ALPHA-GALACTOSYLCERAMIDE ANALOGS, THEIR METHODS OF MANUFACTURE, INTERMEDIATE COMPOUNDS USEFUL IN THESE METHODS , AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

The invention relates to α-galactoceramide analogs, to the methods for preparing them, and to intermediate compounds useful in methods for manufacturing them. It also relates to pharmaceutical compositions containing these α-galactoceramide analogs. Glycolipid ligands from marine origin such as Agelasphins, which are tropical sponges, have been described as activators of a particular line of T lymphocytes, the NKT cells.

The NKT cells have been recently shown as being the main factors in the immune response in various physiopathologic processus such as multiple sclerosis, auto-immune diabetes, and some bacterial or viral infections. They also seem to be involved in the anti-tumoral defence.

Glycolipids extracted from sponges present a great interest due to their anti-tumoral and immuno-modulating activities in vivo. Agelasphins isolated frpm sponges of the Age/as genus, have shown a high activity on B16 melanoma cells in the mouse. All these Agelasphins have an α-anomeric configuration. Their toxicity is low and their immuno-stimulating properties are also high. These Agelasphins are the first known natural α- galactoceramides.

One α-galactosy!ceramide analog, KRN 7000, has been synthesized and is presently under clinical trials as anticancerous drug.

An example of Agelasphin with anti-tumoral and immuno- stimulating activities isolated from Agelas mauritianus sponges, has the following Formula:

The synthetic glycolipid α-galactosy)ceramide analog, presently under clinical trials, KRN 7000, has the following Formula:

As can be seen from the above formulae, KRN 7000 differs from the natural Agelasphin by the absence of an hydroxyl group on position 2' of the acyl chain of the ceramide.

Recent works describe the pharmacologic effect of compounds with a "truncated sphingosin", i.e. of compounds with variable lengths of the fatty chain, on the Th1/Th2 balance.

These works have permitted to evidence, more or less precisely, some parameters which seem to have an influence on the biological activity of this type of galactoceramide analogs .

Thus, these galactoceramide analogs may be described as molecules comprising a galactosyl cycle bounded to the ceramide part by a glycosidic link. In KNR 7000, the glycosidic link is an O atom. The ceramide part comprises an acyl chain and a sphingosyl residue.

The first parameter described as of importance for the biological activity of galactosylceramide analogs is its configuration. Indeed, an α anomeric configuration appears to be required for the activity, although β analogs are also candidates for biological targets such as antiviral and antipaludic infections.

The second parameter described as of importance is the nature of the sugar. A galactosyl cycle with an hydroxyl group on position 2" appears to be the most appropriate sugar.

The third parameter described as of importance is the glycosidic link. The anomeric oxygen which is naturally present on galactoceramides is one of the main sites of the biological activity. Some works have been carried out on KRN 7000 analogs by substituting the anomeric oxygen by a carbon in order to obtain C-galactosylceramide compounds which are 4 to 100 times more active as anticancerous compounds, notably.

The fourth parameter the importance of which has been studied is the acyl chain. It has been shown that the hydroxyl on position 2' which is originally present in the natural compounds is not useful for the targeted activity: it is not present in KNR 7000. However, this acyl chain may be modified for anchoring fluorophor compounds or may be altered by variations of the chain length.

In fact, all these works have demonstrated that the sphingosyl residue is probably one of the masterpiece of the activity of the galactoceramide analogs and, therefore, searches have been focused on this part of the galactoceramide analogs.

According to the works done until now, the presence of two vicinal hydroxyl groups on positions 3 and 4 of the sphingosyl chain is compulsory on the human model. For example, Laurent BROSSAY and al. in the article entitled "Cutting edges: Structural Requirements for Galactosylceramide Recombination by CD 1 -restricted NKT cells published in the "Journal of Immunology", page 5124-5128, in 1998, have reported that the presence of the hydroxyl group on the position 4 of the sphingosyl base is compulsory to have an activity in human model.

In the same manner, the length of the fatty chain of the sphingosyl residue as well as the presence or the absence of unsaturations have been demonstrated as having a great influence on the selectivity of the

biological activity. It has also been demonstrated that important variations could derive from the variation of this length of the fatty chain.

Theses results were recently confirmed in a crystallographic study by Koch, M.; Strange, V. S.; Shepherd, D.; Gadola, S. D.; Mathew, B.; Ritter, G.; Fersht, A. R.; Besra, G. S.; Schmidt, R. R.; Jones, E. Y.; Cerundolo, V. Nat Immunol 2005, 6, 819-826.

In this study, interactions between the human CD1d receptor and the hydroxyl groups on positions 3 and 2" are shown.

Besides, in all works carried out until now on α- galactosceramide analogs, the α-galactosylceramide analogs have been synthesized by a method which consists to first prepare the ceramide or sphingosyl residue and then, to carry out a glygosydic coupling in position α of an activated galactosyl donor.

This method involves the previous synthesis of each sphingosyl residue, which has to be modified before its incorporation on the sugar. The acyl chain, then, may be introduced either before the glycosidic coupling or after this glycosidic coupling starting from the galactosylsphinganin chain.

In contrast to the prejudices existing in the prior art, the inven- tion is based firstly, on the surprising discovery that α -galactosylceramide analogs with no hydroxyl group on position 4 of the sphinganin chain lead to compounds having an excellent biological activity in human models, and secondly, the invention proposes a method for preparing α galactosylceramide analogs in which an ethylenic, i.e., unsaturated, product is used.

Thanks to this ethylenic product, a broad range of α - galactoceramide analogs with a sphyngosin chain may be synthesized by a mere metathesis reaction with fragments of synthesized or commercial saturated alkyl chains. With the method of the invention, any residue having a functional unsaturation (branched alkyl chain, aromatic chain, heterocycle

chains, sugars...) may be incorporated at the end of the aglycone unsaturated chain of this intermediate product.

Thus, this method enables to produce an important number of different analogs by combinatory chemistry starting from different glycosidic precursors.

Accordingly, the invention proposes a compound having the following Formula I:

Formula I wherein:

X is O, S, S(O), S(O ₂ ), or NH,

R ₁ is H or a protecting group such as an isotertbutyloxy- carboxy group (Boc), methoxycarbonyl group, ethoxycarbonyl group, benzy- loxycarbonyl group (Cbz), allyloxycarbonyl group (Aloe),

9-fluorenylmethoxycarbonyl group (Fmoc), 2-(trimethylsilyl)ethoxycarbonyl group (Teoc), 2,2,2-trichloroethoxycarbonyl, benzyl group (Troc), benzyl group

(Bn), diphenylmethyl group (Dpm), trityl group (Tr), 9-phenylfluorenyl group

(PhFI), allyl group, p-methoxybenzyl group (PMB), preferably Ri is an isotert- butyloxycarboxy group (Boc), or a benzyloxycarbonyl group (Cbz), or a

9-fluorenylmethoxycarbonyl group (Fmoc),

R ₅ is H or a protecting group such as a trimethylsilyl group (TMS), triethylsilyl group (TES), fe/t-butyldimethylsilyl group (TBS), tert- butyldiphenylsilyl group (TBDPS), triisopropylsilyl group (TIPS), diethylisopro- pylsilyl group (DEIPS), thexyldimethylsilyl group (TDS), triphenylsilyl group (TPS), d/-fe/t-butylmethylsilyl group (DTBMS), methyl group, te/t-butyl group, benzyl group (Bn), p-methoxybenzyl group (PMB), 3,4-dϊmethoxybenzyl group

(DMB), trityl group (Tr), allyl group, methoxymethyl group (MOM), 2- methoxyethoxymethyl group (MEM), benzyloxymethyl group (BOM), p- methoxybenzyloxymethyl group (PMBM), 2-(trimethylsilyl)ethoxymethyl group (SEM), tetrahydropyranyl group (THP), methylthiomethyl group (MTM), acetate group (Ac), benzoate group (Bz), pivalate group (Pv), methoxyacetate group, chloroacetate group, levulinate group (Lev), benzyloxycarbonyl group (RO-Cbz), p-nitrobenzyloxycarbonyl (RO-CO ₂ PNB), ferf-butoxycarbonyl group (RO-Boc), 2,2,2-trichloroethoxycarbonyl (RO-Troc), 2-

(trimethylsιϊyl)ethoxycarbonyl group (RO-Teoc), allyloxy group (RO-Aloc), preferably R ₅ is a terf-butyldiphenylsilyl group (TBDPS), benzyl group (Bn), acetate group (Ac), more preferably R ₅ is a terbutyldiphenylsilyl group (TBDPS),

Ri and R ₅ may together form a protecting group such as an λ/,O-acetal group, preferably an oxazolidine group or an oxazoline group, - R ₂ is H or a protecting group such as a trimethylsilyl group (TMS), triethylsilyl group (TES), terf-butyldimethylsilyl group (TBS), tert- butyldiphenylsilyl group (TBDPS), triisopropylsilyl group (TIPS), diethylisopro- pylsilyl group (DEIPS), thexyldimethylsilyl group (TDS), triphenylsilyl group (TPS), cf/-fe/?-butylmethylsilyl group (DTBMS), methyl group, te/f-butyl group, benzyl group (Bn), p-methoxybenzyl group (PMB), 3,4-dimethoxybenzyl group (DMB) ₁ trityl group (Tr), allyl group, methoxymethyl group (MOM), 2- methoxyethoxymethyl group (MEM), benzyloxymethyl group (BOM), p- methoxybenzyloxymethyl group (PMBM), 2-(trimethylsilyl)ethoxymethyl group (SEM), tetrahydropyranyl group (THP), methylthiomethyl group (MTM), preferably R ₂ is a benzyl group (Bn), te/t-butyldiphenylsilyl group (TBDPS), te/t-butyldimethylsilyl group (TBS), trityl group (Tr), isopropylidene group or cyclohexylidene group, more preferably R ₂ is a benzyl group (Bn),

R ₃ , R ₄ , and Re are identical or different, and are H or a protecting group such as a trimethylsilyl group (TMS), triethylsilyl group (TES), te/t-butyldimethylsilyl group (TBS), terf-butyldiphenylsilyl group (TBDPS), triisopropylsilyl group (TIPS), diethylisopropylsilyl group (DEIPS), thexyldimethylsilyl group (TDS), triphenylsilyl group (TPS), di-tert-

butylmethylsilyl group (DTBMS), methyl group, te/t-butyl group, benzyl group (Bn), p-methoxybenzyl group (PMB), 3,4-dimethoxybenzyl group (DMB), trityl group (Tr), allyl group, methoxymethyl group (MOM), 2-methoxyethoxymethyl group (MEM), benzyloxymethyl group (BOM), p-methoxybenzyloxymethyl group (PMBM), 2-(trimethylsilyl)ethoxymethyl group (SEM), tetrahydropyranyl group (THP), methylthiomethyl group (MTM), acetate group (Ac), benzoate group (Bz), pivalate group (Pv), methoxyacetate group, chloroacetate group, levulinate group (Lev), benzyloxycarbonyl group (RO-Cbz), p- nitrobenzyloxycarbonyl (RO-CO ₂ PNB), te/t-butoxycarbonyl group (RO-Boc), 2,2,2-trichloroethoxycarbonyl (RO-Troc), 2-(trimethylsϊlyl)ethoxycarbonyl group (RO-Teoc), allyloxy group (RO-Aloc), preferably R ₃ , R ₄ and R ₆ are identical and are a benzyl group (Bn), terf-butyldiphenylsilyl group (TBDPS), terf-butyldimethylsilyl group (TBS), trityl group (Tr), isopropylidene group or, cyclohexylidene group, more preferably R ₃ , R ₄ and R ₆ are identical and are a benzyl group (Bn),

R ₃ and R ₄ may together form an O,O-acetal group such as an isopropylidene group, cyclohexylidene group, cyclopentylidene group, benzylidene group, mesitylmethylene group, p-methoxybenzylidene group, methylene group, diphenylmethylene group, isopropylidene group, or dispoke group, and

R ₄ and R ₆ may together form an O,O-acetal group such as a benzylidene or a paramethoxybenzylidene group.

This compound of Formula I is one of the intermediate enabling to carry out the method of synthesis of different α-galactoceramide compounds which are also the subject matter of the invention.

This intermediate compound enables to synthesize α- galactoceramide analogs having only one hydroxyl group in position 3 of the sphingosyl residue. But it also enables to synthesize α-galactoceramide analogs with hydroxyl groups both in positions 3 and 4 of this sphingosyl residue.

Furthermore, this intermediate compound of Formula I enables to prepare α-galactoceramide analogs with a glycosidic link which can be O, S, (SO), (SO ₂ ), or NH.

The method according to the invention for preparing the compound of Formula I above in which X is O, S or NH comprises the following steps:

(a) providing a compound of the following Formula 1-1 :

Formula 1-1 wherein R ₂ , R ₃ , R ₄ , Re are identical and are a benzyl group,

(b) osidic coupling of the compound of Formula 1-1 with a compound of Formula I-2 :

Formula I-2 wherein :

X is OH, SH, or NH ₂ ,

Ri is an isotertbutyloxycarboxy group (Boc), and R ₅ is a fe/t-butyldiphenylsilyl group (TBDPS). The alternative and powerful method for preparing the compound of Formula I in which X is S comprises the following steps:

(a) providing a compound of the following Formula 1-3 :

Formula 1-3 wherein R ₂ , R3, R ₄ , and R ₆ are identical and are a benzyl group,

(b) treating the compound of Formula 1-3 with NaH, CS ₂ , and adding para-nitrobenzoyl chloride to obtain the 1-thio-para-nitrobenzoyl ester,

(c) saponification of the glycosyl ester obtained in step (b), for example with sodium methanolate (M _e O ^" N _a ⁺ ) and or Cesium carbonate, or potassium carbonate,

(d) nucleophilic substitution with the sphingosyl compound of Formula 1-2

Formula 1-2 wherein :

X is a leaving group, preferably X is an O mesyl, O triflate, O-tosyl, Cl, Br, or I group. In this process, steps (c) and (d) are carried out simultaneously.

For obtaining α-galactoceramide analogs, one may start from the compound of Formula I or from an other intermediate compound, which is also the subject matter of the invention. This second intermediate compound which is the subject matter of the invention is the compound having the following Formula II:

Formula Il wherein:

X is O, S(O), S(O ₂ ), or NH, - Ri is H or a protecting group such as an isotertbutyloxy- carboxy group (Boc), methoxycarbonyl group, ethoxycarbonyl group, benzy- loxycarbonyl group (Cbz), allyloxycarbonyl group (Aloe), 9-fluorenylmethoxycarbonyl group (Fmoc), 2-(trimethylsilyl)ethoxycarbonyl group (Teoc), 2,2,2-trichloroethoxycarbonyl, benzyl group (Troc), benzyl group (Bn), diphenylmethyl group (Dpm), trityl group (Tr), 9-phenylfluorenyl group (PhFI), allyl group, p-methoxybenzyl group (PMB), preferably R-i is an isotert- butyloxycarboxy group (Boc), or a benzyloxycarbonyl group (Cbz), or a 9-fluorenylmethoxycarbonyl group (Fmoc),

R ₅ and R ₈ are independently H or a protecting group such as a trimethylsilyl group (TMS), triethylsilyl group (TES), tert- butyldimethylsilyl group (TBS), te/t-butyldiphenylsilyl group (TBDPS), triiso- propylsilyl group (TIPS), diethylisopropylsilyl group (DEIPS), thexyldimethyl- silyl group (TDS), triphenylsilyl group (TPS), d/-te/f-butylmethylsilyl group (DTBMS), methyl group, terf-butyl group, benzyl group (Bn), p-methoxybenzyl group (PMB), 3,4-dimethoxybenzyl group (DMB), trityl group (Tr), allyl group, methoxymethyl group (MOM), 2-methoxyethoxymethyl group (MEM), benzy- loxymethyl group (BOM), p-methoxybenzyloxymethyl group (PMBM), 2- (trimethylsilyl)ethoxymethyl group (SEM), tetrahydropyranyl group (THP), methylthiomethyl group (MTM) ₁ acetate group (Ac), benzoate group (Bz), pivalate group (Pv), methoxyacetate group, chloroacetate group, levulinate group (Lev), benzyloxycarbonyl group (RO-Cbz), p-nitrobenzyloxycarbonyl (RO-CO ₂ PNB), terf-butoxycarbonyl group (RO-Boc), 2,2,2-

trichloroethoxycarbonyl (RO-T roc), 2-(trimethylsilyl)ethoxycarbonyl group (RO- Teoc), allyloxy group (RO-Aloc), O,O-acetal groups such as an isopropylidene group, cyclohexylidene group, cyclopentylidene group, benzylidene group, mesitylmethylene group, p-methoxybenzylidene group, methylene group, diphenylmethylene group, isopropylidene group or dispoke group, preferably R ₅ and R ₈ are a terf-butyldiphenylsilyl group (TBDPS), benzyl group (Bn), acetate group (Ac), more preferably R ₅ and R ₈ are a terbutyldiphenylsilyl group (TBDPS),

R ₁ and R ₅ may together form a protecting group such as an λ/,O-acetal group, preferably an oxazolidine group or an oxazoline group,

R ₂ is H or a protecting group such as a trimethylsilyl group (TMS), triethylsilyl group (TES), terf-butyldimethylsilyl group (TBS), tert- butyldiphenylsilyl group (TBDPS), triisopropylsilyl group (TIPS), diethylisopro- pylsilyl group (DEIPS), thexyldimethylsilyl group (TDS), triphenylsilyl group (TPS), c//-ferf-butylmethylsilyl group (DTBMS), methyl group, terf-butyl group, benzyl group (Bn), p-methoxybenzyl group (PMB), 3,4-dimethoxybenzyl group (DMB), trityl group (Tr), allyl group, methoxymethyl group (MOM), 2- methoxyethoxymethyl group (MEM), benzyloxymethyl group (BOM), p- methoxybenzyloxymethyl group (PMBM), 2-(trimethylsilyl)ethoxymethyl group (SEM), tetrahydropyranyl group (THP), methylthiomethyl group (MTM), preferably R ₂ is a benzyl group (Bn), terf-butyldiphenylsilyl group (TBDPS), terf-butyldimethylsilyl group (TBS), trityl group (Tr), isopropylidene group or cyclohexylidene group, more preferably R ₂ is a benzyl group (Bn),

R ₃ , R ₄ , and Re are identical or different, and are H or a protecting group such as a trimethylsilyl group (TMS), triethylsilyl group (TES), terf-butyldimethylsilyl group (TBS), terf-butyldiphenylsilyl group (TBDPS), triisopropylsilyl group (TIPS), d fethy I iso propy lsi IyI group (DEIPS), thexyldimethylsilyl group (TDS), triphenylsilyl group (TPS), di-tert- butylmethylsilyl group (DTBMS), methyl group, terf-butyl group, benzyl group (Bn), p-methoxybenzyl group (PMB), 3,4-dimethoxybenzyl group (DMB), trityl group (Tr), allyl group, methoxymethyl group (MOM), 2-methoxyethoxymethyl group (MEM), benzyloxymethyl group (BOM), p-methoxybenzyloxymethyl

group (PMBM), 2-(trimethylsilyl)ethoxymethyl group (SEM), tetrahydropyranyl group (THP), methylthiomethyl group (MTM), acetate group (Ac), benzoate group (Bz), pivalate group (Pv) ₁ methoxyacetate group, chloroacetate group, levulinate group (Lev), benzyloxycarbonyl group (RO-Cbz), p- nitrobenzyloxycarbonyl (RO-CO ₂ PNB), ferf-butoxycarbonyl group (RO-Boc), 2,2,2-trichloroethoxycarbonyl (RO-Troc), 2-(trimethylsilyl)ethoxycarbonyl group (RO-Teoc), allyloxy group (RO-Aloc), preferably R ₃ , R ₄ and R ₆ are identical and are a benzyl group (Bn), terf-butyldiphenylsilyl group (TBDPS), ferf-butyldimethylsilyl group (TBS), trityl group (Tr), isopropylidene group, cyclohexylidene group, more preferably R ₃ , R ₄ and R ₆ are identical and are a benzyl group (Bn),

R ₃ and R ₄ may together form an O.O-acetal group such as an isopropylidene group, cyclohexylidene group, cyclopentylidene group, benzylidene group, mesitylmethylene group, p-methoxybenzylidene group, methylene group, diphenylmethylene group, isopropylidene group, or dispoke group, and

R ₄ and R ₆ may together form an O,O-acetal group such as a benzylidene or a paramethoxybenzylidene group.

To prepare this compound of Formula II, the invention proposes the following methods.

Starting from the compound of Formula I above, the method of the invention for preparing the compound of Formula Il in which X is O, or NH comprises the steps of providing a compound of Formula I in which X is O, or NH, protecting the OH groups of this compound, if they are present, with a protecting group preferably chosen among a ferf-butyldiphenylsilyl group (TBDPS), a benzyl group (Bn), or an acetate group (Ac). Then, the protected compound is epoxidated to obtain a compound of the following Formula 11-1 in which X, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are as in the starting compound of Formula I,

Formula 11-1.

Then an organoacetylenic compound is added to this compound of Formula 11-1 , which enables to obtain the compound of the following Formula II-2.

Formula II-2

Then, the compound of Formula II-2 is partially hydrogenated to obtain the compound of Formula Il in which Re is H. Finally, if desired, Re is introduced after this partial hydrogenation, by methods well known from the man skilled in the art, such as those disclosed in "Protection Groups in Organic Synthesis", John Wiley and Sons, for obtaining the compound of Formula Il in which Rs is different from H.

For example, when R ₈ is a TBDPS group, it can be introduced by using TBDPS Cl and imidazole in dimethyl formamide (DMF).

The method of the invention for preparing the compound of Formula Il in which X is S(O ₂ ) or SO, comprises the steps of preparing the compound of Formula I, X being S in this compound of Formula I, protecting the OH groups of this compound of Formula I, if any, with a tert- butyldiphenylsilyl group, or a benzyl group, or an acetate group, carrying out simultaneously epoxidation and oxidation reactions of the compound of

Formula I with the OH groups protected to obtain compounds of the following Formula 11-1 a and Formula 11-1 b according to the degree of oxidation:

Formula 11-1 a

Formula 11-1 b

Then, an organoacetylenic compound is added to the compounds of Formula 11-1 a or Formula 11-1 b to obtain respectively the compounds of the following Formula ll-2a and Formula ll-2b.

Formula ll-2a

Formula ll-2b

Finally, the compounds of Formula ll-2a and Formula ll-2b are partially hydrogenated to obtain the compound of Formula Il in which X is S(O) or S(O ₂ ).

But another method for preparing a compound of Formula Il in which X may be O, S(O), S(O ₂ ) or NH , comprising the following steps :

(a) providing a compound of Formula I or obtained by the methods described above for obtaining this compound of Formula I,

(b) protecting the OH group, if present, of this compound preferably with a te/t-butyldiphenylsilyl group (TBDPS), benzyl group (Bn), or acetate group (Ac),

(c) epoxidation of the compound obtained in step (b), to obtain a compound of the following Formula 11-1 :

Formula 11-1

(d) opening the compound of Formula 11-1 with a Grignard reactant to obtain the compound of Formula II.

The compound of Formula I enables to obtain α- galactosylceramide analogs having the desired biological activity, i.e.,

monohydroxylated α-galactosylceramide compounds (only one hydroxyl group, protected or not, on position 3 of the sphingosyl chain) as well as dihydroxylated α-galactosylceramide analogs, (having an hydroxyl group on position 3 and on position 4 of the sphingosyl chain, these hydroxyl groups being protected or not) and that, by a particularly short and flexible method of synthesis, which furthermore enables to obtain good yields.

Thus, the monohydroxylated α-galactosylceramide analogs which have, contrarily to the prejudice existing in the art, a biological activity, in particular an immunostimulatory and antitumoral activity as it will be demonstrated hereinafter, are α-galactosylceramide analogs of the following Formula III:

Formula III wherein:

X is O, S, S(O), S(O ₂ ), or NH,

Rn is H or a fatty ester of Formula C _n H _2n +2 with 1<n<15, R-io is a substituted or unsubstituted C ₁ to C ₃₀ alkyl group, substituted or unsubstituted aryl group, or substituted or unsubstituted arylalkyl group, and

R ₉ is CH ₃ or a linear or branched or unsubstituted CrC ₃ O alkyl chain, preferably a C ₃ -C ₇ or C ₁₃ -C _2O alkyl chain which may contain at least one heteroaryl group such as the following groups:

in which R ₁₂ is preferentially H or CH ₃ or a linear or branched C-rC- ₁₀ alkyl chain, or Rg is a linear or branched C ₁ -C ₃₀ alkyl chain containing an heteroatom, such as a chain of the following Formula:

in which:

♦ 0 < q < 10,

♦ 0 < x <30,

♦ 0 < p <30, and

♦ Y is O, S or NH.

A particularly preferred α-galactoceramide compound of the invention is the compound of the following Formula Hl-A:

Another particularly preferred monohydroxylated α-galactoceramide compound of the invention is the compound having the following Formula Hl-B:

Formula Hl-B

Still another particularly preferred monohydroxylated α-galactoceramide compound of the invention is the compound of the following Formula IM-C:

Formula Hl-C

Finally, another particularly preferred monohydroxylated α- galactoceramide compound of the invention is the compound of the following Formula IH-D:

Formula Hl-D.

The method according to the invention for preparing these compounds is a method using the compound of Formula I which is already prepared or which is prepared according to the methods which have been described above. The method of preparing the monohydroxylated α- galactoceramide analogs of the invention having the Formula III comprises the following steps :

(a) providing a compound of Formula I which is already prepared or which is obtained by the above described methods for preparing this compound,

(b) cross-metathesis reaction of this compound of Formula I with a compound of the following Formula 111-1 :

R ₉

Formula 111-1 wherein R ₉ is as defined above,

(c) protection of the alcohol in position 3 of the sphingosyl chain, when R ₅ is H,

(d) deprotection of the amino group,

(e) amidification reaction of the compound obtained in step (c) with a compound of the following Formula 111-2:

Formula 111-2 wherein:

♦ R ₁₀ is the same as defined above, ♦ R ₁₃ is independently H or an activating group such a p- nitrophenol group, /V-hydroxysuccinimide group, acid chloride group, preferably a p-nitrophenol group,

(f) removal of the double liaison and protecting groups, for example by catalytic hydrogenation, for example an hydrogenation with H ₂ /Pd-c,

(g) introduction of R ₁₁ on the galactosyl cycle, for example in presence of DCC (λ/,λ/'-Dicyclohexylcarbodiimide) and '4- Di(methylamino)pyridine) DMAP.

(h) removal of R ₅ and deprotection of the alcohol in position 3 of the sphingosyl chain.

Starting from the compound of Formula I, the invention also proposes a method of preparing α-galactoceramide analogs in which the hydroxyl group in position 3 of the sphingosyl chain is replaced by a cetone group.

This compound has the following Formula IV:

Formula IV wherein:

X iS O ₁ S ₁ S(O) ₁ S(O ₂ ), or NH,

R ₁₁ is H or a fatty ester of Formula C _n H ₂ n+2 with 1 <n<15,

R ₁₀ is a substituted or unsubstituted C ₁ to C ₃₀ alkyl group, substituted or unsubstituted aryl group, or substituted or unsubstituted arylalkyl group,

R ₉ is CH ₃ or a linear or branched or unsubstituted C ₁ -C ₃₀ alkyl chain, preferably a C ₃ -C ₇ or C ₁₃ -C ₂ o alkyl chain, which may contain at least one heteroaryl group such as:

or or

in which Ri ₂ is preferentially H or CH ₃ or a linear or branched C-i-C-io alkyl chain, or R ₉ is a linear or branched C ₁ -C ₃₀ alkyl chain containing an heteroatom, such as a chain of the following Formula:

in which:

♦ 0 < q < 10 ♦ 0< x <30,

♦ 0 < p <30, and

♦ Y is O, S or NH.

The method of preparing these α-galactoceramide analogs of Formula IV comprises the following steps: (a) providing a compound of Formula I or preparing a compound of Formula I by the methods described above,

(b) cross-metathesis reaction of this compound with a compound of the following Formula 111-1 :

R ₉ Formula 111-1 wherein R ₉ is as defined above,

(c) isomerisation of the allylic alcohol into ketone mediated by transition metal complexes

(d) deprotection of the amino group, (e) amidification reaction of this compound with a compound of the following Formula III-2 :

Formula IM-2 wherein:

Rio is the same as defined above, Ri ₃ is independently H or an activating group such a p- nitrophenol group, /V-hydroxysuccinimide group, acid chloride group, preferably a p-nitrophenol group,

(a) removal of the protecting groups. But, as already stated, α-galactoceramide analogs comprising an hydroxyl group both in positions 3 and 4 of the sphingosyl chain, the hydroxyl group being protected or not, can also be prepared by the method of the invention. More precisely, the invention relates to a method of preparing α- galactoceramide analogs having the following Formula V:

Formula V wherein;

X is O, S(O), S(O ₂ ), or NH, Rs and R ₃ are H,

Rn is H or a fatty ester of formula with 1<n<15,

Rio is a substituted or unsubstituted Ci to C ₃₀ alkyl group, substituted or unsubstituted aryl group, or substituted or unsubstituted arylalkyl group,

RECTIFIED SHEET (RULE 91) ISA/EP

R ₉ is CH ₃ or a linear or branched or unsubstituted CrC ₃₀ alkyl chain, preferably C ₃ -C ₇ and C<| ₃ -C ₂ o or which could contain heteroaryl such as

in which R- ₁₂ is preferentially H or CH ₃ or a linear or branched CrC-io alkyl chain, or Rg is a linear or branched CrC _3O alkyl chain containing an heteroatom, such as a chain of the following Formula:

in which:

♦ 0 < q <10,

♦ 0 < x <30,

♦ 0 < p <30, and

♦ Y is O ₁ S or NH, comprising the following steps:

(a) providing a compound of Formula Il or obtained by the methods of the invention,

(b) cross-metathesis reaction of this compound with a compound of the following Formula III-1 :

R ₉

Formula III-1 wherein Rg is as defined above,

(c) protection of alcohols in positions 3 and 4 of the sphingosyl chain when R ₅ and R ₈ are H,

(d) deprotection of the amino group,

(e) amidification reaction of this compound with a compound of the following Formula 111-2:

Formula III-2 wherein: - R _1O is the same as defined above,

R ₁₃ is independently H or an activating group such a p- nitrophenol group, λ/-hydroxysuccinimide group, acid chloride group, preferably a p-nitrophenol group, and

(f) reduction of the double bond and removal of all the protecting groups for obtaining the compound of Formula V in which R ₁₁ is H.

But for obtaining the compound of Formula V in which Rn is different from H, step (f) is replaced by the following steps (f) to (h ¹ ):

(f) reduction of the double bound and removal of the protecting groups of the galactosyl cycle (sugar moiety), only, and (g ¹ ) introduction of R ₁₁ , and

(h') deprotection of alcohols in positions 3 and 4 of the sphingosyl chain.

But the compounds of Formula V may also be prepared starting from the intermediate compound of Formula II. In that case, the method of preparing a compound of Formula

V as defined above comprises the following steps:

(a) providing a compound of Formula Il or preparing a compound of Formula Il by the methods described above,

(b) protecting the OH groups, if present, of this compound with a fe/t-butyldiphenylsilyl group (TBDPS), benzyl group (Bn), or acetate group (Ac),

(c) epoxidation of the compound obtained in step (b), to obtain a compound of the following Formula 11-1 :

Formula 11-1

(d) adding an organoacetylenic compound to the compound of Formula 11-1 to obtain the compound of the following Formula II-2 :

Formula II-2

(e) partial hydrogenation of the compound of Formula II-2,

(f) cross-metathesis reaction of this compound with a compound of the following Formula 111-1 :

R ₉

Formula 111-1 wherein R ₉ is as defined above,

(g) protections of alcohols in positions 3 and 4 of the sphingosyl chain,

(h) deprotection of the amino group,

(i) amidification reaction of this compound with a compound of the following Formula HI-2:

Formula III-2 wherein:

R-io is the same as defined above, - R- _I3 is independently H or activating group such p- nitrophenol group, λ/-hydroxysuccinimide group, acid chloride group, preferably p-nitrophenol group, and

(j) reduction of the double bond and removal of all the protecting groups, for obtaining the compound of Formula V in which Rn is H. But for obtaining the compound of Formula V in which Rn is different from H, step (j) is replaced by the following steps (j ¹ ) to (Y):

(j') reduction of the double bond and removal of the protecting groups on the galactosyl cycle, (sugar moiety), only, and

(k ¹ ) introduction of Rn, (I ¹ ) deprotection of alcohols in positions 3 and 4 of the sphingosyl chain.

But the compounds of Formula V can also be prepared starting from the intermediate compound of Formula II.

In that case, the method of preparing a compound of Formula V as defined above comprises the following steps:

(a) providing a compound of Formula 11 or preparing a compound of Formula Il by the methods described above,

(b) protecting the OH groups, if present, of this compound with a te/t-butyldiphenylsilyl group (TBDPS), benzyl group (Bn), or acetate group (Ac),

(c) epoxidation of the compound obtained in step (b), to obtain a compound of the following Formula 11-1 :

Formula 11-1

(d) opening the compound of Formula 11-1 with a Grignard reactant to obtain the compound of Formula II.

(e) cross-metathesis reaction of this compound with a compound of the following Formula 111-1 :

R ₉

Formula III-1 wherein Rg is as defined above,

(f) protection of alcohols in positions 3 and 4 of the sphingosyl chain,

(g) deprotection of the amino group,

(h) amidification reaction of this compound with a compound of the following Formula III-2:

Formula III-2 wherein:

R-io is the same as defined above R ₁₃ is independently H or activating group such p-nitrophenol group, λ/-hydroxysuccinimide group, acid chloride group, preferably p-nitrophenol group,

(i) reduction of the double bond and removal of all the protecting groups for obtaining the compound of Formula V in which Rn is H.

When the compound of Formula V in which a Rn group different from H is to be introduced, step (i) is replaced by the following steps (D to (k ¹ ):

(i') reduction of the double bond and removal of the protecting groups on the galactosyl cycle (sugar moiety), only, and

(j') introduction of Rn on the galactosyl cycle, and

(k') deprotection of alcohols in positions 3 and 4, if desired.

Obviously, a further object of the invention is the α-galactoceramide analogs having the following Formula V:

Formula V wherein:

X is O, S(O), S(O ₂ ), or NH,

R ₅ and Rs are H,

Rn is H or a fatty ester of Formula C _n H _2n +2 with 1<n<15,

Rio is a substituted or unsubstituted C ₁ to C ₃₀ alkyl group, substituted or unsubstituted aryl group, or substituted or unsubstituted arylalkyl group,

R ₉ is CH ₃ or a linear or branched or unsubstituted Ci-C ₃₀ alkyl chain, preferably C ₃ -C ₇ and C- ₁₃ -C2 ₀ or which could contain heteroaryl such as

H

in which Ri ₂ is preferentially H or CH ₃ or a linear or branched CrC-io alkyl chain, or R ₉ is a linear or branched C ₁ -C ₃₀ alkyl chain containing an heteroatom, such as a chain of the following Formula :

(CH ₂ )X (CH ₂ )P

in which:

♦ 0 < q < 10,

♦ 0 < x < 30, ♦ 0 < p < 30, and

♦ Y is O, S or NH, at the proviso that when X = O, then Rs is not H.

Thanks to the use of the intermediate compounds of Formula I or of Formula II, which permit to prepare, in a very easy and simple manner, numerous analogs of the natural α-galactoceramide or synthetic α-galactoceramide KRN 7000, α-galactoceramides compounds comprising two galatosyl molecules may be prepared.

Thus, a further object of the present invention is a method of preparing α-galactoceramide analogs having the following Formula Vl:

Formula Vl wherein:

- X is O, S, S(O), S(O ₂ ), NH, R ₅ is as defined above,

Rn is H or a fatty ester of Formula C _n H _2n +2 with 1 <n<15, Ri ₄ is a substituted or unsubstituted Ci to C ₃₀ alkyl group, substituted or unsubstituted aryl group, or substituted or unsubstituted arylalkyl group, or a linear or branched Ci-C ₃₀ alkyl chain containing an heteroatom, such as a chain of the following Formula:

in which:

♦ 0 < q < 10,

♦ O < x < 30,

♦ O < p < 30.

Chains of the following Formula

are particularly useful and helpful to introduce cyclic compounds between the amino functionality of the compounds of Formula Vl.

Such compounds having cyclic groups between the two amino functionality of the compounds of Formula Vl are particularly interesting because of their biological potentiality and as ligands for asymmetric catalysis or I cryptand complex. The method of the invention for preparing the compound of

Formula Vl comprises the following steps:

(a) providing a compound of Formula I which is already prepared or which is obtained by the methods of the invention,

(b) cross-metathesis reaction of this compound with itself, (c) protection of alcohol in position 3 of the sphingosyl chain,

(d) deprotection of the amino group,

(e) amidification reaction of this compound with a compound of the following Formula 111-2:

Formula III-2

wherein: - R ₁₀ is the same as defined above,

R- _I3 is independently H or activating group such p-nitrophenol group, λ/-hydroxysuccinimide group, acid chloride group, preferably p-nitrophenol group,

(f) reduction of the double bond and removal of all the protecting groups, for obtaining the compound of Formula Vl in which Rii is H.

When the compound of Formula Vl to be obtained must have Rn groups different from H, step (f) is replaced by the following steps (f ) to (h'):

(f) reduction of the double bond and removal of the protecting groups on the galactosyl cycle (sugar moiety), only, and

(g') introduction of Rn on the sugar moiety, and

(h ¹ ) deprotection of the alcohol in. position 3, of the sphingosyl chain.

A particularly preferred compound falling under the scope of the compound of Formula Vl above is a compound having the following Formula Vl- A:

Formula Vl-A

Compounds having two glycosyl sugars and which are obtained from the compound of Formula Il i.e. comprising a sphingosyl chain with hydroxyl groups both in positions 3 and 4 of the sphingosyl chain are also an object of the invention. They are α-galactoceramide analogs having the following Formula VII:

Formula VII wherein:

RECTIFIED SHEET (RULE 91) ISA/EP

X iS O ₁ S(O) ₁ S(Oa) ₁ Or NH,

R ₅ and R ₈ are H,

Rn is H or a fatty ester of Formula C _n H _2n + ₂ with 1<n<15,

Ri ₄ is a substituted or unsubstituted Ci to C ₃ 0 alkyl group, substituted or unsubstituted aryl group, or substituted or unsubstituted arylalkyl group, or a linear or branched C ₁ -Ca ₀ alkyl chain containing a heteroatom, such as a chain of the following Formula:

in which: ♦ 0 < q < 10,

♦ 0 < x < 30,

♦ 0 < p <30.

When Ri ₄ is a linear or branched Ci-C ₃₀ alkyl chain containing a natural atom, preferably, this Ci-C ₃₀ alkyl chain has the following Formula:

(CH ₂ )X ^' "(CH ₂ )P

in which:

♦ 0 < q < 10,

♦ 0 < x < 30,

♦ 0 < p < 30, which is useful for forming cyclic compounds between the two amino functionality of the sphingosyl chain.

The compounds of Formula VII may be obtained directly from the compound of Formula Il by a method comprising the following steps:

(a) providing a compound of Formula I or preparing this compound by a method of the invention,

(b) cross-metathesis reaction of this compound with itself,

(c) protection of the alcohols in positions 3 and 4 of the sphingosyl chain,

(d) deprotection of the amino group,

(e) amidification reaction of this compound with a compound of the following Formula 111-2:

Formula III-2 wherein: - R-io is the same as defined above,

R ₁₃ is independently H or activating group such p- nitrophenol group, /V-hydroxysuccinimide group, acid chloride group, preferably p-nitrophenol group,

(f) reduction of the double bond and removal of all the protecting groups, for obtaining the compound of Formula VII in which Rn is H.

For obtaining the compound of Formula VII in which R ₁₁ is different from H, step (f) is replaced by the following steps (f ) to (h'):

(f) reduction of the double bond and removal of the protecting groups on the galactosyl cycle (sugar moiety), only,

(g') introduction of R-n on the sugar moiety _, (h ¹ ) deprotection of the alcohols in position 3. In all the methods of manufacturing of the invention, the removal of the protecting groups is carried out according to well known methods, such as those described in "Protection Groups in Organic Synthesis" John Wiley & Sons.

For example, for removing benzyl groups, the compound is stirred at room temperature in a solvent not participating to the debenzylation reaction, such as methanol, ethanol, 2-propanol, ethylacetate, tetrahydrofuran, dimethylformamide, in presence of a catalyst such as Pd-C, Pd (OH) ₂ , Ptθ ₂ , etc.

But they can be also obtained starting from the compound of Formula Il as it clearly appears to the man skilled of the art.

The compounds of Formula III, IV, V, Vl and VII have biological activities in human models, rendering them particularly useful as active ingredients of a pharmaceutical composition. Therefore, an other object of the invention is a pharmaceutical composition comprising at least one compound of formulae III-VII and a pharmaceutical acceptable carrier.

In the same manner, a further object of the present invention is a pharmaceutical composition containing at least one compound obtained by a process according to the invention for manufacturing the compounds of formulae III- VII and a pharmaceutical acceptable carrier.

The invention will be better understood and other characteristics and advantages thereof will be more clearly apparent when reading the following description which refers to the annexed figures in which: - Figure 1 shows the productions of IL-4 by: a) NKT of MAD11 , a polyclonal population of NKTi, alone, b) NKT of MAD11 after incubation with HeLa-CDId cells, c) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 0.1 μg/ml of the synthetic reference α-galactosylceramide KRN7000, d) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 0.1 ug/ml of the compound of Formula III- A of the invention, e) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 1 μg/ml of the compound of Formula IN-A of the invention,

Figure 2 shows the production of IFN-γ by: a) NKT of MAD11 , a polyclonal population of NKTi, alone,

b) NKT of MAD11 after incubation with HeLa-CDId cells, c) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 0.1 μg/ml of the synthetic reference α-galactosylceramide KRN7000, d) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 0.1 μg/ml of the compound of Formula III- A of the invention, e) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 1 μg/ml of the compound of Formula IN-A of the invention, Figure 3 shows the production of TNF-α by: a) NKT of MAD11 , a polyclonal population of NKTi, alone, b) NKT of MAD11 after incubation with HeLa-CDI d cells, c) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 0.1 μg/ml of the synthetic reference α-galactosylceramide KRN7000, d) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 0.1 μg/ml of the compound of Formula IM-A of the invention, e) NKT of MAD11 after incubation with HeLa-CDId cells loaded with 1 μg/ml of the compound of Formula Hl-A of the invention.

Of course, the examples which are given below are in no way invention to the particular embodiments they describe.

Material and methods

Geηeral _. Methpds

Water-sensible reactions were performed under an argon atmosphere in flame-dried glassware. All solvents were reagent grade. THF was freshly distilled from sodium/benzophenone under argon. Et ₂ O was freshly distilled from sodium under argon. MeOH and DCM were freshly distilled from calcium hydride under argon. DMF was distilled under argon prior to use.

Mejting _. p _. olnt

Melting points were determined on a RCH (C. Reichert) microscope equipped with a Koffer heating system.

Chromatography All reactions were monitored by thin layer chromatography

(Kieselgel 60F ₂ 5 ₄ MERCK aluminium sheet).

Flash columm chromatography was performed on silica gel 60 ACC 40-63 μm (Carbo-erba reactifs - SDS).

QptlcaJ _.. rptatj _. on _.. measurements

Optical rotation values were measured in a 100 mm cell on Perkin Elmer 341 polarimeter under Na lamp radiation.

Infra-red spectroscopy IR spectra were recorded with a BRUCKNER Vector 22 spectrometer. The wave numbers are given in cm ^"1 .

Nuclear magnetic resonance _. spectroscopy

NMR spectra were recorded on a BRUCKNER Avance 300 at 300 MHz ( ¹ H) and 75 MHz ( ¹³ C) using the residual solvent as internal standard. The coupling constants are expressed in Hertz. The multiplicity of the signals are abbreviated as : s (singulet), d (doublet), t (triplet), q ( quadru-

plet), m (mu)tiplet), bs (broad singulet), dd (doublet of doublet), dt (doublet of triplet)...

Example 1 : Synthesis of the compound of formula I:

1) Synthesis of 2, 3, 4, 6-tetra-O-benzyl-α-D- galactopyranosyl fluoride of Formula 1-1

Formula 1-1 Synthesis of 1 , 2, 3, 4, 6-penta-O-acetylqalactopyranosyl 7 of formula I-3 To D-galactose (20 g, 111 mmol, 1 eq) dissolved in a mixture of dry CH2CI ₂ and pyridine (60 mL/100 ml_) at 0 ^c C under argon were added 4- dimethylaminopyridine (1.34 g, 11 mmol, 0.1 eq) and dropwise acetic anhydride (59.8 mL, 632.7 mmol, 5.7 eq). The mixture was heated to reflux for 24 h. CH ₂ CI ₂ was evaporated and the crude was diluted with CHCI ₃ (200 mL) and washed with saturated aqueous NaHCO ₃ solution. The organic layer was dried over MgSO ₄ and concentrated. The crude product (39.02 g, 90%) was engaged in the next step.

C ₁₆ H ₂₂ O _{1 1} MoI. Wt.: 390,3393 Synthesis of phenyl 2, 3, 4, 6-tetra-O-acetyl-i-thio-β-D-qalactopyranosyl 8

To the crude 7, dissolved in benzene (430 mL) under argon at room temperature, were added thiophenol (20.4 mL, 200 mmol, 2 eq) and tin tetrachloride (10.6 mL, 90 mmol, 0.9 eq). The solution was heated to reflux for

2 h. The brown mixture was neutralized with saturated aqueous NH ₄ CI solution (300 mL) and diluted with CH ₂ CI ₂ (150 ml_). The aqueous layer was extracted with CH ₂ CI ₂ (3 x 100 mL). The combined organic layers were dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 60/40) afforded 8 as a yellow oil (34.45 g, 78%).

C _2O H ₂₄ O ₉ S MoI. Wt.: 440,4642

¹ H NMR (300 MHz, CPCh) 7.50 (m, 2H), 7.30 (m, 3H), 5.42 (d, J = 3.3 Hz, 1 H), 5.25 (d, J = 10.2, 9.6 Hz, 1 H), 5.05 (dd, J = 10.2, 3.3 Hz, 1 H), 4.70 (d, J = 9.6 Hz, 1 H), 4.20 (dd, J = 11.3, 7.1 Hz, 1H), 4.12 (dd, J = 11.3, 7.1 Hz, 1H), 3.95 (dd, J = 7.1 , 7.1 Hz, 1 H), 2.20-2.00 (4s, 12H.)

¹³ C NMR (75 MHz, CDCh) δ 170.1 , 132.6, 129.0, 128.2, 86.7, 74.5, 72.1 , 67.3, 61.7, 20.7.

Synthesis of phenyl 1-thio-β-D-qalactopyranosyl 9 To 8 (31.71 g, 72.07 mmol, 1 eq) dissolved in dry MeOH (800 mL) under argon was added sodium methanolate (17.3 g, 302.7 mmol, 4.2 eq). After being stirred for 1 h, Amberlite IR 120 (300 g) was added and the mixture was stirred for 15 min up to pH = 7. The solution was filtered through alumina. The cake was washed with MeOH (150 mL) and the organic layer was concentrated. The crude was engaged in the next step.

Synthesis of phenyl 2,3A6-tetra-0-benzyl-1-thio-β-D-galactopyranosyl 10

To the crude 9 (72.07 mmol, 1 eq) dissolved in dry DMF (1500 ml_) under argon at 0 ⁰ C was added sodium hydride (10.72 g, 446.8 mmol, 6.2 eq). After 15 min, were added dropwise benzyl bromide (54 mL, 454 mmol, 6.3 eq) and a catalytic amount of potassium iodide. The mixture was stirred at room temperature for 3 h, diluted with water (500 mL) and Et ₂ O (600 mL). The aqueous phase was extracted with Et ₂ O (3 * 300 mL). The organic extracts were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 90/10) afforded 10 as viscous syrup (27.48 g, 60% over 2 steps).

C ₄₀ H ₄₀ O ₅ S MoL Wt.: 632,8076

¹ H NMR (300 MHz, CDCh) 7.60-7.10 (m, 25H), 4.97 and 4.60 (2d, J = 11.5 Hz, 2H), 4.78 and 4.72 (2d, J = 10.2 Hz, 2H), 4.72 (s, 2H), 4.65 (d, J = 9.6 Hz, 1H), 4.47 and 4.41 (2d, J = 11.6 Hz, 2H), 3.98 (d, J = 2.4 Hz), 3.93 (t, J = 9.6 Hz, 1 H), 3.68-3.53 (m, 4H).

¹³ C NMR (75 MHz, CPCh) δ 138.2. 134.0, 131.3-128.8, 87.5, 84.0, 77.3, 76.5, 75.5, 74.5, 73.4, 72.8, 68.6.

Synthesis of 2, 3, 4. 6-tetra-O-benzyl-α-D-qalactopyranosyl fluoride 11

To a solution of 10 (10 g, 15.82 mmol, 1 eq) in dry CH ₂ CI ₂ (191 mL) under argon at -15°C were added diethylaminosulfur trifluoride (3.14 mL, 23.73 mmol, 1.5 eq) and, after 2 min, λ/-bromosuccinimide (3.66 g, 20.57 mmol, 1.3 eq). After being stirred at -15°C for 30 min the reaction was diluted with CH ₂ CI ₂ (420 mL) and poured into a cold saturated aqueous NaHCO ₃ solution (195 mL). The organic layer was dried over MgSO ₄ and concentrated.

Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 94/6) afforded 11 as viscous syrup (6.29 g, 73%).

C ₃₄ H ₃₅ FO ₅ MoI. Wt.: 542,6371

¹ H NMR (300 MHz, CDCI-O 7.39-7.21 (m, 20H), 5.59 (dd, J = 54.O ₁ 2.7 Hz, 1 H), 4.93 and 4.56 (2d, J = 11.4 Hz, 2H), 4.84 and 4.74 (2d, J = 11.7 Hz, 2H), 4.82 and 4.71 (2d, J = 11.8 Hz, 2H), 4.47 and 4.40 (2d, J = 11.8 Hz, 2H), 4.10 (t, J = 6.6 Hz, 1 H), 4.02 (ddd, J = 24.6, 9.3, 2.7 Hz, 1H), 3.99 (d, J = 2.7 Hz ₁ 1 H), 3.96 (dd, J = 9.3, 2.7 Hz, 1 H), 3.54 (d, J = 6.6 Hz, 2H). ¹³ C NMR (75 MHz, CDCh) δ 138.4-137.8, 128.5-127.6, 106.3 (d, J = 224 Hz), 78.6, 75.9 (d, J = 23.3 Hz), 75.0, 74.4, 73.8, 73.6, 73.2, 71.9, 68.4.

2) Synthesis of (2S, 3R)-2-(tert- butyloxycarbonylamino)-3-O-(tert-butyldiphenylsiIyl)-pent-4- ene-1-ol of Formula I-2

.Boc

HN ^'

OTBDPS

Formula 1-2 ethyl πα,23,5α1-1-r(2-(2R)-hvdroxy-2,6,6,-trimethylbicvclor3.1 ,nhept-3- ylidene)aminoT ethanoate 1

Glycinethylester hydrochochlorid (16.59 g, 118.88 mmol, 2 eq) was dissolved in 93 ml_ of benzene and neutralized by ammoniac gas for 15 min. Ammonium salts were eliminated by filtration and the solution was added on (+)-(1 R,2R,5R)-2-hydroxy-3-pinanone (10 g, 59.44 mmol, 1 eq). A catalytic amount of boron trifluoride-diethyl etherate was added and the resulting solution was heated to reflux in a Dean-Stark apparatus for 4 h. Without treatment, benzene was evaporated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 50/50) afforded imine 1 as a yellow oil (14.19 g, 94%).

C _]4 H ₂₃ NO ₃ MoI. Wt: 253,3373

¹ H NMR (300 MHz. CDCh) δ 4.21 (q, J = 7.1 Hz, 2H), 4.15 (t, J = 2.0 Hz, 1 H), 4.14 (t, J = 2.0 Hz, 1 H), 2.77 (bs, 1 H), 2.46 (s, 2H), 2.32 (ddt, J = 10.7, 6.1 ,

2.0 Hz, 1 H), 2,11-2.00 (m, 2H), 1.56 (d, J = 10.7 Hz, 1 H), 1.49 (s, 3H), 1.32 (s, 3H), 1.27 (cl, J = 7.1 Hz, 3H), 0.88 (s, 3H).

¹³ C NMR (75 MHz. CDCh) δ 180.0, 170.2, 76.5, 60.9, 52.6, 50.4, 38.6, 38.3, 33.6, 28.2, 28.1 , 27.3, 22.8, 14.2.

Aldolisation 2

To a solution of iminoglycinate 1 (14.19 g, 56.01 mmol, 1.3 eq) dissolved in dry CH ₂ CI ₂ (26 ml_) at 0 ⁰ C under argon was added chlorotita- nium triisopropoxide (14.60 g, 56.01 mmol, 1.3 eq) in dry CH ₂ CI ₂ (42 ml_). The addition of acrolein (2.96 ml_, 43.09 mmol, 1 eq) dissolved in dry CH ₂ CI ₂ (21 ml_) causes a color change from yellow to orange. Finally, triethylamine (13.2 ml_, 94.80 mmol, 2.2 eq) was added and a precipitate appears. The reaction was stirred at 0 ⁰ C for 4 h and diluted with addition of brine (300 ml_). The mixture was diluted with EtOAc (600 mL) and water (500 ml_) and filtered through Celite. The cake was washed with EtOAc (2 x 100 mL). The aqueous solution was extracted with EtOAc (2 * 100 mL). The combined organic extracts were dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 50/50) afforded a mixture of ethyl and isopropyl esters in a ratio of 25/75 as an oil (8.42 g, 60%).

Isopropyl (1 R-Hα,2β,3(2R,3R),5α1)-3-hvdroxy-2-r(2-hvdroxy-2,6,6,- trimethlbi- cvclo[3,1 ,11hept-3-ylidene)amino]-4-pentanoate.

C ₁₈ H ₂₉ NO ₄ MoI. Wt.: 323,4272

¹ H NMR (300 MHz, CDCU) δ 5.94 (ddd, J = 17.2, 10.7, 5.6 Hz, 1 H), 5.35 (d, J = 17.2 Hz, 1 H), 5.18 (d, J = 10.7 Hz, 1 H) ₁ 5.05 (hept, J = 6.3 Hz, 1H), 4.60 (t, J

= 6.2 Hz, 1 H), 4.26-4.10 (m, 2H), 3.25 (bs, 1 H), 3.01 (bs, 1 H), 2.52 (m, 1 H), 2.31 (m, 1 H), 2.09-2.01 (m, 2H), 1.58 (d, J = 11.0 Hz, 1H), 1.50 (s, 3H), 1.31 (s, 3H), 1.26 (d, J = 6.3 Hz, 3H), 1.24 (d, J = 6.3 Hz, 3H), 0.87 (s, 3H). ¹³ C NMR (75 MHz, CPCh) δ 180.5, 169.5, 136.6, 116.9, 76.8, 73.7, 68.5, 67.0, 50.4, 38.6, 38.5, 34.1 , 28.2, 28.0, 27.3, 22.8, 21.8, 21.7.

Ethyl (1 R-riα,2β,3(2R,3R),5α]}-3-hvdroxy-2-r(2-hvdroxy-2,6,6,- trimethlbicv- clo[3,1 ,1]hept-3-ylidene)amino1-4-pentanoate.

C ₁₇ H ₂₇ NO ₄ MoI. Wt: 309,4006

¹ H NMR (300 MHz. CDCh) δ 5.94 (ddd, J = 17.2, 10.7, 5.6 Hz, 1 H), 5.35 (d, J = 17.2 Hz, 1H), 5.18 (d, J = 10.7 Hz, 1 H), 4.60 (t, J = 6.2 Hz, 1 H), 4.26-4.10 (m, 4H), 3.25 (bs, 1H), 3.01 (bs, 1 H), 2.52 (m, 1 H), 2.31 (m, 1 H), 2.09-2.01 (m, 2H), 1.58 (d, J = 11.0 Hz, 1 H), 1.50 (s, 3H), 1.31 (s, 3H), 1.25 (d, J = 6.3 Hz, 3H), 0.87 (s, 3H).

¹³ C NMR (75 MHz. CPCi ₃ ) δ 180.5, 169.5, 136.6, 116.9, 76.8, 73.7, 61.0, 67.0, 50.4, 38.6, 38.5, 34.1 , 28.2, 28.0, 27.3, 22.8, 14.2.

Preparation of compound 5

Acid hydrolysis of imines 3

The imines 2 (8.42 g, 26.03 mmol, 1 eq) were dissolved in

THF (46 ml_) and 1.0 M aqueous HCI solution (182 mL, 182 mmol, 7 eq) was added. The mixture was stirred for 3 days at room temperature. THF and water were partially evaporated and the crude was engaged directly in the next step.

C ₇ H ₁₃ NO ₃ C ₈ H _{] 5} NO ₃ MoL Wt: 159,183 MoI. Wt: 173,2096

Protection of amine by a Boc group 4

The amines 3 (26.03 mmol) were dissolved in DMF (93 ml_) at O ⁰ C under argon followed by the addition of triethylamine (57.9 mL, 416.5 mmol, 16 eq) and di-te/f-butyldicarbonate (11.93 g, 54.66 mmol, 2.1 eq) dissolved in DMF (93 mL). The mixture was stirred at room temperature for 24 h before addition of water (450 mL) and dilution with Et ₂ θ (250 mL). The aqueous phase was extracted with Et ₂ O (3 * 100 mL). The organic layers were combined, dried over MgSO ₄ and concentrated. The crude product was engaged in the next step.

BocHN

C ₁₂ H ₂₁ NO ₅ C ₁₃ H ₂₃ NO ₅ MoI. Wt.: 259,2988 MoI. Wt.: 273,3254

Protection of alcohol by a silyl group 5

The esters 4 (26.03 mmol) were dissolved in DMF (73 mL) at room temperature under argon followed by the addition of imidazole (4.43 g, 65.08 mmol, 2.5 eq) and ferf-butyldiphenylsilyl chloride (8.59 g, 31.24 mmol, 1.2 eq). The mixture was stirred at room temperature for 24 h before addition of satured aqueous NaHCO ₃ solution (250 mL) and dilution with Et ₂ O (250 mL). The aqueous phase was extracted with Et ₂ O (2 * 125 mL). The organic extracts were combined, dried over MgSO ₄ , filtered through silica to eliminate excess of DMF and concentrated. Purification by flash chromatography on

silica gel (petroleum ether/EtOAc: 95/5) afforded a mixture of silyl by-products and 5 (11.15 g).

BocHN

C ₂₈ H ₃₉ NO ₅ Si C ₂₉ H ₄₁ NO ₅ Si MoI. Wt.: 497,6985 MoI. Wt.: 511,725

Reduction of ester group 6

(2S,3R)-2-(te/t-butyloxycarbonylamino)-3-0-(te/t-butyldip henylsilyl)-pent-4- ene-1-ol

The previous crude mixture was dissolved in dry Et ₂ O (128 ml_) at O ⁰ C under argon. To this solution were added distilled methanol (2.22 ml_, 54.65 mmol, 7 eq) and lithium borohydride 2M in THF (27 ml_, 54.65 mmol, 7 equiv). The reaction mixture was allowed to warm to room temperature over 24 h and diluted with addition of satured aqueous NH ₄ C) solution (200 ml_) and diluted with EtOAc (300 mL). The aqueous phase was extracted with EtOAc (3 * 100 mL). The organic extracts were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 80/20) afforded 6 as a syrup (3.53 g, 30% over 4 steps).

. Boc

HN ^'

OTBDPS C ₂₆ H ₃₇ NO ₄ Si MoI. Wt: 455,6618

Formula 1-2

Igβa +6.6 (c 1.0, CHCI ₃ ).

JR 3409, 3072, 3050, 2961 , 2931 , 2896, 2857, 2362, 1694, 1504, 1472, 1456,

1428, 1392, 1366, 1248, 1171 , 1112 cm ^'1 .

¹ H NMR (300 MHz, CDCU) 7.68-7.61 (m, 4H), 7.45-7.34 (m, 6H), 5.80 (ddd, J = 17.1 , 10.2, 6.6 Hz, 1 H), 5.05 (m, 3H), 4.41 (bs, 1 H), 3.86 (dd, J = 7.3, 3.0 Hz, 1 H), 3.63-3.56 (m, 2H), 2.65 (bs, 1 H), 1.40 (s, 9H), 1.08 (s, 9H). ¹³ C NMR (75 MHz, CDCI ₃ ) δ 156.3, 137.0, 136.1 , 136.0, 133.2, 130.2, 130.0, 127.9, 127.7, 117.5, 79.6, 76.6, 62.8, 56.5, 28.5, 27.2, 19.5.

3) Glycosidic coupling of the compound of formula 1-1 with the compounds of formula I-2 obtained in step 2 above

(2S.3RV2-(fe/t-butyloxycarbonylaminoV3-Q-(ferf-butyldiphe nylsilylV1-(2.3,4.6- tetra-O-benzyl-α-D-qalactopyranosyl)pent-4-ene 12

Molecular sieves (30 g) was stirred for 2 h under vacuum at 600 ⁰ C and placed under argon at 0 ⁰ C. Tin chloride (4.41 g, 23.24 mmol, 3 eq) and silver perchlorate (5.25 g, 23.24 mmol, 3 eq) were dissolved in dry THF (54 ml_). The mixture was stirred for 2.5 h in the dark at 0 ⁰ C under argon. To a solution of sphingosine 6 (3.53 g, 7.75 mmol, 1 eq) dissolved in dry Et ₂ θ (84 ml_) was added the fluorosugar 11 (6.29 g, 11.61 mmol, 1.5 eq) dissolved in dry Et ₂ O (99 ml_). The mixture was added through a cannula into the lewis acid solution. The resulting solution was stirred at 0 ⁰ C for 20 min. The mixture was filtered through Celite and the cake washed with EtOAc (100 ml_). The organic layer was washed with a saturated aqueous NaHCO ₃ solution (3 * 100 ml_), dried over MgSO ₄ and concentrated. Purification by flash chroma- tography on silica gel (petroleum ether/EtOAc: 92/8) afforded 12 as a syrup (2.51 g, 33%).

C ₆₀ H ₇₁ NO ₉ Si MoI. Wt.: 978,2925

25

M D +26.9 (c 1.2, CHCU). IR : 3414, 2930, 2857, 2360, 1715, 1502, 1454, 1428, 1391 , 1365, 1159, 1 103, 1053 cm ^"1 .

¹ H NMR (300 MHz, CDCI _? ) 7.67-7.63 (m, 4H), 7.40-7.26 (m, 26H), 5.81 (ddd, J= 17.1 , 10.4, 6.7 Hz, 1 H), 4.98-4.89 (m, 4H), 4.81 and 4.71 (2d, J = 11.7 Hz, 2H), 4.79 (d, J = 3.6 Hz, 1 H), 4.75 and 4.60 (2d, J = 11.9 Hz, 2H), 4.56 (d, J = 11.4 Hz, 1 H), 4.46 (d, J = 11.8 Hz, 1 H), 4.38-4.35 (m, 2H), 4.01 (dd, J = 10.0, 3.6 Hz, 1 H), 3.88-3.77 (m, 5H), 3.67 (dd, J = 10.8, 4.4 Hz ₁ 1 H), 3.45 (dd, J = 9.3, 9.2 Hz, 1 H), 3.43 (dd, J = 9.3, 9.1 Hz, 1 H), 1.39 (s, 9H), 1.07 (s, 9H). ¹³ C NMR (75 MHz. CDCh) δ 155.6, 138.9-138.0, 137.2, 136.0, 134.0, 133.6, 129.7-127.4, 117.3, 98.9, 78.9, 78.6, 76.6, 75.0, 74.7, 73.4, 73.1, 72.9, 69.6, 69.0, 68.3, 55.4, 28.4, 27.1 , 19.5.

(2S,3R)-2-(terf-butv[oxycarbonylamino)-3-hvdroxy-1-(2,3,4 ,6-tetra-0-benzyl-α- D-qalactopyranosyl)pent-4-ene 13

To 12 (2.51 g, 2.56 mmol, 1 eq) dissolved in THF (11 mL) at room temperature was added tetrabutylammonium fluoride trihydrate (2.02 g, 6.41 mmol, 2.5 eq). The mixture was stirred at room temperature for 16 h and diluted with saturated aqueous NaHCO ₃ solution (30 mL). The aqueous layer was extracted with EtOAc (3 * 100 mL). The organic layers were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on

silica gel (petroleum ether/EtOAc: 65/35) afforded 13 as white solid (1.62 g, 85%).

C ₄₄ H ₅₃ NO ₉ MoI. Wt.: 739,8929

Formula I

M ²⁵ D +46.7 (c 1.0, CHCI ₃ ). mp 84-85°C. !R : 3486, 3391 , 3030, 2929, 1705, 1500, 1455, 1392, 1367, 1353, 1237,

1162, 1096, 1058, 1026 cm ^"1 .

¹ H NMR (300 MHz, CDCh) 7.39-7.28 (m, 20H), 5.88 (ddd, J = 17.0, 10.8, 6.3

Hz, 1 H), 5.48 (d, J = 8.4 Hz, 1 H), 5.28 (d, J = 17.0 Hz, 1H), 5.18 (d, J = 10.8

Hz, 1H), 4.94 and 4.58 (2d, J = 11.3 Hz, 2H), 4.87 and 4.69 (2d, J = 11.7 Hz, 2H), 4.79-4.74 (m, 3H), 4.49 and 4.40 (2d, J = 11.3 Hz ₁ 2H), 4.27 (m, 1 H),

4.05 (dd, J = 10.2, 3.6 Hz, 1 H), 4.02-3.85 (m, 4H), 3.77 (m, 1 H), 3.69-3.48 (m,

4H), 1.48 (s, 9H).

¹³ C NMR (75 MHz, CDCh) δ 155.7, 138.6-138.4, 138.0, 137.8, 128.4-127.5,

115.7, 98.9, 79.6, 79.3, 75.7, 74.8, 75.5, 74.1 , 73.6, 72.8, 69.7, 69.2, 68.3, 53.2, 28.4.

Example 2: Synthesis of the compound of formula IH-A

Formula Ml-A

(2S.3R)-2-(terf-butyloxycarbonylamino)-3-hvdroxy-1-(2,3,4 ,6-tetra-O-benzyl-α-

D-galactopyranosyl)heptadec-4-ene 14

To 13 (compound of formula I) (450 mg, 0.608 mmol, 1 eq) dissolved in dry CH ₂ CI ₂ (4.6 ml_) at room temperature under argon were added tetradecene (1.56 mL, 6.08 mmol, 10 eq) and Grubbs Il catalyst (25.8 mg, 0.030 mmol, 0.05 eq). The mixture was heated to reflux for 17 h. Tetradecene (1.56 ml, 6.08 mmol, 10 eq) and Grubbs Il (25.8 mg, 0.030 mmol, 0.05 eq) were added and the solution continued to stir for 7 h. Without treatment, CH ₂ CI ₂ was evaporated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 85/15) afforded 14 as an oil (249.8 mg, 45%).

C ₅₆ H ₇₇ NO ₉ MoI. Wt: 908,2119

TaI ²⁵ D +31.4 (c 0.7, CHCh).

IR : 3435, 3064, 3031, 2925, 2855, 1711, 1497, 1454, 1392, 1366, 1245, 1209, 1164, 1136, 1100, 1057, 1028 cm ^"1 .

¹ H NMR (300 MHz. CDCh) 7.37-7.28 (m, 20H), 5.66 (dt, J = 15.4, 6.8 Hz, 1H), 5.45 (m, 2H), 4.96 and 4.58 (2d, J = 11.4 Hz, 2H), 4.87 and 4.73 (2d, J = 11.8 Hz, 2H), 4.83-4.75 (rn, 3H), 4.50 and 4.41 (2d, J = 11.8 Hz, 2H), 4.20 (m, 1H), 4.07 (dd, J = 10.0, 3.6 Hz, 1H), 4.03 (m, 1H), 3.95-3.86 (m, 3H), 3.70 (m, 2H), 3.60-3.49 (m, 3H), 2.01 (m, 2H), 1.48 (s, 9H), 1.29 (s, 20H), 0.91 (t, J = 6.7 Hz, 3H). ¹³ C NMR (75 MHz, CDCh) δ 155.7, 138.6-137.8, 132.8, 129.5, 128.4-127.5, 98.9, 79.5, 79.3, 75.7, 74.8, 74.5, 74.2, 74.0, 73.6, 72.8, 69.7, 69.3, 68.6, 53.6, 32.4, 31.9, 29.7-29.1, 28.4, 22.7, 14.2.

Deprotection of BOC 15 14 (130 mg, 0.143 mmol, 1 eq) was dissolved in dry THF (24 ml_) and HCI gas bubbled up to total consumption of starting material on TLC. Without treatment, THF was evaporated and the crude was engaged in the next step.

Preparation of 4-p-nitrophenyl hexacosanoate 16

To hexacosanoic acid (200 mg, 0.50 mmol, 1 eq) dissolved in CH ₂ CI ₂ (6 mL) under argon at room temperature were added p-nitrophenol (70 mg, 0.50 mmol, 1 eq), DCC (104 mg, 0.50 mmol, 1 eq) and a catalytic amount of 4-dimethylaminopyridine (6.1 mg, 0.05 mmol, 0.1 eq). The mixture was stirred for 16 h in the dark. The reaction was filtered through silica gel and the filtrate was concentrated. Purification by flash chromatography on silica gel (petroleum ether/CH ₂ CI ₂ : 75/25) afforded 16 as white solid (215 mg, 83%).

C ₃₂ H ₅₅ NO ₄ MoI. Wt: 517,7834

¹ H NMR (300 MHz. CDCk) 8.37 (d, J = 9.1 Hz ₁ 2H), 7.27 (d, J = 9.3 Hz, 2H), 2.60 (d, J = 7.5 Hz, 2H), 1.76-1.09 (m, 46H) ₁ 0.89 (t, J = 6.5 Hz, 3H). ¹³ C NMR (75 MHz, CPCI ₃ ) δ 171.3, 155.7, 125.2, 122.4, 34.4, 31.9, 29.7, 29.4, 29.2, 29.1 , 24.8, 22.7, 14.1.

(2S.3R)-2-(λ/-hexacosanoylamino)-3-hvdroxy-1-(2,3,4,6-te tra-O-benzyl-α-D- galactopyranosyl)heptadec-4-ene 17

To the chlorhydrate 15 (0.143 mmol, 1 eq) dissolved in THF (5.7 ml_) under argon at room temperature were added 16 (73.9 mg, 0.143 mmol, 1 eq), triethylamine (24 μL, 0.172 mmol, 1.2 eq) and a catalytic amount of 4-dimethylaminopyridine. The mixture was heated to reflux for 16 h and diluted with saturated aqueous NaHCO ₃ solution (10 ml_). The aqueous layer was extracted with Et ₂ O (2 * 15 ml_). The organic layers were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 86/14) afforded 17 as white oil (74.7 mg, 44% over 2 steps).

C ₇₇ H _{1 19} NO ₈ MoI. Wt: 1186,7707

Igfij +27.5 (c 1.2, CHCI ₃ ) mp 80-81 °C.

IR : 3324, 2919, 2850, 1639, 1546, 1497, 1471 , 1350, 1103, 1046 cm ^"1 .

¹ H NMR (300 MHz. CDCI ₃ ) 7.37-7.24 (m, 20H), 6.45 (d, J = 8.1 Hz, 1 H), 5.65

(dt, J = 15.6, 6.9 Hz ₁ 1 H), 5.41 (dd, J = 15.6, 5.4 Hz, 1 H), 4.91 and 4.55 (2d, J

= 11.4 Hz ₁ 2H), 4.87 and 4.70 (2d, J = 11.7 Hz, 2H), 4.75 (m, 3H), 4.47 and 4.37 (2d, J = 11.4 Hz, 2H) ₁ 4.14 (m, 1 H), 4.03 (dd, J = 10.2, 3.6 Hz, 1 H), 4.01 (m, 2H), 3.89-3.82 (m, 4H), 3.69 (dd, J = 10.2, 3.3 Hz, 1 H), 3.50 (m, 2H), 2.12 (t, J = 7.5 Hz, 2H), 1.98 (m, 2H), 1.60-1.10 (m, 66H), 0.88 (t, J = 6.3 Hz, 6H). ¹³ C NMR (75 MHz. CDChi δ 173.4, 138.4-137.6, 133.0, 129.1 , 128.4-127-5, 99.1, 79.2, 75.8, 74.8, 74.4, 74.2, 74.0, 73.6, 72.7, 69.8, 69.1 , 68.7, 52.8, 36.7, 32.4, 32.0, 29.7, 29.4, 25.8, 22.4, 14.2.

(2S,3R)-1 -(α-D-galactopyranosvD^-hexacosanoylaminohθptadecan^-ol 18 compound of formula Hl-A

To 17 (64.2 mg, 0.054 mmol, 1 eq) dissolved in MeOH (4.7 ml_) and THF (2.3 ml_) at room temperature was added palladium (10%) on activated carbon (64.2 mg) in one portion. The mixture was stirred under H2. After 3.5 days, the mixture was filtered through Celite and filter cake washed with a combination of MeOH and CHCI ₃ . The filtrate was concentrated and the residue was purified on silica gel (CHCI ₃ /MeOH: 100/0 to 95/5) to provide a white solid (30.1 mg, 67%).

C ₄₉ H ₉₇ NO ₈ MoI. Wt.: 828,2964

Formula Hl-A

M ²⁵ Q +32.7 (c 1.0, Pyridine) mp 170-171 °C.

JR : 3267, 2919, 2850, 1647, 1550, 1469, 1261, 1096 cm ^"1 .

¹ H NMR (300 MHz, CDCI ₃ ) 8.56 (d, J = 8.7 Hz, 1 H) ₁ 5.46 (d, J = 3.9 Hz, 1 H),

5.10 (bs, 5H) ₁ 4.74 (m, 1 H), 4.65 (dd, J = 9.9, 3.9 Hz, 1 H) ₁ 4.57-4.29 (m, 8H),

2.48 (t, J = 7.2 Hz, 2H), 1.95-1.82 (m, 6H), 1.26 (s, 66H), 0.87 (t, J = 6.3 Hz,

6H).

¹³ C NMR (75 MHz, CDCI ₃ ) δ 173.4, 102.1 , 73.1 , 71.9, 71.6, 71.0, 70.5, 69.6,

62.7, 54.9, 36.8, 35.1 , 32.1 , 30.0, 29.6, 26.6, 26.4, 22.9, 14.3.

Example 3: Synthesis of the compound of Formula Hl-B

Formula Hl-B

(2S.3R)-2-(teAt-butyloxycarbonylamino)-3-hvdroxy-1-(2,3,4 .6-tetra-O-benzyl-α- D-qalactopyranosyl)non-4-ene 19

To 13 (300 mg, 0.405 mmol, 1 eq) dissolved in dry CH ₂ CI ₂ (4 ml_) at room temperature under argon were added hex-1-ene (502 μl, 4.05 mmol, 10 eq) and Grubbs Il (17.2 mg, 0.020 mmol, 0.05 eq). The mixture was heated to reflux for 24 h. Without treatment, CH ₂ CI ₂ was evaporated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 80/20) afforded 19 as a white solid (240.2 mg, 75%).

C ₄₈ H ₆₁ NO ₉ MoI. Wt.: 795,9992 rαf ⁵ n +39.6 (c 0.9, CHCI ₃ ). mp 57-58°C.

IR : 3855, 3448, 3064, 3031, 2927, 1715, 1497, 1455, 1367, 1243, 1166,

1100, 1058 cm ^"1 .

¹ H NMR (300 MHz. CDCh) 7.38-7.25 (m, 20H), 5.64 (dt, J = 15.6, 6.6 Hz, 1H),

5.43 (m, J = 15.6, 5.4 Hz, 2H), 4.92 and 4.55 (2d, J = 11.4 Hz, 2H), 4.84 and 4.70 (2d, J = 11.8 Hz, 2H), 4.76 (s, 2H), 4.74 (d, J = 3.9 Hz, 1H), 4.46 and 4.38 (2d, J = 11.7 Hz, 2H), 4.19 (m, 1H), 4.02 (dd, J = 9.9, 3.3 Hz, 1H), 4.00 (m, 1H), 3.92-3.80 (m, 3H), 3.66 (m, 2H), 3.54-3.46 (m, 3H), 1.99 (m, 2H),

1.44 (s, 9H), 1.28 (s, 4H), 0.88 (t, J = 6.6 Hz, 3H).

¹³ C NMR (75 MHz. CDCk) δ 155.8, 138.6-137.9, 132.7, 129.7, 128.5-127.6, 98.9, 79.5, 79.3, 75.8, 74.9, 74.6, 74.2, 74.0, 73.6, 72.8, 69.7, 69.2, 68.7, 32.1,31.4,28.5,22.3, 14.0.

Deprotection of BOC 20

19 (237.9 mg, 0.299 mmol, 1 eq) was dissolved in dry THF (21 mL) and HCI gas bubbled up to total consumption of starting material on TLC. Without treatment, THF was evaporated and the crude was engaged in the next step.

(2S,3R)-2-(/V-hexacosanoylamino)-3-hvdroxy-1-(2,3A6-tetra -O-benzyl-α-D- galactopyranosyl)non-4-ene 21

To the chlorhydrate 20 (0.299 mmol, 1 eq) dissolved in THF (11.9 ml_) under argon at room temperature were added 16 (154.6 mg, 0.299 mmol, 1 eq), triethylamine (50 μl_, 0.359 mmol, 1.2 eq) and a catalytic amount of 4-dimethylaminopyridine. The mixture was heated to reflux for 27 h and diluted with saturated aqueous NaHCO ₃ solution (20 ml_). The aqueous layer was extracted with Et2θ (2 ^χ 20 ml_). The organic layers were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 78/22) afforded 21 as white oil (185.2 mg, 58% over 2 steps).

C ₆₉ H ₁₀₃ NO ₈ MoI. Wt: 1074,558

Fa ir25 n +30.9 (c 1.2, CHCI ₃ )

IR : 3328, 3032, 2852, 1640, 1546, 1467, 1338, 1292, 1105 cm ^"1 . ¹ H NMR (300 MHz, CDCh) 7.34-7.24 (m, 20H), 6.44 (d, J = 7.8 Hz, 1H), 5.65 (ddd, J = 15.3, 7.2, 6.6 Hz, 1 H), 5.42 (dd, J = 15.3, 5.1 Hz, 1 H), 4.91 and 4.55 (2d, J = 11.4 Hz, 2H), 4.86 and 4.70 (2d, J = 11.7 Hz, 2H), 4.75 (s, 3H), 4.47 and 4.37 (2d, J = 11.7 Hz, 2H), 4.14 (m, 1 H), 4.04 (dd, J = 9.9, 3.3 Hz, 1 H), 4.01-3.95 (m, 2H), 3.88-3.82 (m, 4H), 3.68 (dd, J = 10.5, 3.6 Hz, 1 H), 3.51 (m, 2H), 2.12 (t, J = 7.5 Hz, 2H), 2.01 (m, 2H), 1.58 (m, 2H), 1.25 (m, 48H), 0.88 (t, J = 6.6 Hz, 6H).

¹³ C NMR (75 MHz, CDCh) δ 173.4, 138.4-137.6, 132.9, 129.3, 128.5-127-5, 99.1 , 79.2, 75.9, 74.8, 74.4, 74.2, 74.0, 73.6, 72.7, 69.8, 69.1 , 68.7, 52.9, 36.7, 32.0, 31.4, 29.7, 29.4, 25.8, 22.7, 22.3, 14.2, 14.0.

(2S,3R)-1-(α-D-qalactopyranosyl)-2-hexacosanoylanrιinon on-3-ol 22: compound of Formula Hl-B

To 21 (162.2 mg, 0.151 mmol, 1 eq) dissolved in MeOH (13 ml_) and THF (6.5 ml_) at room temperature was added palladium (10%) on activated carbon (162.2 mg) in one portion. The mixture was stirred under H ₂ . After 3 days, the mixture was filtered through Celite and filter cake washed with a combination of MeOH and CHCI ₃ . The filtrate was concentrated and the residue was purified on silica gel (CHCI ₃ /MeOH: 95/5 to 85/15) to provide a white solid (56.1 mg, 52%).

C ₄₁ H ₈₁ NO ₈ MoI. Wt: 716,0837

Formula Hl-B i25 M D +23.6 (c 1.0, Pyridine) _mp i44-145°C, IR : 3427, 3274, 2919, 2850, 1642, 1557, 1466, 1371 , 1141 , 1080, 1049, 1028 cm -1

¹ H NMR (300 MHz, CDCk) 8.53 (d, J = 8.7 Hz, 1 H), 5.46 (d, J = 3.6 Hz, 1H), 5.06 (bs, 5H), 4.74 (m, 1 H), 4.67 (dd, J = 9.9, 3.6 Hz, 1 H), 4.59-4.28 (m, 8H),

2.50 (t, J = 7.5 Hz, 2H), 1.86 (s, 6H) ₁ 1.31 (s, 50H), 0.86 (t, J = 6.6 Hz, 3H), 0.82 (t, J = 7.2 Hz, 3H).

¹³ C NMR (75 MHz, CDCU) δ 173.5, 102.1, 73.1 , 71.9, 71.7, 71.0, 70.6, 69.6, 62.7, 54.9, 36.8, 35.1 , 32.1 , 30.0, 29.6, 26.4, 22.9, 14.3.

Example 4 : Synthesis of compound of Formula Hl-C

Formula Hl-C

(2S.3R)-2-(terf-butyloxycarbonylamino)-3-hvdroxy-7.7-dime thyl-1-(2,3,4,6- tetra-O-benzyl-α-D-qalactopyranosyl)oct-4-ene 23

To 13 (410.1 mg, 0.554 mmol, 1 eq) dissolved in dry CH ₂ CI ₂ (5.5 mL) at room temperature under argon were added 4,4-dimethylpent-1- ene (798 μl, 5.54 mmol, 10 eq) and Grubbs Il (23.5 mg, 0.028 mmol, 0.05 eq). The mixture was heated to reflux for 12 h. 4,4-dimethylpent-1-ene (798 μl, 5.54 mmol, 10 eq) and Grubbs Il (23.5 mg, 0.028 mmol, 0.05 eq) were added and the solution continue to stir for 12 h. Without treatment, CH ₂ CI ₂ was evaporated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 82/18) afforded 23 as a white oil (233.1 mg, 52%).

C ₄₉ H ₆₃ NO ₉ MoI. Wt.: 810,0258

M ,25n +35,4 (c 0.9, CHCI ₃ ).

IR : 3031, 2928, 1713, 1497, 1454, 1392, 1266, 1242, 1165, 1099, 1059, 1028 cm ^"1 .

¹ H NMR (300 MHz, CDCi ₃ ) 7.36-7.25 (m, 20H), 5.71 (ddd, J = 15.0, 7.5, 7.2 Hz, 1 H), 5.43 (dd, J = 15.0, 5.7 Hz, 1 H), 5.42 (d, J = 8.4 Hz, 1 H), 4.92 and 4.55 (2d, J = 11.4 Hz, 2H), 4.82 and 4.71 (2d, J = 11.7 Hz, 2H), 4.75 (m, 3H), 4.44 and 4.38 (2d, J = 11.7 Hz, 2H), 4.22 (m, 1 H), 4.02 (dd, J = 9.9, 3.3 Hz, 1 H), 3.98 (m, 1H), 3.92-3.84 (m, 3H), 3.68 (m, 2H), 3.57-3.47 (m, 3H), 1.90 (d, J = 7.2 Hz, 1 H), 1.88 (d, J = 7.5 Hz, 1 H), 1.44 (s, 9H), 0.85 (s, 9H). ¹³ C NMR (75 MHz, CDOg) δ 155.8, 138.5-137.8, 132.1 , 129.9, 128.4-127.5, 99.9, 79.6, 79.2, 75.7, 74.8, 74.5, 74.3, 73.9, 73.6, 72.8, 69.7, 69.3, 68.6, 53.8, 46.9, 30.9, 29.3, 28.4.

Deprotection of BOC 24

23 (262.4 mg, 0.324 mmol, 1 eq) was dissolved in dry THF (23 mL) and HCI gas bubbled up to total consumption of starting material on TLC. Without treatment, THF was evaporated and the crude was engaged in the next step.

(2S,3R)-2-(λ/-hexacosanoylamino)-3-hvdroxy-7,7-dimethyl- 1-(2,3,4,6-tetra-0- benzyl-α-D-qalactopyranosyl)oct-4-ene 25

To the chlorhydrate 24 (0.324 mmol, 1 eq) dissolved in THF (12.9 ml_) under argon at room temperature were added 16 (167.5 mg, 0.324 mmol, 1 eq), triethylamine (54 μl_, 0.389 mmol, 1.2 eq) and a catalytic amount of 4-dimethylaminopyridine. The mixture was heated to reflux for 20 h and diluted with saturated aqueous NaHCO ₃ solution (20 ml_). The aqueous layer was extracted with Et ₂ O (2 * 20 ml_). The organic layers were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 80/20) afforded 25 as white wax (229.2 mg, 65% over 2 steps).

C ₇₀ H ₁₀₅ NO ₈ MoL Wt.: 1088,5846

TaI ²⁵ D +27.2 (c 1.6, CHClV) mp 53-54°C.

IR : 3326, 3031, 2849, 1639, 1538, 1497, 1470, 1349, 1243, 1055 cm ^"1 .

¹ H NMR (300 MHz, CDCk) 7.29-7.12 (m, 20H) ₁ 6.36 (d, J = 7.8 Hz ₁ 1 H), 5.64

(ddd, J = 15.3, 7.8, 7.5 Hz, 1 H), 5.34 (dd, J = 15.3, 5.4 Hz, 1 H) ₁ 4.82 and 4.47

(2d, J = 11.7 Hz, 2H), 4.75 and 4.61 (2d, J = 12.0 Hz ₁ 2H), 4.69 (d, J = 3.6 Hz,

1H), 4.66 (s, 2H) ₁ 4.38 and 4.28 (2d, J = 11.4 Hz, 2H), 4.09 (m, 1H), 3.96 (dd,

J = 10.2, 3.6 Hz ₁ 1 H) ₁ 3.94-3.76 (m, 5H), 3.64 (dd, J = 10.5, 3.9 Hz, 1 H), 3.48-

3.40 (m, 3H), 2.02 (t, J = 7.2 Hz, 2H), 1.82 (m, 2H) ₁ 1.49 (m, 2H), 1.17 (m,

44H), 0.79 (m, 12H).

¹³ C NMR (75 MHz, CDCh) δ 173.4, 138.5-137.7, 131.9, 130.0, 128.5-127.5, 99.1 , 79.2, 76.0, 74.8, 74.5, 74.1 , 74.0, 73.6, 72.7, 69.9, 69.0, 68.8, 53.2, 47.0, 36.7, 32.0, 31.0, 29.8, 29.8, 29.4, 25.8, 22.8, 14.3.

(2S,3R)-1-(α-D-qalactopyranosyl)-2-hexacosanoylamino-7,7 -dinnethyloct-3-ol 26- compound of Formula Hl-C

To 25 (205.8 mg, 0.189 mmol, 1 eq) dissolved in MeOH (16 mL) and THF (8 ml_) at room temperature was added Palladium (10%) on activated carbon (206 mg) in one portion. The mixture was stirred under H ₂ . After 3.5 days, the mixture was filtered through Celite and filter cake washed with a combination of MeOH and CHCI ₃ . The filtrate was concentrated and the residue was purified on silica gel (CHCI ₃ /MeOH: 99/1 to 95/5) to provide a white solid (35.5 mg, 26%).

C ₄₂ H ₈₃ NO ₈ MoI. Wt.: 730,1103

Formula Hl-C

rαl ²⁵ η +45.9 (c 0.7, Pyridine) mp 118-119°C.

IR : 3421, 2919, 2850, 1646, 1559, 1472, 1363, 1079 cm -1

¹ H NMR (300 MHz, CDCh) 8.54 (d, J = 8.7 Hz, 1 H), 5.45 (d, J = 3.9 Hz ₁ 1 H), 5.03 (bs, 5H), 4.72 (m, 1 H), 4.64 (dd, J = 9.9, 3.9 Hz, 1 H), 4.56-4.26 (m, 8H), 2.48 (t, J = 7.2 Hz, 2H), 1.82 (m, 6H), 1.24 (s, 46H), 0.82 (s, 12H). ¹³ C NMR (75 MHz, CDCIs) δ 173.5, 102.0, 72.7, 71.5, 71.3, 70.6, 70.2, 69.2, 62.3, 54.7, 44.3, 36.5, 35.7, 31.8, 30.4, 30.0, 29.6, 29.3, 29.2, 26.4, 22.9, 21.6, 14.3.

Example 5: Synthesis of the compound of Formula Hl-D

Formula Hl-D

(2S.3R)-2-(te/t-butyloxycarbonylamino)-3-hvdroxy-7-phenyl-1- (2.3,4,6-tetra-O- benzyl-α-D-galactopyranosyl)hept-4-ene 27 To 13 (300 mg, 0.405 mmol, 1 eq) dissolved in dry CH ₂ CI ₂ (4 ml_) at room temperature under argon were added 4-phenyl-but-1 -ene (608 μl, 4.05 mmol, 10 eq) and Grubbs Il (12.7 mg, 0.020 mmol, 0.05 eq). The mixture was heated to reflux for 24 h. 4-phenyl-but-1-ene (608 μl, 4.05 mmol, 10 eq) and Grubbs Il (12.7 mg, 0.020 mmol, 0.05 eq) were added and the solution continue to stir for 36 h. Without treatment, CH ₂ CI ₂ was evaporated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 81/19) afforded 27 as a white oil (139.5 mg, 41 %).

C ₅₂ H ₆₁ NO ₉ MoI. Wt.: 844,042

i25

MZD +31.3 (c 1.2, CHCI ₃ ). !R : 3446, 3063, 3029, 2929, 1713, 1604, 1497, 1454, 1392, 1366, 1246, 1165, 1096 cm ^"1 .

¹ H NMR (300 MHz, CDCU) 7.26-7.04 (m, 25H), 5.58 (ddd, J = 15.6, 7.2, 6.9 Hz, 1 H), 5.34 (dd, J = 15.6, 5.4 Hz, 2H), 4.84 and 4.48 (2d, J = 11.4 Hz, 2H), 4.75 and 4.59 (2d, J = 12.0 Hz, 2H), 4.68 (s, 2H), 4.61 (d, J = 3.6 Hz, 1 H), 4.39 and 4.31 (2d, J = 11.7 Hz, 2H), 4.09 (m, 1H), 3.93 (dd, J = 10,2, 3.6 Hz, 1 H), 3.91 (m, 1 H), 3.79, (m, 2H), 3.69 (d, J = 9.9 Hz, 1 H), 3.57 (m, 1 H), 3.50- 3.38 (m, 4H), 2.55 (t, J = 7.5 Hz, 2H), 2.23 (m, 2H), 1.37 (s, 9H). ¹³ C NMR (75 MHz, CDCU) δ 155.7, 141.6, 138.5-137.8, 131.4, 130.6, 128.4- 127.5, 125.9, 99.8, 79.6, 79.3, 75.8, 74.8, 74.5, 74.1, 74.0, 73.6, 72.8, 69.6, 69.1 , 68.6, 53.6, 35.4, 33.9, 28.4.

Deprotection of BOC 28

27 (136.7 mg, 0.162 mmol, 1 eq) was dissolved in dry THF (11.5 ml_) and HCI gas bubbled up to total consumption of starting material on TLC. Without treatment, THF was evaporated and the crude was engaged in the next step.

(2S,3R ^' )-2-(λ/-hexacosanov)aminoV3-hvdroxy-7-pheπyl-1-(2,3,4.6-te tra-O- benzyl-α-D-galactopyranosyl) hept-4-ene 29

To the chlorhydrate 28 (0.162 mmol, 1 eq) dissolved in THF (6.5 inL) under argon at room temperature were added 16 (83.8 mg, 0.162 mmol, 1 eq), triethylamine (27 μl_, 0.194 mmol, 1.2 eq) and a catalytic amount of 4-dimethylaminopyridine. The mixture was heated to reflux for 16 h and diluted with saturated aqueous NaHCO ₃ solution (10 mL). The aqueous layer was extracted with Et ₂ O (2 x 10 mL) and CHCI ₃ (2 x 10 mL). The organic layers were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc 76:24) afforded 29 as white powder (83.1 mg, 46% over 2 steps).

C ₇₃ H ₁₀₃ NO ₈ MoI. Wt.: 1122,60

rα1 ²⁵ p +28.2 (c 0.8, CHCI ₃ ) mp 65-66°C.

IR : 3328, 3062, 3031, 2918, 2850, 1636, 1617, 1540, 1472, 1113 Cm ^"1 . ¹ H NMR (300 MHz. CDCk) 7.36-7.23 (m, 23H), 7.17-7.12 (m, 2H), 6.38 (d, J = 8.1 Hz, 1 H), 5.64 (ddd, J = 15.6, 7.2, 6.6 Hz, 1H), 5.41 (dd, J = 15.6, 5.1 Hz, 1 H), 4.91 and 4.55 (2d, J = 11.4 Hz, 2H), 4.84 and 4.66 (2d, J = 11.7 Hz, 2H), 4.75 (s, 3H), 4.46 and 4.37 (2d, J = 11.7 Hz, 2H), 4.12 (m, 1 H), 4.02 (dd, J = 9.9, 3.6 Hz, 1 H), 3.99-3.94 (m, 6H), 3.55-3.46 (m, 3H), 2.63 (t, J = 6.9 Hz, 2H), 2.12 (m, 2H), 2.11 (t, J = 7.8 Hz, 2H), 1.58 (m, 2H), 1.26 (s, 44H), 0.88 (t, J = 6.6 Hz, 3H).

¹³ C NMR (75 MHz, CDCh) δ 173.3, 141.5, 138.5-137.7, 131.6, 130.3, 128.4- 127.5, 125.9, 99.0, 79.2, 75.9, 74.8, 74.4, 74.2, 73.9, 73.6, 72.6, 69.8, 68.9, 68.7, 52.8, 36.7, 35.4, 33.9, 31.9, 29.7, 29.4, 25.8, 22.7, 14.1.

(2S,3R)-1-(α-D-qalactopyranosyl)-2-hexacosanoylamino-7-p henylhept-3-ol 30 - compound of Formula Hl-D

To 29 (78.5 mg, 0.070 mmol, 1 eq) dissolved in MeOH (6 ml_) and THF (3 ml_) at room temperature was added palladium (10%) on activated carbon (40 mg) in one portion. The mixture was stirred under H ₂ . After 17 h, the mixture was filtered through Celite and filter cake washed with a combination of MeOH and CHCI ₃ . The filtrate was concentrated and the residue was purified on silica gel (CHCI ₃ /MeOH: 99/1 to 95/5) to provide a white solid (38.7 mg, 72%).

C ₄₅ H ₈₁ NO ₈ MoI. Wt.: 764,13

Formula Hl-D rα1 ²⁵ n +43.4 (c 0.7, Pyridine) _mp 144-145°C.

IR : 3265, 2918, 2850, 1652, 1538, 1472, 1456, 1071 cm ^"1 .

¹ H NMR (300 MHz, CDCI ₃ ) 8.51 (d, J = 8.7 Hz, 1H), 7.32-7.16 (m, 5H), 5.43

(d, J = 3.9 Hz, 1H), 5.07 (bs, 5H), 4.69 (m, 1H), 4.63 (dd, J = 9.9, 3.6 Hz, 1H),

4.53-4.23 (m, 8H), 2.56 (t, J = 6.9 Hz, 2H), 2.47 (t, J = 7.2 Hz, 2H), 1.85 (m,

6H), 1.63 (m, 2H), 1.26 (s, 44H), 0.87 (t, J = 7.2 Hz, 3H).

¹³ C NMR (75 MHz, CDCI ₃ ) δ 173.5, 143.1 , 128.8, 128.6, 125.9, 102.0, 73.0,

71.7, 71.6, 70.9, 70.5, 69.4, 62.6, 54.9, 36.8, 36.2, 34.8, 32.1 , 32.0, 30.0,

29.6, 26.4, 26.3, 22.9, 14.2.

Example 6: Synthesis of dimer of Formula Vl

Formula Vl

(2S.3R.6R,7S)-2,7-(di-terf-butyloxycarbonylamino)-3,6-dihvdr oxy-1 ,8- di(2,3,4,6-tetra-O-benzyl-α-D-qalactopyranosyl)oct-4-ene 31 To 13 (100 mg, 0.135 mmol, 1 eq) dissolved in dry CH ₂ CI ₂

(1.3 ml_) at room temperature under argon was added Grubbs-Hoveda I! (4.2 mg, 0.007 mmol, 0.05 eq). The mixture was heated to reflux for 16 h. Grubbs- Hoveda Il (4.2 mg, 0.007 mmol, 0.05 eq) were added and the solution continue to stir for 3 days. Without treatment, CH ₂ CI ₂ was evaporated. Purification by flash chromatography on silica gel (petroleum ether/EtOAc: 60/40) afforded 31 as a brown oil (60.7 mg, 31 %).

IcQf 25o +39.2 (c 1.2, CHCI ₃ ).

IR : 3446, 3030, 2927, 1710, 1497, 1454, 1367, 1217, 1164, 1097, 1057, 1028 cm ^"1 .

¹ H NMR (300 MHz. CDCU 7.34-7.21 (m, 40H), 5.67 (bs, 2H), 5.33 (d, J = 8.4

Hz, 2H), 4.90 and 4.53 (2d, J = 11.4 Hz, 4H), 4.79 and 4.63 (2d, J = 12.0 Hz,

4H), 4.74 (m, 6H), 4.46 and 4.37 (2d, J = 11.7 Hz, 4H), 4.16 (m, 2H), 4.00 (dd,

J = 9.9, 3.6 Hz, 2H), 3.98 (m, 2H), 3.90-3.85 (m, 4H), 3.78 (dd, J = 9.9, 2.7 Hz,

2H), 3.58-3.46 (m, 10H), 1.43 (s, 18H).

¹³ C NMR (75 MHz, CPCI ₃ ) δ 155.8, 138.6-137.9, 131.6, 128.5-127.6, 99.8,

79.7, 79.3, 75.9, 74.9, 74.6, 74.0, 73.6, 73.5, 72.9, 69.7, 68.8, 53.9, 28.5.

Deprotection of BOC 32 31 (124.6 mg, 0.086 mmol, 1 eq) was dissolved in dry THF

(12 ml_) and HCI gas bubbled up to total consumption of starting material on TLC. Without treatment, THF was evaporated and the crude was engaged in the next step.

(2S,3R,6R,7S)-2J-(di-λ/-hexacosanoylamino)-3,6-dihvdroxy -1 ,8-di(2.3.4,6- tetra-O-benzyl-α-D-galactopyranosyl)oct-4-ene 33

To the chlorhydrate 31 (0.086 mmol, 1 eq) dissolved in THF (6.8 ml_) under argon at room temperature were added 16 (88.7 mg, 0.172 mmol, 2 eq), triethylamine (29 μl_, 0.206 mmol, 2.4 eq) and a catalytic amount

of 4-dimethylaminopyridine. The mixture was heated to reflux for 19 h and diluted with saturated aqueous NaHCO ₃ solution (10 ml_). The aqueous layer was extracted with CHCI ₃ (2 x 15 ml_). The organic layers were combined, dried over MgSO ₄ and concentrated. Purification by flash chromatography on silica gel (CHCI ₃ /MeOH: 99/1 ) afforded 33 as white powder (100.1 mg, 58% over 2 steps).

TaI ²⁵ D +32.5 (c 1.0, CHCh) mp 129-130°C. IR : 3321 , 3063, 3031 , 2919, 2850, 1637, 1539, 1497, 1468, 1453, 1347, 1209, 1156, 1111 , 1054, 1027 cm ^"1 .

¹ H NMR (300 MHz, CDCk) 7.32-7.26 (m, 40H), 6.41 (d, J = 7.8 Hz, 2H), 5.68 (s, 2H), 4.92 and 4.54 (2d, J = 11.4 Hz, 4H), 4.82 and 4.64 (2d, J = 11.7 Hz, 4H), 4.75 (d, J = 3.3 Hz, 2H), 4.73 (s, 4H), 4.46 and 4.36 (2d, J = 11.7 Hz, 4H), 4.15 (m, 2H), 4.00 (dd, J = 9.9, 3.3 Hz, 2H), 3.94-3.84 (m, 10H), 3.74 (dd, J = 10.2, 3.6 Hz, 2H), 3.60 (dd, J = 10.2, 3.6 Hz, 2H), 3.50 (m, 4H), 2.07 (t, J = 7.5 Hz, 4H), 1.55 (m, 4H), 1.26 (s, 88H) ₁ 0.88 (m, 6H).

¹³ C NMR (75 MHz, CDCI3) δ 173.6, 138.5-137.6, 131.3, 128.5-127.5, 99.0, 79.2, 75.6, 74.8, 74.5, 74.1 , 73.6, 73.2, 72.7, 69.9, 68.9, 68.6, 53.2, 36.6, 31.9, 29.7, 29.4, 25.7, 22.7, 14.1.

Example 7: Evaluation of the biological properties of the compounds of the invention

The biological properties of the compounds of the invention were evaluated as follows:

Cell culture

HeLa cells were established from cervix tumor cells in 1951. Transfected HeLa-CDId cells were kindly provided by Mitchell Kronenberg (La JoIIa Institute for Allergy and Immunology, La JoIIa, CA). These cells were maintained in DMEM medium containing 1000 mg/ml of glucose (Biowest) supplemented with 10% foetal bovine serum (FBS) (Eurobio), 2 mM of L- glutamine (Invitrogen), 0.5 Ul/ml of penicillin and 0.5 mg/ml of streptomycin (Invitrogen).

Wehi 164 clone 13 cells were established from a fibrosarcoma of Balb/c mouse induced by injection of methylcholanthrene. These cells were maintained in RPMI medium (Biowest) supplemented with 10% FBS, 2 mM of L-glutamine, 0.5 Ul/ml of penicillin and 0.5 mg/ml of streptomycin (hereafter referred as CM).

PBMCs (Peripheral Blood Mononuclear Cells) were separated by Ficoll density centrifugation (LMS Eurobio) and incubated for a week with immature dendritic cells loaded with synthetic alpha galactosylceramide (KRN 7000). NKT cells were positively selected by magnetic cell sorting from PBMC using anti-Vα24 and anti-Vβ11 monoclonal antibodies (Beckman Coulter). They were expanded and maintained in RPMI medium supplemented with 10% FBS, 2 mM of L-glutamine, 0.5 Ul/ml of penicillin, 0.5 mg/ml of streptomycin and 300 U/ml recombinant interleukin 2 (IL-2) (Chiron).

Cytokine release assays

Glycolipids were obtained in solid form, suspended in DMSO and solubilised by two successive incubations: first at 56°C during 10 minutes then at 37°C for at least 1 hour.

HeLa-CDId cells were incubated with the glycolipid at various concentrations at 37°C for 16 hours and washed three times with CM. The NKT cells, washed twice in CM to eliminate IL-2, were added to HeLa-CDId cells. 15000 NKT cells were incubated with 30000 HeLa-CDId cells for 6 hours in 150 μl of CM for interferon (IFN)-γ and IL-4 production or 100 μl for tumor necrosis factor (TNF)-α production (in triplicate). Then, supernatants were washed twice and stored at -80 ⁰ C until cytokine concentration evaluation.

The amount of TNF-α released in the supernatant was estimated by the Wehi 164 cytotoxicity assay (Hoffmann et al., 1997).

The amount of IFN-γ and IL-4 in the supernatant was evaluated by ELlSA (Enzyme Linked Immunosorbent Assay) with the BD OptiEIA IFN-γ set and BD OptiEIA IL-4 set (BD Biosciences) respectively. Tests were performed following supplier's instructions.

Results of the biological tests carried out with the compound of Formula Hl-A

Formula Hl-A The results of the tests carried out with the compound of

Formula IH-A are shown in Figures 1-3 in which the compound of Formula Hl-A is noted VL 335.

NKTi cell activation is evaluated by production of three cytokines: tumour necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-4. Cytokine production by NKTi MAD11 (polyclonal population) alone or after loading of HeLa-CDId cells, or with glycolipid KRN 7000 or the

compound of Formula Hl-A (noted VL 335) of the invention, at various concentrations, was evaluated.

As can seen from Figures 1-3, the production of cytokines by

NKTi MAD11 after incubation with HeLa-CDId loaded with 0.1 μg/ml of the compound of Formula Hl-A was superior to the production of cytokines by

NKTi MAD11 after incubation with HeLa-CDId loaded with 0.1 μg/ml of synthetic reference alpha-galactosylceramide KRN 7000.

These results show that the compounds of the invention, and more particularly the compound of Formula Hl-A, have superior immunomodulating activities as compared to the reference KRN7000. These superior activities render the compounds of the invention of high interest for the control of, in particular, anti-cancerous process.

Furthermore, the compounds of the invention may be synthesized with a very simple method, i.e. at low cost as compared to KRN7000.