this is coupled to (S-Ia) or (la) by reacting an activated ester of BODIPY with (S-Ia) or (la), in particular the ester of N-hydroxy.

 The invention also relates to a process for the preparation of molecules corresponding to the formula (S-I), respectively (I).

 The process according to the invention is broken down into several distinct stages, one of these stages consisting in preparing a product according to formula (la), this process comprising at least one stage (coupling stage in the SD or D scheme) at during which a glycoside (S-III), respectively an [alpha] - galactoside (III), is reacted with a ceramide (IV) then a step of deprotection of the functional groups:

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Schéma S-D

SD scheme

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Scheme D
In formula (III): - Y'1, Y'2, Y'3 and Y'4 represent a protective group for the hydroxyls or a monosaccharide protected by suitable protective groups so that the hydroxyls of the galactoside (III) are not involved in the coupling reaction with ceramide (IV). In known manner, Y'1, Y'2, Y'3, Y'4 can be chosen from benzyl, acetyl groups or benzylated, acetylated monosaccharides ... The same strategy can be envisaged for the glycosyl residue designated Su in the formula (S-III).

 In formula (IV): - Y'5 represents a protective group for the amines so that the amine of the ceramide (IV) does not intervene in the coupling reaction with the glycoside (S-III), respectively the galactoside (III). In a known manner, Y′5 can be chosen from benzyloxycarbonyl, terbutyloxycarbonyl, etc. groups; - Z represents the group -CH = CH- or -CH2-CH2-;

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 - X and A have the same definition as in formula (S-I), respectively (I). When X or A contain a functional group capable of reacting during the coupling reaction of galactoside with ceramide, the latter is protected by an appropriate protective group.

 Preferably, the protecting groups for the galactoside, Y ',, Y'2, Y'3, Y'4, and the ceramide, Y'5, are chosen so that they can be removed simultaneously in a single step. When the molecule (IV) does not contain unsaturations, protective groups are preferably chosen which can be removed by hydrogenation in the presence of an appropriate catalyst, such as the benzyl and benzyloxycarbonyl groups. When the molecule (IV) contains one or more unsaturations which it is desired to keep in the final compound (SI) or (I), protective groups are chosen which can be removed without damaging the integrity of the molecule, such as as for example: the acetyl group.

 In formulas (S-V) and (V): - Z represents the group -CH = CH- or -CH2 = CH2-; - X, R and A have the same definition as in formulas (S-I) and (I) above; - Y'1, Y'2, Y'3, Y'4, represent, as in formula (III), a protective group for hydroxyls or a monosaccharide protected by appropriate protective groups; - Y'5 represents, as in formula (IV) above, a group protecting amines.

 When R = H, the galactoside (III) (or glycoside (S-III)) and ceramide (IV) are coupled with a mixture in equimolar amounts of the galactoside (III) (or glycoside (S-III) ) and ceramide (IV). When R represents an α-galactosyl radical, the reaction is carried out by introducing an at least double molar amount of galactoside (III) relative to the ceramide (IV).

 Advantageously, the coupling of the glycoside (S-III) or of the galactoside (III) with the ceramide (IV) is carried out under conditions described by Mukayama et al., Chem. Lett., 1981, 431-432, in the presence of AgClO4 and SnCl2. Preferably, the reaction is carried out in a mixture of tetrahydrofuran (THF) and

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 diethyl ether. It can be seen by NMR analysis that the configuration of the glycosidic cycle is in accordance with what was expected.

 In formulas (S-Ia) and (la): the groups Su, Y1, Y2, Y3, Y4, X, R, Z and A have the same definition as in formulas (S-I), respectively (I).

 The compounds corresponding to formula (III) are known in the prior art, their mode of preparation has been described, in particular by K. L. Nicolaou, R. E.

Dolle, P. P. Papahatjis, (J. Am. Chem. Soc., 1984, 106, 4189 and references cited).

In the case where Y'1, Y'2, Y'3 and Y'4 represent a benzyl group, the synthetic scheme is as follows (scheme C):

Scheme C
In this scheme, the abbreviations DAST and NBS mean DiethylAminoSulfurTrifluoride and N-BromoSuccinimide respectively (Encyclopedia of Reagents for Organic Synthesis, Ed: Paquette, 1995, JohnWiley & sons, Ltd, p.1787 and 768). In the case where it is desired to obtain the 6-fluorinated peracetylated ocgalactoside compound, the procedure is the same as above, eliminating the deacetylation and benzylation steps.

 In the case of products of formula (S-I), a person skilled in the art will know how to adapt the preparation of the fluorinated derivative to the glycosidic cycle concerned.

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 In the case where one or more of the substituents Y'1, Y'2, Y'3 and Y'4 represent a benzylated or acetylated monosaccharide group, the procedure is the same.

The products corresponding to formula (IV) were prepared by a process characterized in that an activated derivative (VI) is reacted with an analog or a derivative of D-erythro-sphingosine (VII) or with D -erythro-sphingosine itself according to scheme B below:

Scheme B
In the molecule (VI), the group X has the same meaning as in the formula (I). Y'5 has the same meaning as in formula (IV) and represents a protective group for the amines so that the amine of (VI) does not react in the reaction with the compound (VII) and that the amine ceramide (IV) does not intervene in the coupling reaction with galactoside (III). Y'5 can be chosen from benzyloxycarbonyl, terbutyloxycarbonyl groups, etc.

 The activated derivative (VI) can be an acid chloride, an ester or an amide. Preferably, it is an activated ester, such as for example a paranitrophenyl ester. W represents any activating group as described for example in Protective Groups in Organic Synthesis, T. W. Green, P.G.M., Wuts, 3rd Ed., 1999, JohnWiley & sons, Ltd. Preferably, W is chosen from nitrophenyl groups and activated succinimidyl esters. Compounds corresponding to formula (VI) can be easily prepared by methods well known to those skilled in the art, these are compounds comprising a protected amine function and an activated acid function, as well as possibly one or more other functions

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 which must not intervene in the coupling reaction with (VII). For the preparation of such molecules, reference may be made to the works relating to the chemistry of amino acids, in particular to the chapters relating to the protection, deprotection and activation of functional groups. The preparation of compounds corresponding to formula (VI) is illustrated later in the description.

 In the molecule (VII), A has the same definition as in the formula (I), Z represents the group -CH = CH-.

Selon l'invention : - A représente : un atome d'hydrogène ; un groupement alkyle en C1-C30, linéaire ou ramifié, ou un groupement aryle ;
X représente un radical alcane di-yle, en C1-C30, linéaire ou ramifié, ou un groupement aryle ; - Z représente -CH2-CH2- ou -CH=CH- ;
Y'5 est choisi parmi : l'atome d'hydrogène, un groupement benzyloxycarbonyle, un groupement ter butyloxycarbonyle. The compound corresponding to formula (IV) below, in which Z and A have the same definition as in formula (I) and Y'5 represents a group chosen from the hydrogen atom and the protective groups for amines , constitutes another object of the invention:

According to the invention: - A represents: a hydrogen atom; a linear or branched C1-C30 alkyl group, or an aryl group;
X represents a linear or branched C1-C30 di-yl alkane radical, or an aryl group; - Z represents -CH2-CH2- or -CH = CH-;
Y'5 is chosen from: the hydrogen atom, a benzyloxycarbonyl group, a ter butyloxycarbonyl group.

 Preferably: - A represents: a C4-C15 alkyl group, linear or branched, or an aryl group; - X represents a linear or branched C6-C15 di-yl alkane radical; - Z represents -CH2-CH2- or -CH = CH-; - Y'5 is chosen from: the hydrogen atom, a benzyloxycarbonyl group.

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Even more preferably:
A represents: a linear or branched C4-C15 alkyl group; - X represents a linear or branched C6-C15 di-yl alkane radical; - Z represents -CH2-CH2- or -CH = CH-;
Y'5 is chosen from: the hydrogen atom, a benzyloxycarbonyl group.

Advantageously: - A represents: a linear C10-C15 alkyl group;
X represents a linear C 6 -C 15 alkane di-yl radical; - Z represents -CH = CH-;
Y'5 represents a benzyloxycarbonyl group.

The compound (Vlla) (compound (VII) with Z = -CH = CH-) is obtained by a synthetic route which is described in scheme A below:

Diagram A
In this scheme A, R 'represents an ethyl radical and R "represents an ethyl or isopropyl radical.

 The iminoester (VIII) derived from (+) -hydroxypinanone is condensed with the aldehyde (IX) via an aldolization reaction to give the compound (X). Depending on the reagent used for the aldolization reaction (Solladié-Cavallo et al., J. Org. Chem., 1994,59, 3240-3242), a mixture of esters can be obtained, which is

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 irrelevant since the acid function is deprotected two steps later.

 Preferably, the reagent ClTi (OiPr) 3 was used combined with triethylamine, a system which gives better yields than CITi (OEt) 3 and gives the mixture of ethyl and propyl esters (X) in a ratio 7 / 3.

 The imine (X) is then hydrolyzed to amine (XI) and the ester function of (XI) is reduced to alcohol (VII). The hydrolysis reaction is carried out in known manner in an acid medium, preferably in a dilute acid medium.

 The reaction for reducing the ester (XI) to alcohol (VII) is carried out in the presence of a mixed hydride, preferably lithium and boron.

H3C H3C COOL' ?3''<C NH2CH2C02R' Hs ' H 3 C / COOR' CH3 BF3Et2O CH3 HsCr"'. HC H3c EH H3c H oh H 16 14 (VIII) Schéma F
La (+) -hydroxypinanone est un produit commercial, de même que l'amino-2 acétate d'éthyle. La réaction est effectuée en présence de BF3Et20 dans un solvant aromatique, tel que le benzène ou le toluène par exemple, de préférence au reflux du solvant. The compound corresponding to formula (VIII) is prepared according to a process summarized in the synthetic scheme F below and in which R 'has the same meaning as that indicated above in scheme A:

H3C H3C COOL '? 3''<CNH2CH2C02R'Hs' H 3 C / COOR 'CH3 BF3Et2O CH3 HsCr "'. HC H3c EH H3c H oh H 16 14 (VIII) Diagram F
(+) -Hydroxypinanone is a commercial product, like 2-amino ethyl acetate. The reaction is carried out in the presence of BF3Et20 in an aromatic solvent, such as benzene or toluene for example, preferably at reflux of the solvent.

 The compound of formula (IX) can be easily prepared by means well known to those skilled in the art, in particular by following the synthetic scheme G described below:

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Scheme G
The compound (XII) is treated with ethyl triethylphosphonoacetate in the presence of a mixed hydride, preferably a lithium and boron hydride, in an anhydrous solvent, to give the ester (XIII). The conversion to trans configuration olefin is almost complete.

 Compound (XIII) is then subjected to a double treatment: a) reduction in alcohol by treatment with DIBAH (di-isobutyl aluminum hydride), then b) oxidation to aldehyde by pyridinium chlorochromate.

 The invention also relates to pharmaceutical compositions characterized in that they comprise at least one compound corresponding to the formula (S-I) or (I) described above. A molecule of formula (SI) or (I) according to the invention, which is provided with an activity for regulating the activity of NKT lymphocytes (orientation TH1 or TH2, cytotoxicity, release of cytokines), can be used for the preparation of a medicament intended for the treatment or prevention of a disease which can be chosen from autoimmune diseases, cancer and in particular metastases from melanomas located in the lungs or the liver, infectious diseases, especially infections with parasites, protozoa, viruses and intracellular bacterial infections, diabetes, especially type 1 diabetes, chronic and acute inflammatory diseases.

Among the autoimmune diseases there may be mentioned in particular uveitis, arthritis, atherosclerosis, lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis and autoimmune diabetes. These compounds can also be used as a radioprotective agent, to prevent or treat the pathological effects of irradiations, such as irradiations by a, ss rays, y ultraviolet irradiations or x-ray irradiations, proton irradiations, neutrons or

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electron beams that are commonly used in the treatment of cancer. These compounds can also be used to prevent or treat deterioration of the bone marrow, as an accelerator of the proliferation of spinal cells.
Among the infectious diseases capable of being treated with the product according to the invention, mention may in particular be made of AIDS, hepatitis B and cytomegalovirus infections.

 The pharmaceutical compositions according to the invention can also comprise other therapeutic active agents.

 The compounds according to the invention are generally formulated in the form of a sterile, pharmaceutically acceptable composition. This pharmaceutical composition can be in any form, provided that it is suitable for the chosen mode of administration. Such a composition can for example be administered parenterally, for example by intravenous, intramuscular or subcutaneous route; it can be given orally or nasally by means of a spray, in the form of drops or suppositories etc .... It can be formulated in the form of solutions in water or in oil or in the form of emulsions. Among the excipients which can be used, mention may be made of: water, alcohol, polyols, glycerin, vegetable oils, preserving agents, stabilizing agents, solubilizing agents, wetting agents, emulsifiers, dyes, salts, buffers, antioxidants.

 The dosage of the compound (S-I) or (I) according to the invention varies according to different factors such as the disease to be treated, the route of administration, the age and the weight of the individual to be treated.

 The invention further relates to a reagent for the in vitro evaluation of the activity of NKT lymphocytes, characterized in that it contains at least one compound of formula (S-I) or (I) as described above.

 A subject of the invention is also the use of such a reagent for carrying out, in vitro, a test for evaluating the activity of NKT lymphocytes, as well as the kit used during this use. and comprising at least one compound of formula (SI) or (I) as defined above.

 Said evaluation test generally comprises the following steps:

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 - the preparation of hepatic or splenic cell suspensions from a mammal, such as for example mice, - the introduction of a compound of formula (SI) or (I) as described previously in this sample of cells, - Evaluating the activity of the compounds of formula (SI) or (I) by measuring the apoptosis of hepatic or splenic NKT lymphocytes.

 The invention further relates to the use of a product of formula (S-I) or (I) for sorting active products in the modulation of lymphocyte activity.

 Other advantages and characteristics of the invention will appear on reading the additional description which follows and which refers to examples of the preparation of substituted quinolines falling within the scope of the invention and of demonstration of their biological activity, as well as 'to the appended figures in which: - Figure 1 represents the structure of the product KRN 7000 1, - Figure 2 represents the number of NKT cells measured after injection of 1.5 or 10 g of fluorescent a-galactosylceramide 4 or NKR 7000 1, - Figure 3 represents the cellular and sub-cellular distribution of the fluorescent α-galactosylceramide 4 compound in the macrophages of the peritoneum in mice, - Figure 4 represents the image cytometry of adherent fluorescent spleen and liver cells after intravenous injection of the fluorescent agalactosylceramide 4 compound in mice, - Figure 5 represents the histological localization of fluorescent cells s on sections of mouse liver after intravenous injection of the fluorescent α-galactosylceramide compound 4 or of compound KRN 7000 1 in mice.

 It goes without saying, however, that these examples are given solely by way of illustrations of the invention and in no way constitute a limitation thereof.

EXAMPLES
The preparations detailed below are based on the principle of the convergent synthesis mode (Diagram 1). Compared with previous work

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by T. Sakai et al., (Org. Lett. 1999,1, 359) the originality of the process described below is due to the convergent synthetic approach, to the forms of protection, activation and introduction of the arm spacer, to the variations made in the mode of synthesis of D-erythro-sphingosine 6 and to the optimizations with modifications of the experimental protocols throughout the synthetic scheme.

Diagram 1
In scheme 1 are described the four main steps AD of the process according to the invention for the preparation of a particular compound corresponding to formula (la). The key compound of this project, glycolipid 3 can thus be used to achieve molecular extension via the functionalization of the primary amine. The

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 transformation E is an example of derivatization intended to provide new biologically active probes.

I - PREPARATION OF α-GALACTOSIDE CERAMIDE 3 AND FLUORESCENT GLYCOLIPIDE 4:
1- Synthesis of glycolipid 4
The synthesis method used takes up certain already known modes of preparation ((a) A. Solladié-Cavallo, JL Koessler, J. Org. Chem., 1994, 59, 3240; b) T. Sakai, 0. V. Naidenko, H. Iijima, M. Kronenberg, Y. Koezuka, J.

Med. Chem., 1999,42, 1836). They have been modified for the sake of simplification or improvement of performance.

1. 1- Sequence A: Preparation of D-erythro-sphingosine 6
It is carried out via an aldolization reaction of the iminoester 5 derived from (+) -hydroxypinanone 13, with 2- (E) -hexadecenal 12 in accordance with the teaching of A. Solladié-Cavallo, JL Koessler, (J. Org. Chem., 1994, 59, 3240) (Schemes 2 and 3). The unsaturated aldehyde 12 is obtained in 3 steps from the tetradecanal 10 (Diagram 2). The formation of the ester 11 is carried out here at ambient temperature but could be carried out at a lower temperature (it is possible to provide for this reaction between 0 C and 30 C, preferably between 10 C and 25 C). The addition of tetradecanal to the ylide derived from triethylphosphonoacetate then ensures an almost instantaneous and practically total conversion into trans olefin. During the oxidation of the corresponding alcohol by pyridinium chlorochromate, the addition of celite to the reaction medium makes it possible to significantly improve the yield by facilitating the treatment. The overall yield over the 3 stages is thus 35%.

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Diagram 2 With regard to the aldolization of the iminoester 5, the modification of the protocol consists in replacing the titanium chlorotriethoxide by its isopropoxy equivalent, easier to prepare and to use (Diagram 3).

Diagram 3
A mixture of esters 14a, b is then obtained, the isopropyl ester 14a being clearly in the majority. The stereochemistry of the 2 asymmetric centers created is respected and the mixture can be treated in the same way as ethyl ester 14b in the rest of the reaction scheme.

 Another major improvement, compared to the methods of the prior art, consists in reducing the mixture of aminoesters 15a, b resulting from the hydrolysis of imines 14a, b by lithium borohydride suspended in THF (2M).

Compared to the use of sodium borohydride, the slow addition of hydride is perfectly controlled, which ensures the homogeneity of the reaction medium and the good reproducibility of the results.

 According to this process, D-erythro-sphingosine 6 is obtained with an overall yield of 28% compared to the commercial (+) - 2-hydroxy-3-pinanone 13 (Diagram 1).

1. 2- Sequence B: Preparation of ceramide 7 with carbamate function
Compound 7 is obtained via the condensation of sphingosine 6 with an activated ester 18 (Scheme 4).

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Diagram 4
According to a similar synthetic objective, Sakai et al. (Org. Lett., 1999, 1, 359) previously used a trifluoroacetamide derivative as an activated ester. According to the invention, the amine function of compound 18 is protected in the form of carbamate; which allows to ensure at the end of synthesis and in a single step, all 0- and N-deprotections, as well as the reduction of the double bond. The amidation of sphingosine 6 and the purification of the resulting ceramide 7 are carried out under standard conditions.

1. 3- Sequence C: Preparation of a-fluorinated perbenzylated D-Galactose 9
The reaction sequence described in the prior art by Nicolaou et al. (J. Am. Chem. Soc., 1984, 106, 4189) has been modified (Scheme 5).

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Diagram 5
The addition of acidic amberlite resin during the deprotection of the acetates of the derivative 20 ensures total conversion and allows a direct successive basic treatment for the O-benzylation of 21. In this same reaction carried out in dimethylformamide, the addition of 'a substoichiometric amount of potassium iodide considerably improves the reaction. The overall yield of the transformation of D-galactose 8 into fluorine derivative 9 according to this process is 14%.

1. 4- Sequence D: a-galactoceramide 3
For the first stage of O-glycosylation, it is essential to introduce into the reaction medium pre-activated pulverulent micro-screens. A change of solvent had to be made with respect to the conditions already described on this type of coupling (T. Sakai, 0. V. Naidenko, H. Iijima, M. Kronenberg, Y. Koezuka, J. Med. Chem., 1999 , 42, 1836), (Diagram 6). A purification step is necessary at this stage (chromatography on silica).

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Diagram 6
The second reaction is carried out under a hydrogen atmosphere in the presence of a palladium catalyst. It provides direct conversion to the sought-after glycolipid 3. The solubility problems of this derivative require the use of a dichloromethane / methanol mixture gradient for its purification on silica.

1. 5- Sequence E: fluorescent glycolipid 4
This final step requires a simple agitation of the two components 3 and 24 in dimethylformamide.

Diagram 7

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The final product sought 4 is directly purified by high performance liquid chromatography (HPLC), (Column C18, solvent gradient: (methanol / water).

2- Biological evaluation of the fluorescent probe 4
Tested under experimental conditions similar to those used for α-galactosylceramides, this probe shows an activity equivalent to that of the compound KRN 7000 described by Y. Osman, et al., Eur. J. Immunol., 2000,
30, 1919.

II- EXPERIMENTAL DATA:
1- GENERAL INFORMATION ON THE SEPARATION METHODS AND
ANALYSIS
The solvents are purified and dried by distillation in the presence of LiAIH4 for tetrahydrofuran (THF), sodium and benzophenone for diethyl ether (Et20), in the presence of phosphorus pentachloride for dichloromethane (CH2Cl2), in the presence of calcium hydride for methanol, acetonitrile and toluene.

 Thin layer chromatographies (TLC) are carried out on 0.2 mm thick silica gel plates 60-F-254 (Merck, Art. 5554), then observed under UV light and revealed by a Dragendorff solution (in possibly combining an acid treatment for the amino compounds) or with a solution of potassium permanganate.

 The preparative separations are carried out using flash chromatography techniques, under medium pressure on columns of Kieselgel 60 silica (230-400 Mesh, Merck).

 Infrared (IR) spectra are recorded on a Nicolet 205 Fourier transform device. The samples are deposited in film on an NaCl plate.

 The mass spectra (MS) by chemical ionization were obtained using a NERMAG R10-10 spectrometer, the reactive gas being ammonia.

 The 1 H NMR spectra are recorded on a BRUKER AC-300P device (300.13 MHz). The chemical shifts 8 are expressed relative to tetramethylsilane (TMS).

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 The 13C NMR spectra are recorded on the same device (75.43 MHz). The chemical shifts 8 are expressed relative to the deuterated chloroform (77.14 ppm).

 The analyzes and purifications by HPLC were carried out on Waters 2690 and 996 devices (module separation and photodiode array detector).

2- OPERATING MODES AND SPECTRAL DATA
All of the spectral data corresponding to aldehyde 12, to D-erythrosphingosine 6, to perbenzylated alphafluorogalactose 9 and to their precursors are similar to those described in the literature. Only entirely original compounds will be presented here.

2. 1- Preparation of the N-protected amino acid 1
10 g (49.75 mmol) of 11-amino-undecanoic acid 16 and 4.25 g (25 mmol) of benzyl chloroformate are added to 200 ml of dichloromethane. The mixture is left under stirring at reflux of the solvent overnight. After evaporation of the solvent, the crude product is purified on a silica column (eluent: 50% cyclohexane - 50% AcOEt) to yield 2.5 g of pure carbamate 17 in the form of a white solid (yield 15%).

 1 H NMR (8, CDC13): 1.15-1.6 (m, 16H), 2.28 (t, J = 7.5Hz, 2H, H-2), 3.12 (q, J = 6 , 3Hz, 2H, H-11), 4.62 (s, 2H, OCH2Ph, 5.05 (s, 1H, NH), 7.20-7.45 (m, 5H, Ph).

 13C NMR (8, CDCl3): 24.7; 26.7; 29.0 - 29.3; 29.8; 34.0; 41.2; 66.7 (CH2); 127.0 - 128.4 (CH); 136.5 (Cq); 156.5 (Cq); 178.1 (Cq).

2. 2- Preparation of the activated ester 18
To a solution of 2.5 g (7.46 mmol) of carbamate 17 in 90 ml of dichloromethane are added 1 g (7.46 mmol) of p-nitrophenol, 1.54 g (7.46 mmol) of dicyclohexylcarbodiimide and 30 mg dimethylaminopyridine. The mixture is left stirring for 24 hours at room temperature. After evaporation of the solvent, the crude product is purified by flash chromatography on a silica column (eluent: cyclohexane / ethyl acetate: 70/30) to yield the pure activated ester 18 in the form of a white solid (1.6 g, yield 47%).

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 1 H NMR (8, CDCb): 1.15-1.60 (m, 16H), 2.55 (t, J = 7.5 Hz, 2H, H- 2), 3.12 (q, J = 6.3 Hz, 2H, H-11), 4.95 (wide, 1H, NH), 5.10 (s, 2H, OCH2Ph), 7.20 (d, J = 9.1Hz, 2H, arom.), 7.30 (m, 5H, arom.), 8.20 (d, J = 9.1 Hz, 2H, arom.).

 13C NMR (8, CDCl3): 24.4; 26.6; 28.9 - 29.3; 29.8; 34.2; 41.0; 66.4 (CH2); 122.3; 125.0, 127.9, 128.4 (CH); 136.7; 145.1; 155.4 (Cq); 156.5 (Cq); 171.2 (Cq).

2. 3- Preparation of ceramide 7
To a solution of 110 mg (0.37 mmol) of sphingosine 6 and 170 mg (0.37 mmol) of activated ester 18 in 15 ml of THF are added 5 mg of dimethylaminopyridine. The mixture is left stirring at room temperature for 48 hours. After evaporation of the solvent, the crude product is purified by flash chromatography on a silica column (eluent: dichloromethane / methanol 95/5) to yield pure ceramide 7 in the form of white wax (160 mg, 70% yield).

SM (IC): 617
1 H NMR (8, CDCI 3): 0.90 (t, J = 6 Hz, 3H, Me), 1.2 - 1.65 (m, 30H), 1.80 (m, 1H, OH), 2 , 07 (q, J = 7 Hz, 2H, H-6), 2.25 (t, J = 7.5 Hz, 2H), 3.10 (m, 1H, OH), 3.18 (q, J = 6.5 Hz, 2H), 3.7 (m, 1H), 3.95 (m, 2H), 4.30 (m, 1H), 4.80 (m, 1H, NH), 5 , 11 (s, 2H), 5.52 (dd, J = 15.3; 6.4 Hz, 1H), 5.75 (dt, J = 15.3; 6.6 Hz, 1H), 6, 35 (d, J = 7 Hz, 1H, NH), 7.35 (m, 5H, aroma).

 13C NMR (#, CDCl3): 14.2 (CH3); 22.8; 25.7; 26.7; 29.2; 29.7; 30.0; 32.0; 32.4; 36.9; 41.2 (CH2); 54.7 (CH); 62.6 (CH); 66.7 (CH2); 74.6 (CH2); 127.2 (CH); 128.6-128.9 (aromatic CH); 134.2 (CH); 156.5 (Cq); 174.1 (Cq).

Elementary analysis for C37H64N2O5;

<Tb>
<tb>% <SEP> C <SEP> H <SEP> N
<tb> Calculated <SEP> 72.04 <SEP> 10.46 <SEP> 4.54
<tb> Found <SEP> 71.91 <SEP> 10.67 <SEP> 4.38
<Tb>

2. 4- Preparation of protected a-galactosyl-ceramide 23
To 3 ml of freshly distilled diethyl ether and placed under argon are added 80 mg (0.15 mmol) of the fluorinated glycoside 9 then 350 mg of microtamis 4A previously activated. Then 60.5 mg (0.30 mmol) of perchlorate are introduced.

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 silver, 55.4 mg (0.30 mmol) of tin chloride and then 60 mg (0.097 mmol) of ceramide 7 in 3.5 ml of an Et20 / THF mixture (3 / 0.5). The mixture is left stirring for 4 hours at room temperature and then diluted with 10 ml of acetone.

After 15 minutes of stirring, the reaction medium is filtered through celite. After evaporation of the solvent, the crude product is purified by flash chromatography on a silica column (eluent: cyclohexane / ethyl acetate mixture 80/20) to yield pure compound 23 in the form of white wax (30 mg, yield 27%) .

 1H NMR (#, CDC13): 0.87 (t, J = 6.8 Hz, 3H, Me), 1.20 - 1.60 (m, 42H), 1.95 (m, 2H), 2, 12 (td, J = 7.4; 2.4 Hz, 2H), 3.17 (q, J = 6.6 Hz, 2H), 3.50 (m, 2H), 3.68 (dd, J = 10.5; Hz, 1H), 3.75 - 3.88 (m, 3H), 3.95 (m, 2H), 4.03 (dd, J = 9.9; 3; 6 Hz, 1H ), 4.13 (m, 1H), 4.35; 4.45 (2d, J = 11.7 Hz, 2H), 4.55; (2d, J = 11.4 Hz, 2H), 4.20; 4.85 (2d, J = 11.1 Hz, 2H), 4.73 (m, 3H), 5.10 (s, 2H), 5.42 (dd, J = 15.4; 5.4 Hz , 1H), 5.66 (dt, J = 16.4; 6.7 Hz, 1H), 6.38 (d, J = 8.1 Hz, 1H, NH), 7.30 (m, 5H, arom.).

 13C NMR (#, CDCl3): 14.2 (Me); 22.8; 25.7; 26.7; 29.2-29.9; 31.9; 32.4; 36.7, 41.1 (CH2); 52.8 (CH); 66.7 (CH2); 68.7 (CH2); 69.1 (CH2); 69.8 (CH); 72.7; 73.4; 74.0; 74.5 (CH2); 74.2; 74.8 (CH); 75.9; 79.3; 99.1 (CH); 127.4-128.5 (aromatic CH); (Oak) ; 133.0 (CH ene); 136.7; 137.7; 138.1; 138.4; 138.5 (Cq); 156.4 (Cq); 173.2 (Cq).

2. 5- Preparation of a-galactosyl-ceramide 3
A solution of α-galactosylceramide 23 (30 mg, 0.046 mmol) is prepared from a mixture of 4 ml of methanol and 2 ml of tetrahydrofuran.

After the addition of 30 mg of 5% palladium on carbon, the mixture is stirred for 3 days at room temperature under a hydrogen atmosphere. The reaction medium is then filtered through celite and the solid residue washed with chloroform. After distilling off the solvents, the crude product is purified by flash chromatography on silica (dichloromethane / methanol mixture 98/2 then mixture 95/5). Compound 3 is thus obtained pure in the form of a white wax (16 mg, yield 85%).

MS (IC) 647
1 H NMR (#, CD30D): 0.8 (t, 3H, Me), 1.11-1.61 (m, 44H), 2.05 (m, 2H), 2.70 (m, 2H) , 3.5-3.85 (m, 10H), 4.8 (d, J = 3 Hz, 1H), 7.2 (d, J = 8 Hz, 1H, NH).

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13C NMR (8, CD30D): 24.2 (CH3); 27.0 - 32.4; (CH2); 37.7 (CH2); 41.2 (CH); 48.3 (CH2); 55.8 (CH); 63.2 (CH2); 68.7 (CH2); 70.7-72.8; 101.6 (CH); 176.6 (Cq).

2.6- Preparation of a-galactosyl-ceramide-BODIPYOO 4
7 mg (0.011 mmol) of α-galactosyl-ceramide 3 are dissolved in 0.5 ml of dimethylformamide (DMF). After introduction of the activated ester 24 (5 mg, 0.011 mmol dissolved in 0.2 m of DMF), the mixture is stirred for 36 hours under an inert atmosphere. After evaporation of the solvent, the crude product is purified directly by high performance liquid chromatography, (Novapak C18 column, methanol then methanol / water gradient, 80/20). 2.5 mg of a red solid are thus isolated (yield 25%) which must be kept cold (-20 ° C.) and protected from light.

MS (FAB) M + 23: 943
Exact mass, High Resolution Analysis 943.6107 for C49H83O9N4F2BNa
1H NMR (8, CD30D): 0.8 (t, J = 6 Hz, 3H, Me), 1.20-1.60 (3m, 44H), 2.15 (m, 2H), 2.30 ( s, 3H), 2.50 (s, 3H), 2.60 (m, 2H), 3.05-3.15 (m, 2H), 3.40- 3.90 (5m, 8H), 6 , 20 (wide s, 1H), 6.30 (wide s, 1H), 7.0 (wide s, 1H), 7.40 (wide s, 1H).

111- Demonstration of the biological activity of the fluorescent product [alpha] -galactosylc ramide 4
3.1 Materials and methods
The C57BL / 6 mice used are 6 to 8 weeks old and are supplied by IFFA-Credo (L'Arbresle, France).

 The a-galactosylceramide 4 fluorescent probe (1.5, 10 or 50 g in 150 ml of a 0.025% Tween 20 PBS solution) or the vehicle alone (0.025% Tween 20 PBS) are injected intraperitoneally. The mice are sacrificed 15 hours after the injection.

 Preparation of cell suspensions:

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They are prepared from the liver of the injected mice. The liver cells are resuspended in an 80% Percoll isotonic solution (Pharmacia,
Uppsala, Sweden) then are covered with a 40% Percoll isotonic solution.

 Centrifugation for 20 minutes at 3000 rpm makes it possible to concentrate the mononuclear cells at the 40-80% interface. The collected cells are finally washed once with a PBS solution.

Marking and analysis in flow cytometry:
Antibodies are supplied by PharMingen (San Diego, CA, USA).

The cells are pre-incubated for 10 minutes with anti-FCyIII / II antibodies (Fcblock, 2.4G2), then they are exposed for 15 minutes to anti-TCRap-FITC (H57-597) and NK1.1-PE antibodies (PK136). After washing, the cells are resuspended in a PBS solution and analyzed by flow cytometry using a FACS Calibur @ BD Becton Dickinson 5San Jose, CA, USA).

Results:
The relative proportion of NK1.1 + and TCR [alpha] [alpha] + lymphocytes of the liver is evaluated by analysis by flow cytometry: # control mouse: 23.65 10.26 # treated mouse: 1.13 0.54
No significant difference is found between the relative proportions of these lymphocytes (NKI.I + and TCR [alpha] ss +) prepared from mice treated with 1.5 or 10 g of the fluorescent probe 4.

 First of all, the biological activity of the a-galactosylceramide ~ 4 molecule was tested in vivo and compared with that of the product KRN 7000 1, the structure of which is shown in Figure 1. As a reagent for activation in vivo of NKT cells, the control (KRN 7000 1) is usually injected intraperitoneally in the form of a suspension in 0.5% of Tween @ 20 (polysorbate) in PBS. This test is based on the property of a-galactosylceramides to induce apoptosis of NKT cells of liver and spleen, restricted by the molecule CDldl, in the hours following the intraperitoneal injection.

 3.2 Influence of KRN 7000 1 and of α-galactosylceramide BODIPY 4 on the number of NKT cells

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1.5 or 10 g of the fluorescent α-galactosylceramide derivative 4 was injected intraperitoneally, while identical concentrations of KRN 7000 1 were injected as a control. The monocytes from the liver were isolated according to the method described above and analyzed for NK1.1 + TCR [alpha] ss int + cells by analysis by flow cytometry.

 The results obtained are shown in the attached FIG. 2 in which the number of NKT cells is expressed as a function of the concentration of fluorescent agalactosylceramide 4 or of KRN 7000 1 injected in g.

 These results show that an amount injected as low as 1 g of the fluorescent α-galactosylceramide 4 molecule is capable of inducing the disappearance of almost all the NKT cells from the liver, as is the control KRN 7000 1. It is remarkable that the remaining NK1.1 + TCR [alpha] ss int + cells are not restricted to the CDldl molecule and therefore do not meet the original definition of NKT cells. The fluorescent α-galactosylceramide 4 molecule according to the invention therefore has an activity comparable to that of KRN 7000 1 in the in vivo apoptosis test and in this range of concentrations.

3. 3 Capture of cells after intraperitoneal injection
In a second step, we looked for the first cells that captured the fluorescent compound after intraperitoneal injection of 10 g of the fluorescent α-galactosylceramide compound 4. The cells of the peritoneum were recovered by washing the peritoneal cavity and looking for the compound fluorescent labeled was performed.

 The results obtained are shown in Figure 3 attached, which shows the cellular and subcellular distribution of the fluorescent agalactosylceramide 4 compound in the macrophages of the peritoneum.

 These results show that an hour after the injection almost all the macrophages provided a positive response to the fluorescence test; the marking being either made up of discrete spots along the membrane or located in vesicles in the cytoplasm. These results also show that the marker binds to a receptor, and that the complex then migrates to the membrane where it is absorbed.

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 Monocytes were recovered from the liver and spleen of mice administered the fluorescent a-galactosylceramide 4 marker intraperitoneally. Adherent and non-adherent cells were collected separately and analyzed for labeling. No labeled cells could be detected in either of the two organs. This shows that the fluorescent a-galactosylceramide 4 molecule was mainly captured by macrophages of the peritoneum. We can formulate two hypotheses explaining the biological activity observed on the liver either by the fact that an undetectable quantity of this molecule escapes capture by the peritoneum and migrates to the liver and peripheral organs, or by the fact that macrophages which have absorbed the molecule migrate to the liver where they release the fluorescent α-galactosylceramide compound 4. This hypothesis is supported by the observation which has been made that macrophages of the peritoneum washed and reinjected intravenously can induce activation in these organs.

3.4 Study of cell capture after intravenous injection
In a third step, it was sought which cells were capable of capturing the fluorescent α-galactosylceramide compound 4 after intravenous injection of 50 μg of this product in mice. Monocytes from the liver and spleen were isolated one hour after injection and the adherent cells were separated from the non-adherent cells. The fraction of cells responding positively was determined by image cytometry. The results are shown in Figure 4.

 These results show that approximately 10% of the adherent cells gave a positive response in both organs with a subcellular localization similar to that observed in the macrophages of the peritoneum. Nonadherent cells (NK, NKT and lymphocytes) gave a negative response: they had not absorbed a significant amount of the labeled molecule. The location of the fluorescent marker in the liver was examined using a fluorescence microscope and frozen sections counter-labeled with Evans blue, and also by confocal microscopy using counter-labeling with rhodaminephalloidine-PI. The results of these tests are shown in Figure 5.

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 These results show that no labeling of hepatocytes could be noted, as well as no labeling of the endothelial cells lining the blood vessels. By their appearance and their location in the sinusoids, the marked cells are assimilated to macrophages or adherent cells, which can be Kupffer cells. The fluorescent marker is stored in vesicles, in the same way as in macrophages of the peritoneum, some of the particles being located near the nucleus.

 Consequently, it can be deduced therefrom that the fluorescent agalactosylceramide compound 4 in accordance with the invention does not behave in the same way depending on the mode of injection used. Although a fraction of the molecules injected into the peritoneum reach the liver (directly or indirectly through the macrophages), which explains the biological activity observed, most of the product injected into the peritoneum remains stored in the macrophages of the peritoneum.

 On the contrary, the product injected intravenously was found massively in cells of the Kuppfer cell type in the liver and in APC or "Antigen Presenting Cells" (dendritic cells and probably macrophages) of the spleen.

 Finally, all the cells not having been affected by the fluorescent α-galactosylceramide compound 4, it can be concluded that there is a specificity in the capture of α-galactosylceramide type compounds leading to their integration in significant quantities in the APC.

Claims (31)

 in which: - Su represents a residue chosen from glycosides and glycoside derivatives; - X represents a linear or branched C1-C30 di-yl alkane radical or an aryl group; - x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents a hydrogen atom; a linear or branched C 1 -C 20 alkyl group or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or a glycoside residue which may be the same or different from the radical Su; - B represents a group chosen from: a hydrogen atom, and then x = y = 0; or B represents a group chosen from: a single bond, a C1-C30 alkyl, alkenyl or alkynyl radical, linear, branched or cyclic, optionally substituted by one or more groups — OH, -NH2, halogen, -COOH, -CONH2 , with an aryl group optionally substituted or with a heteroaryl group, with a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; an aryl group

 CLAIMS 1. A-glycosylceramide derivative corresponding to the general formula (S-1) <Desc / Clms Page number 39>  optionally substituted or a heteroaryl group, a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; a peptide or pseudopeptide chain. - K represents a pharmacophore group.  2. Derivative according to claim 1, characterized in that it is an α-galactosylceramide corresponding to the general formula (I):

 (I) in which: - YI, Y2, Y3 and Y4 represent a hydrogen atom, a benzyl group or a monosaccharide; - X represents a linear or branched C1-C30 di-yl alkane radical or an aryl group; - x is an integer equal to 0 or 1; y is an integer equal to 0 or 1; - A represents a hydrogen atom; a linear or branched C1-C20 alkyl group or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical corresponding to formula (II) below: <Desc / Clms Page number 40>  (II) in which Y [, Y2, Y3, Y4 have the same definitions as those indicated above in formula (I), - B represents a group chosen from: a hydrogen atom, and then x = y = 0; or B represents a group chosen from: a single bond, a C1-C30 alkyl, alkenyl or alkynyl radical, linear, branched or cyclic, optionally substituted by one or more groups — OH, -NH2, halogen, -COOH, -CONH2 , with an aryl group optionally substituted or with a heteroaryl group, with a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; an optionally substituted aryl group or a heteroaryl group, a heterocycle comprising from 5 to 10 members and one or more hetero atoms chosen from 0, N, S; peptide or pseudopeptide chain.

- K represents a pharmacophore group.  3. Derivative according to claim 1, characterized in that x = y = O and B = H and in that it corresponds to the formula (S-Ia): <Desc / Clms Page number 41> Su represents a residue chosen from glycosides and glycoside derivatives; - X represents a linear or branched C1-C30 di-yl alkane radical or an aryl group; - A represents a hydrogen atom; a linear or branched C1-C20 alkyl group or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or a glycoside residue which may be identical to or different from the radical Su.  in which

 4. Derivative according to claim 2, characterized in that x = y = 0 and B = H and in that it corresponds to the formula (la):

 in which: Y1, Y2, Y3 and Y4 represent a hydrogen atom, a benzyl group or a monosaccharide; <Desc / Clms Page number 42>  in which Y1, Y2, Y3, Y4 have the same meanings as those indicated above in the formula (la).

 - X represents a linear or branched C1-C30 di-yl alkane radical or an aryl group; - A represents a hydrogen atom; a linear or branched C1-C20 alkyl group or an aryl group; - Z represents -CH2 -CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical corresponding to formula (II) below:  5. Derivative according to any one of claims 1 to 4, characterized in that in the formula (SI) or in the formula (I) one or more of the conditions below are met: - Y1, Y2, Y3 and Y4 represent the hydrogen atom; - X represents an alkane di-yl radical, C6-C15, linear or branched or an aryl group; - x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents: a C4-C15 alkyl group, linear or branched or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical: <Desc / Clms Page number 43> B represents a linear or branched C1 or C12 alkyl or alkenyl group.  - B represents a group chosen from: the hydrogen atom, and then x = y = 0, or

 6. Derivative according to claim 5, characterized in that in the formula (SI) or in the formula (I) one or more of the conditions below are met: - Y1, Y2, Y3 and Y4 represent the atom hydrogen; - X represents a linear or branched C6-C15 di-yl alkane radical; - x is an integer equal to 0 or 1; - y is an integer equal to 0 or 1; - A represents: a linear or branched C4-C15 alkyl group; - Z represents -CH2-CH2- or -CH = CH-; - R represents a hydrogen atom or an a-galactosyl radical; - B represents a group chosen from: a hydrogen atom and then x = y = 0, or B represents a C1-C12 alkyl group, linear or branched.  7. Derivative according to claim 6, characterized in that in the formula (SI) or in the formula (I) one or more of the conditions below are met: - Y1, Y2, Y3 and Y4 represent the atom hydrogen; - X represents a linear di-yl C6-C15 alkane radical; - x is equal to 1; - y is an integer equal to 0 or 1; - A represents: a linear C10-C15 alkyl group; - Z represents -CH2-CH2-; - R represents a hydrogen atom; <Desc / Clms Page number 44> B represents a group chosen from: a hydrogen atom, and then x = y = 0, or B represents a linear C1-C6 alkyl group.  8. A derivative according to claim 1 or claim 2, characterized in that in the formula (S-I) or in the formula (I): - y = 1; - K represents a pharmacophore group chosen from: boron dipyrromethene difluoride, nitrobenzoxadiazole, groups with azirine function, iodinated heterocyclic groups, radioactive groups and dansyl groups.  9. A derivative according to claim 8, characterized in that in the formula (S-I) or in the formula (I): - y = 1; - K represents a dipyrromethene boron difluoride group.  10. A method of preparing a derivative corresponding to the formula (SI) according to claim 1 in which x = l or y = l, and BH, this method being characterized in that a compound corresponding to the formula (S-Ia) according to claim 3, in which the parameters Su, X, Z and A have the same meaning as that indicated in formula (SI), with a compound corresponding to formula (Ib) below:

 (Ib) in which x, y, B and K have the same meaning as that indicated in the formula (SI) and T is chosen to carry out the alkylation (x = 0) or the acylation (x = l) of the free amine function of (S-Ia), according to the SE scheme below: <Desc / Clms Page number 45>

 Diagram E

 (Ib) in which x, y, B and K have the same meaning as that indicated in formula (I) and T is chosen to carry out the alkylation (x = 0) or the acylation (x = l) of the free amine function of (la), according to scheme E below:

11. Process for the preparation of a derivative corresponding to formula (I) according to claim 2 in which x = l or y = l, and BH, this process being characterized in that a compound corresponding to the formula (la) according to claim 2, in which the parameters Y1, Y2, Y3, Y4, X, Z and A have the same meaning as that indicated in formula (I), with a compound corresponding to formula (Ib) below : S-E diagram <Desc / Clms Page number 46>

12. Method according to claim 10 characterized in that the compound corresponding to the formula (S-Ia), is prepared by a method comprising at least one step during which an α-glycoside corresponding to the formula (S is reacted -III) in which Su has the same meaning as in formula (SI), with a ceramide corresponding to formula (IV) in which Y'5 represents an amine protecting group; Z represents the group -CH = CH- or -CH2CH2-; X and A have the same definition as in formula (S-I). this coupling step being followed by a deprotection step of the functional groups according to the S-D scheme: 13. Method according to claim 11 characterized in that the compound corresponding to formula (la), is prepared by a method comprising at least one step during which an α-galactoside corresponding to formula (III) is reacted in which Y'1, Y'2, Y'3 and Y'4 represent a hydroxyl protective group or a monosaccharide protected by appropriate protective groups, with a ceramide corresponding to formula (IV) in which Y'5 represents a S-D diagram <Desc / Clms Page number 47>

 protective group for amines; Z represents the group -CH = CH- or -CH2CH2-; X and A have the same definition as in formula (I), this coupling step being followed by a deprotection step of the functional groups according to scheme D: 14. Method according to claim 13, characterized in that Y'1, Y'2, Y'3, Y'4 and Y'5 are chosen so that they can be removed simultaneously in a single step. Scheme D  15. The method of claim 13, characterized in that Y'1, Y'2, Y'3, Y'4 and Y'5, are chosen from benzyl and benzyloxycarbonyl groups.  16. Product corresponding to formula (IV): <Desc / Clms Page number 48> A represents: a hydrogen atom; a linear or branched C1-C20 alkyl group or an aryl group; - X represents a di-yl alkane radical, C1-C30, linear or branched or an aryl group; - Z represents -CH2-CH2- or -CH = CH-; - Y'5is chosen from: the hydrogen atom, a benzyloxycarbonyl group, a ter butyloxycarbonyl group.  in which :

 17. Product according to claim 16, characterized in that: - A represents: a linear C10-C15 alkyl group; - X represents a linear di-yl C6-C15 alkane radical; - Z represents -CH = CH-; - Y'5 represents a benzyloxycarbonyl group.  18. Method according to claim 12 or claim 13, characterized in that the product corresponding to formula (IV) is prepared by reaction of an activated derivative corresponding to formula (VI) in which W represents an activating group with a analog or a derivative of D-erythro-sphingosine corresponding to formula (VII) or with D-erythro-sphingosine, according to scheme B below: <Desc / Clms Page number 49>

Scheme B in which X, Y'5, Z and A have the same definition as in formula (IV).  19. The method of claim 18, characterized in that the compound corresponding to formula (VIIa), is obtained by reaction of an iminoester corresponding to formula (VIII) in which the radical R 'represents an ethyl radical and R " represents an ethyl or isopropyl radical with the aldehyde corresponding to formula (IX) via an aldolization reaction to give the compound (X) then that the imine (X) is then hydrolyzed to amine (XI) and the ester function of (XI) is reduced to alcohol (VII) according to scheme A below:

20. The method of claim 19, characterized in that the compound corresponding to formula (VIII) is prepared by reaction of (+) hydroxypinanone with 2-amino ethyl acetate, in the presence of BF3Et2O in a Diagram A <Desc / Clms Page number 50>

 aromatic solvent, preferably at reflux of the solvent, in accordance with synthesis scheme F below: Scheme F in which R 'represents an ethyl radical.  21. The method of claim 19, characterized in that the compound corresponding to formula (IX) is prepared by reaction of the compound (XII) with ethyl triethylphosphonoacetate in the presence of a mixed lithium and boron hydride, in an anhydrous solvent, to give the ester (XIII), which is then subjected to a double treatment: a) reduction to alcohol by treatment with DIBAH, then b) oxidation to aldehyde by pyridinium chlorochromate, according to the synthetic scheme G described below:

Scheme G 22. Pharmaceutical composition characterized in that it comprises at least one compound corresponding to the formula (S-I) according to claim 1 in a pharmaceutically acceptable carrier.  23. Pharmaceutical composition characterized in that it comprises at least one compound corresponding to formula (I) according to claim 2 in a pharmaceutically acceptable carrier. <Desc / Clms Page number 51>  24. Use of a compound of formula (S-1) or of formula (I) according to any one of claims 1 to 9, for the preparation of a medicament intended for regulating the activity of NKT lymphocytes.  25. Use of a compound of formula (S-1) or of formula (I) according to any one of claims 1 to 9, for the preparation of a medicament intended for the treatment or prevention of a disease which can be chosen from autoimmune diseases, cancer, infectious diseases, diabetes, especially type 1 diabetes, chronic and acute inflammatory diseases.  26. Use according to claim 25, characterized in that the medicament is intended for the treatment of a disease chosen from: arthritis, atherosclerosis, lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, and autoimmune diabetes.  27. Reagent for the in vitro evaluation of the activity of NKT lymphocytes, characterized in that it contains at least one compound of formula (S-I) or of formula (I) according to any one of claims 1 to 9.  28. Use of a reagent according to claim 27, for carrying out, in vitro, a test for evaluating the activity of NKT lymphocytes.  29. Use according to claim 28, characterized in that the said test comprises the following stages: - the preparation of hepatic or splenic cell suspensions from a mammal, - the introduction of a compound of formula (SI) or (I) in this sample of cells, - evaluation of the activity of the compounds of formula (SI) or (I) by measuring the apoptosis of hepatic or splenic NKT lymphocytes.  30. Use according to claim 28 or 29, for sorting active products in the modulation of lymphocyte activity.  31. Kit for implementing an in vitro test for evaluating the activity of NKT lymphocytes, characterized in that it comprises at least one compound of formula (S-1) or (I) according to l any of claims 1 to 9.