Synthetic variants of ganglioside NGcGM3 and their use in cancer treatment

Abstract

The present invention relates to the field of biotechnology, especially in cancer immunotherapy.Synthetic variants of glycosylated sialic acid containing GM3 ganglioside (NGcGM3) and compositions and methods related to these derivatives for use in the treatment of malignant tumors and their metastasis are described.According to the present invention, the anti-tumor effects of these derivatives are at least partly due to their ability to stimulate iNKT cells.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, particularly in cancer immunotherapy. Synthetic variants of N-glycolylated sialic acid of GM3 ganglioside (NGcGM3), and compositions and methods of their derivatives for the treatment of malignant tumors and their metastases are described. [Background technology]

[0002] The existence of glycosphingolipids that have antitumor activity and act as stimulators of invariant NKT (iNKT) cells has been convincingly described in the state of the art from natural or synthetic compounds derived from α-galactosylceramide (αGalCer) (U.S. Pat. No. 5,936,076).

[0003] In contrast, evidence that other types of glycolipids, particularly gangliosides, are iNKT cell-activating compounds is conflicting and poorly understood, and to our knowledge, there is no evidence that they have antitumor activity. Experimental results are available showing that ganglioside GD3 is a tumor-associated glycolipid capable of inducing CD1d-restricted iNKT cell responses (Park et al., Immunology, 2008, 123, 145-155), but ganglioside GM3 appears to be inhibitory to this response, as coimmunization of mice with this glycolipid and GD3-pulsed antigen-presenting cells (APCs) prevented iNKT cell activation.

[0004] Conversely, our results showing how GD3 acts as a suppressor of the innate immune response in ovarian cancer by inhibiting iNKT cell activation (Webb et al., Can. Res., 2012, 72, 3744-3752) indicate how these tumors circumvent the antitumor effects of iNKT cells via this ganglioside, providing an early escape mechanism.

[0005] The same contradiction is evident from Paget et al., namely, that gangliosides GM3 and GD3 can become endogenous activators of iNKT cells in dendritic cells (DCs) stimulated with Toll receptor (TLR) agonists, depending on the structural changes undergone in the ceramide (Paget et al., PLoS Biology, 2019, 17(3), e3000169). They found that both synthetic variants of GM3 and GD3 with acetylated sialic acid (NAcGM3 and NAcGD3) and synthetic variants with d18:1-C24:1 ceramide could activate iNKT cells in a CD1d-dependent manner. However, Paget et al. did not demonstrate the iNKT cell-activating ability of either the synthetic variant of GM3 containing glycosylated sialic acid (NGcGM3) or provide evidence of antitumor activity of NAcGM3 or NAcGD3 d18:1-C24:1.

[0006] On the other hand, NGcGM3 is an evolutionarily fixed tumor neoantigen and is therefore of interest for cancer immunotherapy, proving to be the basis for various described strategies (Labrada et al., Semin Oncol, 2018, 45, 41-51). Prior art supports the validation of NGcGM3 as a tumor antigen based on experimental evidence that reduced expression of this ganglioside in tumor cell lines reduces solid tumor growth and metastatic dissemination in mice. Of particular interest to the present invention is the reduction observed in subcutaneous tumors generated by implanting P3X63 mouse myeloma cells (which have high expression of NGcGM3) pretreated with a glucosylceramide synthase inhibitor, demonstrating how this ganglioside can be a stimulator of tumor progression (de Leon et al., Cancer Immunol Immunother, 2006, 55, 443-450).

[0007] Based on this evidence, no direct antitumor application of NGcGM3 ganglioside has been described, while previously described immunotherapeutic approaches have identified NGcGM3 as an antigen. Among these, the one closest to the present invention is the GlycoVaxGM3 vaccine, a nanoparticulate product obtained by combining NGcGM3 with the outer membrane protein complex of Neisseria meningitidis. This vaccine induces specific antibodies against gangliosides in both experimental animals and cancer patients (Labrada et al., Semin Oncol, 2018, 45, 41-51). However, prior art indicates that this vaccine has always been obtained using a natural mixture of different molecular species of NGcGM3 obtained from horse erythrocytes (Estevez et al., Vaccine, 2000, 18, 190-197).

[0008] Evidence that NGcGM3 can bind to CD1d has been previously documented (Gentilini et al., Cancer Immunol Immunother, 2016, 65, 551-562), which is consistent with the findings of the present authors. However, this ganglioside binds only to CD3 purified from human peripheral mononuclear cells activated in culture with αGalCer and IL-2. + Because this study started with a cell population, it does not provide evidence that this ganglioside can directly activate iNKT cells. This procedure increased the number of iNKT cells and demonstrated that they could bind to a CD1d-IgG1 fusion protein containing NGcGM3. Because these experiments were performed with a natural mixture of NGcGM3, it is not possible to define whether any particular molecular species is responsible for the described effects, or whether iNKT NGcGM3 is involved. + It does not teach any anti-tumor effect of the population. Summary of the Invention

[0009] The object of the present invention is a synthetic ganglioside of NGcGM3 of formula A, which has antitumor and antimetastatic activity. Formula A [ka] (Wherein R is -C 23 H 47 and -C23H45 (selected from the group including

[0010] Furthermore, the present invention relates to pharmaceutical compositions comprising a ganglioside designated A as an active ingredient and a pharmaceutically acceptable vehicle. In particular, these compositions may contain another immunomodulator. In addition, they may carry an antigen. Such compositions may be in the form of nanoparticles or liposomes. In particular, nanoparticles are formed by hydrophobic insertion of one or more gangliosides described herein into hydrophobic outer membrane proteins of Gram-negative bacteria, particularly Neisseria meningitidis.

[0011] Another embodiment of the present invention is the use of gangliosides of formula A or pharmaceutical compositions containing them in the manufacture of a medicament for the treatment of cancer and its metastases.

[0012] In a further embodiment, the present invention comprises administering to a mammal, particularly a human, having cancer a therapeutically effective amount of a ganglioside described herein or a pharmaceutical composition comprising the same in nanoparticle or liposomal form. The tumor to be treated may be CD1d positive or CD1d negative. In particular, liver metastases are treated.

[0013] In another embodiment, the present invention describes an in vitro method for preparing dendritic cells loaded with gangliosides of formula A or pharmaceutical compositions comprising them, based on incubating dendritic cells obtained from a mammal, in particular a human, with said gangliosides and using the cells obtained in adoptive cell transfer therapy. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention Interesting synthetic variants of ganglioside NGcGM3 according to the present invention and described above are represented by formula A (i.e., formulas X and XI). The ceramide composition of both compounds is sphingosine (an amino alcohol with 18 carbon atoms and an unsaturated hydrocarbon chain), but they differ in the fatty acid, which is lignoceric acid in compound X and nervonic acid in compound XI.

[0015] Synthesis method The procedure developed by the present inventors was used to synthesize NGcGM3 ganglioside of formula A. First, sialylation of a hexabenzylated lactose acceptor was carried out using a thiophenyl-type N-glycolylated sialic acid donor. After isolation of the corresponding α-anomeric trisaccharide by forming its 1→4 lactone (anomeric trisaccharide β) (the anomeric trisaccharide β is not lactonized), it remains as a protected functional group during the remaining sequence of synthetic steps. The synthetic process used includes the following steps: a) Removal of the benzyl group by hydrogenolysis b) Per-O-acetylation of the resulting derivative c) Selective removal of the acetyl group attached to the anomeric carbon of the trisaccharide glucose unit d) Preparation of trichloroacetimidate trisaccharide donors e) Glycosylation of azidosphingosine benzoate f) Reduction of the azide group of the synthesized glycoside g) Acylation of the amino function formed with the carrier reagent of the second lipid chain and final removal of the protecting group from the resulting product (including the lactone). The above synthetic steps for compounds of formula A are better understood with reference to Figures 1a and 1b, in which the following abbreviations have been used:

[0016] Chemical structure abbreviations: Ac acetyl, Bn benzyl, Bz benzoyl, Me methyl, SPh thiophenyl. Abbreviations for reagents used in the synthesis: AcOH acetic acid, Ac2O acetic anhydride, BF3-OEt2 boron trifluoride-diethyl ether complex, CH3CN acetonitrile, DBU 1,8-diazabicyclo[5.4.0]undec-7-ene, DMF N,N-dimethylformamide, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) hydrochloride, Et3N triethylamine, MeOH methanol, NaOMe sodium methoxide, NHOAc ammonium acetate, NIS N-iodosuccinimide, TfOH triflic acid.

[0017] Source and preparation of lipid antigens used Gangliosides used were quantified by a colorimetric resorcinol assay for detecting lipid-bound sialic acid according to the methodology described by (Svennerholm L, Biochem Biophys Acta 1957, 24 604-11). αGalCer (KRN7000), used as a reference, was purchased from either Enzo Life Science (Farmingdale, NY, USA) or Avanti Polar lipids (Alabaster, AL, USA). All lipids were first dissolved in a 2:1 chloroform:methanol mixture and divided into aliquots of an appropriate amount for the day's use. The solvent was evaporated, and the dried aliquots were stored at -20°C until use. For in vitro experiments, glycolipid stock solutions were prepared in anhydrous dimethyl sulfoxide (DMSO) with sonication. For in vivo experiments, lipids were dissolved in a vehicle solution containing 5.6% sucrose, 0.75% L-histidine, and 0.5% Tween-20.

[0018] iNKT cell hybridoma The FF13 mouse iNKT cell hybridoma, previously described by Schumann J. (Schumann J. Eur J Immunol. 2007, 37, 1431-41), was provided by Dr. Lucia Mori (University Hospital Basel, Switzerland) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin, and 55 μM 2-mercaptoethanol containing GlutaMAX-I and 25 mM HEPES.

[0019] Obtaining mature dendritic cells Mature bone marrow precursor-derived dendritic cells (bmDCs) were used as APCs for in vitro iNKT cell activation assays and in vivo adoptive transfer experiments. bmDCs were obtained following a previously described protocol for differentiation from bone marrow progenitors in the presence of granulocyte / macrophage colony-stimulating factor (GM-CSF) (Inaba K, J Exp Med 1992, 176 1693-1702), with some modifications. Briefly, bone marrow precursors were isolated from the femurs and tibias of C57BL / 6 mice and cultured in complete RPMI-1640 medium (RPMI-1640 containing GlutaMAX-I, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and 55 μM 2-mercaptoethanol) supplemented with 20 ng / mL GM-CSF (R&D Systems). Cultures were fed with fresh medium and bmDC maturation was induced with 1 μg / ml LPS on day 3. On day 7, cells were harvested and used for iNKT cell activation assays or adoptive transfer.

[0020] Obtaining mixed nanoparticles of NGcGM3 d18:1-C24:1 or NGcGM3 d18:1-C24:0 with bacterial outer membrane protein complexes (OMPCs) OMPCs of Gram-negative bacteria, including N. meningitidis, Salmonella typhi, Salmonella enteritis, Haemophilus influenzae, Bordetella pertussis, or Escherichia coli, were dispersed in a 0.01 M Tris-HCl buffer solution (pH 8.5) containing 10-15 mM sodium deoxycholate (DOC) and 0.25-5 mM sodium dodecyl sulfate (SDS) to a final concentration of 0.5-3 mg / ml in a shaking reactor for 12 hours. Next, a mass of synthetic ganglioside NGcGM3 18:1-24:1 or NGcGM3 18:1-24:0 equivalent to the mass of OMPCs added was added, and stirring was extended for 3-10 hours. The detergent is then removed using a tangential filtration system with a 10-100 kDa membrane. The ultrafiltered solution is then subjected to ultracentrifugation at 100,000 g for 0.5-2 hours. The supernatant is concentrated to the desired concentration and sterilized by filtration into a sterile capsule with a pore size of 0.2 μm.

[0021] The dosage of nanoparticles used for treating a subject suffering from cancer or its metastasis is in the range of 10 μg to 2 mg, preferably 30 μg to 1 mg per injection.

[0022] Obtaining mixed nanoparticles of NGcGM3 d18:1-C24:1 or NGcGM3 d18:1-C24:0 with bacterial outer membrane protein complexes (OMPCs), which may also contain immunomodulators and / or tumor antigens. In another embodiment of the present invention, the above-described mixed nanoparticles are produced, further comprising an immunomodulatory agent selected from Toll-like receptor (TLR) agonists 3, 7, or 9.

[0023] Mixed nanoparticles containing tumor antigens, preferably neoantigens from public and private mutations, can also be obtained as previously described.

[0024] The compounds of the present invention represented by Formula A (ie, Formulas X and XI) have antitumor, antimetastatic and immunostimulatory activities and can be used in the treatment of tumors and their metastases.

[0025] (1) Antitumor activity Compounds of formulas X and XI, formulations having nanoparticles as vehicles obtained by inserting these gangliosides into a mixture of hydrophobic lipids or proteins of microbial origin, and preparations of dendritic cells pulsed with an effective amount of ganglioside of formula A exhibit antitumor activity against the murine myeloma P3X63 Ag 8.653 (ATCC NCRL 1580) (X63) when the cells are inoculated SQ into mice, as shown in Examples 5, 10, and 14.

[0026] (2) Anti-metastatic activity Compounds of formulas X and XI, formulations having nanoparticles as vehicles obtained by inserting these gangliosides into a mixture of microbial lipids or hydrophobic proteins, and preparations of dendritic cells pulsed with an effective amount of ganglioside of formula A have anti-liver metastatic activity against murine EL4 thymoma when the cells are inoculated via the tail vein of mice, as shown in Examples 2, 3, 9, 12, 13 and 18.

[0027] (3) Efficacy against CD1d-expressing human tumors Compounds of formulas X and XI, formulations containing nanoparticles obtained by inserting these gangliosides into a mixture of microbial lipids or hydrophobic proteins as vehicles, and preparations of dendritic cells pulsed with an effective amount of ganglioside of formula A can be successfully used not only against CD1d-positive hematologic malignancies such as multiple myeloma, but also against CD1d-expressing human solid tumors and their metastases. These include lung, head and neck, prostate, neuroblastoma and other brain tumors, melanoma, colon cancer, and kidney cancer (Ingram, Z. Cells 2021, 10, 1329). Similarly, these treatments may also be effective against CD1d-negative tumors and metastases. [Brief explanation of the drawings]

[0028] [Figure 1a] Diagram of total ganglioside synthesis. a) NGcGM3 d18:1-C24:1, b) NGcGM3 d18:1C24:0. [Figure 1b] Diagram of total ganglioside synthesis. a) NGcGM3 d18:1-C24:1, b) NGcGM3 d18:1C24:0. [Figure 2] Effect of treatment with NGcGM3 extracted from natural sources and synthetic NGcGM3 d18:1-C24:1 on liver metastatic spread of EL-4 tumor cells. [Figure 3] Effect of treatment with NGcGM3 extracted from natural sources and synthetic NGcGM3 d18:1-C24:1 on the survival rate of C57BL / 6 mice bearing EL-4 tumor cells. [Figure 4] Expression of CD1d molecules on the cell surface of P3X63 mouse myeloma cells as measured by flow cytometry. [Figure 5] Antitumor effect of NGcGM3 d18:1-C24:1 treatment on subcutaneously implanted P3X63 myeloma cells. [Figure 6] Effect of different molecular species of NGcGM3 on the in vitro activation of iNKT cells. [Figure 7] Effect of different molecular species of NGcGM3 on the in vivo activation of iNKT cells as measured by serum levels of IFNγ. [Figure 8] Comparison of the effects of different molecular species of NGcGM3 and GM3, which have the same ceramide structure, on the in vitro activation of iNKT cells, as measured by IL-2 secretion in culture. [Figure 9] Effect of nanoparticle treatment with NGcGM3 molecular species and OMPC of Neisseria meningitidis on liver metastatic spread of EL-4 tumor cells. [Figure 10]Antitumor effect of treatment of subcutaneously implanted P3X63 myeloma cells with nanoparticles of NGcGM3 d18:1-C24:1 or NGcGM3 d18:1-C24:1 plus N. meningitidis OPMC. [Figure 11] Effect of nanoparticles of NGcGM3 species and N. meningitidis OPMC on the in vitro activation of iNKT cells as measured by IL-2 secretion in culture. [Figure 12] Effect of adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3 on the liver metastatic spread of EL-4 tumor cells. [Figure 13] Effect of adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3 on survival of C57BL / 6 mice in the EL-4 tumor model. [Figure 14] Effect of adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3 on tumor growth of P3X63 cells. [Figure 15] IFNγ levels in the serum of P3X63 tumor-bearing mice after adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3. [Figure 16] IFNγ levels in the serum of EL-4 tumor-bearing mice after adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3. [Figure 17] Comparison of specific antibody responses to NGcGM3 induced by injection of nanoparticles containing molecular species of NGcGM3 and OPMC of N. meningitidis in EL-4 tumor-bearing mice. [Figure 18a] Effect of dual depletion of NK cells and NKT cells on the action of NGcGM3 d18:1-C24:1 in the EL-4 liver metastasis spread model. [Figure 18b] Effect of NK cell depletion on the action of NGcGM3 d18:1C24:1 in an EL-4 cell liver metastasis expansion model. [Example]

[0029] The present invention will be described in detail below with reference to experimental examples, but the present invention is not limited to these examples.

[0030] Example 1. Chemical synthesis of NGcGM3 mutants Methods for synthesizing the compounds of the present invention and their chemical and physical properties are presented below (see Figures 1a and 1b).

[0031] A. Synthesis of benzyl O-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-didesoxy-D-glycero-α-D-galacto-2-nonulopyranosyl-1→4-lactone)-(2→3)-O-(2,6-di-O-benzyl-βD-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α / β-D-glucopyranoside (Formula III) (Figure 1a) [ka]

[0032] A solution of the sialic acid donor, methyl (I) 5-acetoxyacetamido-4,7,8,9,10-tetra-O-acetyl-3,5-didesoxy-2-thiophenyl-D-glycero-α-D-galacto-2-nonulopyranosilonate (I) (7.25 g, 11.3 mmol), and the lactose acceptor, benzyl-O-(2,6-di-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6 tri-O-benzyl-α,β-D-glucopyranoside (II) (5.8 g, 6.6 mmol), in dry CH3CN (56 mL) was prepared, and 3 Å powdered molecular sieves (8.7 g) were added. The mixture was stirred at room temperature for 10 min. The mixture was then cooled to -30 °C, and NIS (3.48 g, 15.5 mmol) was added, followed by TfOH (67 μL, 0.76 mmol), and stirred at the same temperature for 2 h. The reaction was monitored by CCD. Upon completion, CHCl (120 mL) was added and the mixture was filtered through a layer of Celite 545. The filtrate was washed with a saturated aqueous solution of NaSO until the red color disappeared. The organic phase was separated, neutralized with EtN, dried over anhydrous NaSO, and the solvent was evaporated under reduced pressure. The resulting residue contained unreacted acceptor II, the α- and β-isomers of the formed trisaccharide, and the elimination product of donor I.

[0033] The product mixture obtained in the above reaction was dissolved in dry CHCl (180 mL). The solution was cooled to 0 °C, DBU (2.16 mL, 14.47 mmol) was added, and the mixture was stirred at the same temperature for 2 h. After neutralization with glacial acetic acid diluted in CHCl (1:4 v / v), the solvent was evaporated to dryness under reduced pressure, and the residue was purified by column chromatography (toluene / acetone 15:1) to recover the remaining unreacted acceptor II and subsequently the pure α[1→4]-lactone (III). After evaporation of the solvent, compound III was purified by thin-layer chromatography (CCD) (R f 0.56, toluene / acetone 2:1) as a homogeneous amorphous white solid. Yield 5.6 g (62%).

[0034] 1H NMR (600 MHz, CDCl3): δ2.15; 2.14; 2.03; 2.02; 1.93 [(3H, s) x 5]; 7.45-7.13 (30H, m, Ar); 4.94 (2H, d, J = 11.8 Hz); 4.75 (1H, d, J = 10.7 Hz); 4.73 (1H, d, J = 10.9 Hz,); 4.65 (2H, d, J = 12.7 Hz,); 4.59 (1H, d, J = 12.1 Hz,); 4.44 (1H, d, J = 12.1 Hz); 30 4.43 (1H, d, J = 12.1 Hz); 4.31 (1H, d, J = 12.3 Hz); 4.90 (1H, d, J = 10.9 Hz); 4.89 (1H, d, J = 10.7 Hz); [12H, benzyl]; Glc:4.49 (1H, d, J = 7.7 Hz, H-1); 3.46 (1H, dd, J = 9.2; 7.7 Hz, H-2); 3.56 (1H, m, H-3); 3.99 (1H, t, J = 9.4 Hz, H-4); 3.38 (1H, ddd, J = 9.8; 4.1; 1.8 Hz, H-5); 3.80 (1H, dd, J = 11.0; 4.0 Hz, H-6a); 3.73 (1H, m, H-6b); Gal:4.47 (1H, d, J = 7.8 Hz, H-1); 3.26 (1H, dd, J = 9.3; 7.6 Hz, H-2); 4.09 (1H, dd, J = 9.4; 4.1 Hz, H-3); 4.90 (1H, m, H-4); 3.56 (1H, m, H-5); 3.73 (1H, m, H-6a); 3.44 (1H, m, H6b); Neu:2.13 (1H, dd, J = 13.8; 5.4 Hz, H-3ec); 1.82 (1H, dd, J = 13.5; 11.5 Hz, H3ax); 5.48 (1H, m, H-4); 4.22 (1H, q, J = 10.4 Hz, H-5); 3.76 (1H, dd, J = 10.5; 2.1 Hz, H-6); 5.21 (1H, dd, J = 5.7; 2.1 Hz, H-7); 5.07 (1H, ddd, J = 7.2; 5.8; 3.0 Hz, H-8); 4.48 (1H, m, H-9a); 3.90 (1H, m, H-9b); 6.54 (1H, d, J = 10.3 Hz, 5-NH); 4.57 (1H, d, J = 15.2 Hz, H-10a); 4.33 (1H, d, J = 15.3 Hz, H-10b). MALDI-TOF MS: [M+Na] + m / z 1404.37 (calculated 1404.52).

[0035] B. Synthesis of acetyl O-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-didesoxy-D-glycero-α-D-galacto-2-nonulopyranosyl-1→4-lactone)-(2→3)-O-(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α / β-D-glucopyranoside (Formula IV) (Figure 1a) [ka]

[0036] α[1→4]-lactone (III) (5.6 g, 4.05 mmol) was dissolved in 230 mL of a MeOH / AcOH mixture (9:1 v / v), 10% Pd / C (0.95 g) was added, and the balloon was purged with a stream of Ar and then filled with H2 gas. The mixture was stirred at room temperature for 24 h. After this time, the catalyst was removed by filtration through a layer of Celite 545, and the solvent was evaporated to dryness under reduced pressure. The resulting solid was suspended in Ac2O (5 mL), and the mixture was stirred at 0 °C for 10 min. Predistilled BF3OEt2 (0.8 mL, 6.3 mmol) was added, followed by stirring at the same temperature for 1 h. After completion of the reaction, the resulting solution was neutralized with a saturated aqueous solution of NaHCO3, CHCl2 was added, and the phases were separated. After separation of the organic phase, the aqueous phase was washed twice with CH2Cl2, the organic extracts were combined, dried over anhydrous Na2SO4, and the solvent was removed by evaporation under reduced pressure to give compound IV as a CCD (R f0.73, toluene / acetone 1:1) as a homogeneous white amorphous solid. Yield 4.1 g (93%). An α / β anomeric mixture (7.6:3.4) was obtained. NMR data indicated that the α-anomer was the major anomer.

[0037] 1H NMR (600 MHz, CDCl3): δ 2.18; 2.16; 2.12; 2.11 (2CH3); 2.10; 2.09; 2.05; 2.00; 1.99; 1.98 [(3H, s, CH3) x 11]; Gluc: 6.24 (1H, d, J = 3.7 Hz, H-1); 5.00 (1H, dd, J = 10.3; 3.8 Hz, H-2); 5.42 (1H, dd, J = 10.3; 9.4 Hz, H-3); 3.76 (1H, dd, J = 9.8 Hz, H-4); 3.99 (1H, ddd, J = 10.2; 4.6; 2.1 Hz, H-5); 4.39 (1H, m, H-6a); 4.18 (1H, dd, J = 12.2; 4.6 Hz, H-6b); Gal: 4.38 (1H, d, J = 8.2 Hz, H-1); 4.85 (1H, dd, J = 9.9; 8.0 Hz, H-2); 4.12 (1H, m, H-3); 4.95 (1H, dd, J = 3.8; 1.1 Hz, H-4); 3.91 (1H, m, H-5); 4.64 (1H, dd, 5J = 12.1; 3.7 Hz, H-6a); 4.26 (1H, m, H-6b); Neu: 2.45 (1H, dd, J = 13.8; 5.4 Hz, H3ec); 1.79 (1H, dd, J = 13.8; 11.5 Hz, H-3ax); 5.54 (1H, td, J = 10.8; 5.3 Hz, H-4); 4.12 (1H, m, H-5); 3.68 (1H, dd, J = 10.5; 1.9 Hz, H-6); 5.15 (1H, dd, J = 9.2; 1.8 Hz, H-7); 5.19 (1H, m, H-8); 4.26 (1H, m, H-9a); 3.91 (1H, m, H-9b); 6.05 (1H, dd, J = 10.2; 1.8 Hz, 5-NH); 4.59 (1H, d, J = 15.4 Hz, H-10a); 4.26 (1H, m, H-10b). MALDI-TOF MS: [M+Na]+ m / z 1116.28 (calculated value 1116.30).

[0038] C. Synthesis of 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5'-didesoxy-D-glycero-αD-galacto-2-nonulopyranosyl-1→4-lactone-(2→3)-O-(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α / β-D-glucopyranose (Formula V) (Figure 1a). [ka]

[0039] A solution of IV (4.1 g, 3.74 mmol) in dry DMF (20 mL) was prepared, NHOAc (0.47 g, 6.19 mmol) was added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, AcOEt (100 mL) was added and washed with saturated aqueous NaCl (5 × 18 mL). The organic phase was separated, dried over anhydrous NaSO, and the solvent was removed under reduced pressure to dryness. Compound V was purified by CCD (R f 0.59, toluene / acetone 1:1) as a homogeneous amorphous white solid. Yield 3.55 g (90%). A mixture of α / β anomers (7:3) was obtained. NMR data indicated that the α-anomer was the major anomer.

[0040] 1H NMR (600 MHz, CDCl3): δ 2.19; 2.12 (3CH3); 2.10; 2.09; 2.06; 2.04; 2.00; 1.99 [(3H, s, CH3) x 10]; Glc: 5.35 (1H, d, J = 3.6 Hz, H-1); 4.83 (1H, m, H-2); 5.48 (1H, dd, J = 9.7 Hz, H-3); 3.71 (1H, m, H-4); 4.18 (1H, m, H-5); 4.43 (1H, m, H-6a); 4.18 (1H, m, H-6b); Gal: 4.42 (1H, d, J = 7.9 Hz, H-1); 4.83 (1H, m, H-2); 4.12 (1H, m, H-3); 4.94 (1H, dd, J = 3.7; 1.4 Hz, H-4); 3.92 (1H, m, H-5); 4.63 (1H, dt, J = 12.0; 3.4 Hz, H-6a); 4.27 (1H, m, H-6b); Neu: 2.46 (1H, dd, J = 13.8; 5.2 Hz, H-3ec); 1.78 (1H, dd, J = 13.9; 11.5 Hz, H-3ax); 5.53 (1H, td, J = 11.1; 5.4 Hz, H-4); 4.12 (1H, m, H-5); 3.71 (1H, m, H-6); 5.17 (2H, m, H-7 and H-8); 4.27 (1H, m, H-9a); 3.92 (1H, m, H-9b); 6.06 (1H, d, J = 10.2 Hz, 5-NH); 4.59 (1H, d, J = 15.4 Hz, H-10a); 4.27 (1H, m, H-10b). MALDI-TOF MS: [M+Na] + m / z 1074.11 (calculated value 1074.29).

[0041] Synthesis of DO-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-didesoxy-D-glycero-α-D-galacto-2-nonulopyranosyl-1→4-lactone)-(2→3)-O-(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl (Formula VI) (Figure 1a). [ka]

[0042] A solution of V (3.55 g, 3.37 mmol) was prepared in dry CHCl (40 mL), trichloroacetonitrile (10.9 mL, 109 mmol) was added, and DBU (0.52 mL, 3.46 mmol) was added dropwise. The mixture was stirred at room temperature for 2 h. Upon completion of the reaction, the mixture was evaporated to dryness under reduced pressure, and the resulting residue was purified by column chromatography (CHCl / acetone 6:1) to give compound VI as a homogeneous amorphous white solid (Rf 0.61, toluene / acetone 1:1) by CCD. Yield 2.95 g (73%).

[0043] 1H NMR (600 MHz, CDCl3): δ 2.19; 2.12; 2.11; 2.10; 2.09; 2.08; 2.05; 2.00; 1.99; 1.98 [(3H, s, CH3) x 10]; Glc: 6.47 (1H, d, J= 3.8 Hz, H-1); 5.06 (1H, dd, J= 10.1; 3.8 Hz, H-2); 5.53 (1H, m, H-3); 3.82 (1H, dd, J= 9.8 Hz, H-4); 4.11 (1H, m, H-5); 4.44 (1H, dd, J= 12.2; 2.0 Hz, H-6a); 4.19 (1H, dd, J= 12.2; 4.8 Hz, H-6b); 8.65 (1H, s, C=N); Gal:20 4.41 (1H, d, J = 7.9 Hz, H-1); 4.86 (1H, dd, J= 9.9; 8.0 Hz, H-2); H-3); 4.95 (1H, dd, J=3.8; 1.2 Hz, H-4); 3.91 (1H, m, H-5); 4.65 (1H, dd, J= 12.1; 3.5 Hz, H-6a); 4.26 (1H, m, H-6b); Neu:2.47 (1H, dd, J= 13.9; 5.4 Hz, H-3ec); 1.79 (1H, dd, J= 14.0; 11.5 Hz, H-3ax); 5.53 (1H, m, H-4); 4.14 (1H, d, J = 10.4 Hz, H-5); 3.69 (1H, dd, J= 10.4; 1.8 Hz, H-6); 5.15 (1H, dd, J= 9.2; 1.8 Hz, H-7); 5.19 (1H, ddd, J= 8.9; 6.0; 2.7 25 Hz, H-8); 4.26 (1H, m, H-9a); 3.91 (1H, m, H-9b); 6.02 (1H, d, J= 10.1 Hz, 5-NH); 4.60 (1H, d, J= 15.4 Hz, H-10a); 4.26 (1H, m, H-10b). MALDI-TOF MS: The decomposition of the product in the analysis, the compound V ([M+Na] +Only the peak corresponding to m / z 1074.19 was observed in the spectrum.

[0044] Synthesis of EO-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-didesoxy-D-glyceroα-D-galacto-2-nonulopyranosyl-1→4-lactone)-(2→3)-O-(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl-(1→1)-(2S,3R,4E)-2 azido-3-benzoyl-4-octadecene-1,3-diol (Formula VII) (Figure 1b). [ka]

[0045] A solution of VI (2.95 g, 2.47 mmol) and azidosphingosine benzoate (2.11 g, 4.94 mmol) was prepared in dry CHCl (25 mL). Powdered 4 Å molecular sieves (5 g) were added and stirred at room temperature for 30 min. The mixture was cooled to 0 °C, and freshly distilled BF-OEt (0.65 mL, 5.17 mmol) was added and stirred at the same temperature for 2 h. After completion of the reaction, the mixture was filtered through a layer of Celite 545, and the filtrate was neutralized with EtN and evaporated to dryness under reduced pressure. The residue was purified by column chromatography (CHCl / acetone 15:1) to recover the remaining unreacted azidosphingosine benzoate, followed by CHCl / acetone 10:1 to obtain pure glycoside VII. f 0.58) as a homogeneous amorphous white solid. Yield 2.02 g (56%).

[0046] 1H NMR (600 MHz, CDCl3 ):δ 2,19; 2,13; 2,12; 2,10; 2,08; 2,06; 2,03 (2CH3); 2,01; 1,98 [(3H, s, CH3) x 10]; 8,05-8,00 (2H, m, orto Ar); 7,56 (1H, t, J = 7,4 Hz, para Ar); 7,44 (2H, t, J = 7,8 Hz, meta Ar); Glc:4,49 (1H, d, J = 7,7 Hz, H-1); 4,94 (1H, m, H-2); 5,15 (1H, m, H-3); 3,77 (1H, dd, J = 9,5 Hz, H-4); 3,50 (1H, m, H-5); 4,44 (1H, dd, J = 11,9; 2,1 Hz, H-6a); 4,10 (1H, m, H-6b); Gal:4,37 (1H, d, J = 8,0 Hz, H-1); 4,84 (1H, dd, J = 9,9; 7,9 Hz, H-2); 4,10 (1H, m, H-3); 4,94 (1H, m, H-4); 3,91 (1H, m, H-5); 4,64 (1H, dd, J = 12,0; 3,5 Hz, H-6a); 4,26 (1H, m, H-6b); Neu:2,45 (1H, dd, J = 13,9; 5,4 Hz, H-3ec); 1,79 (1H, dd, J = 13,9; 11,6 Hz, H-3ax); 5,53 (1H, m, H-4); 4,14 (1H, q, J = 10,4 Hz, H-5); 3,68 (1H, dd, J = 10,5; 1,8 Hz, H-6); 5,15 (1H, m, H-7); 5,19 (1H, ddd, J = 9,0; 6,1; 2,7 Hz, H-8); 4,26 (1H, m, H-9a); 3,91 (3H, m, H-9b); 5,97 (d, J = 10,2 Hz, 5-NH); 4,60 (d, J = 15,3 Hz, H-10a); 4,26 (1H, m, H-10a); Sph:3,85 (1H, dd, J = 10,5; 6,6 Hz, H-1a); 3,57 (1H, dd, J = 10,6;6,1 Hz, H-1b); 3,91 (1H, m, H-2); 5,59 (1H, dd, J = 8,1; 4,2 Hz, H-3); 5,53 (1H, m, H-4); 5,91 (1H, dt, J = 15,3; 6,7 Hz, H-5); 2,06 (2H, m, H-6);1,40 - 1,18 (22H, m, H-7 hasta H-17); 0,86 (3H, t, J = 7,0 Hz, H-18).; MALDI-TOF MS: [M+Na] + m / z 1485.93 (calculated 1485.58).

[0047] Synthesis of FO-[5-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-didesoxy-D-glycero-α-D-galacto-2-nonulopyranosyl-1→4-lactone]-(2→3)-O-(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl-(1→1)-(2R,3S,4E)-3-benzoyl-2-tetracosanamido-4-octadecene-1,3-diol (Formula VIII) (Figure 1b). [ka]

[0048] A solution of glycoside VII (2.02 g, 1.38 mmol) was dissolved in 70 mL of pyridine / HO / Et 23The resulting solid was prepared in a mixture of 10:1:0.3 v / v / v / v / v N (10:1:0.3 v / v / v / v / v), cooled to 0 °C, and a stream of HS gas was passed through for 1 h. The reactor was sealed, and the mixture was stirred at the same temperature. After 6 h, HS gas was bubbled through the mixture for another 1 h. After sealing the reactor, the mixture was stirred at 0 °C for 16 h. The progress of the reaction was monitored by CCD. Upon completion, the solvent was evaporated under reduced pressure, and the residue was coevaporated several times with toluene to remove the HO and pyridine residues it contained. The resulting solid was dissolved in CHCl (100 mL), and tetracosanoic acid (1.0 g, 2.75 mmol) was added, followed by EDC-HCl (0.79 g, 4.13 mmol), and the mixture was stirred at room temperature for 2 h. After completion of the reaction, the mixture was washed with water (5 × 20 mL), the organic phase was dried over anhydrous NaSO, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography using successive mixtures with different ratios of CHCl / acetone (15:1 → 10:1) to give VIII via CCD (toluene / acetone 2:1, R f 0.58) to give a homogeneous amorphous white solid. Yield 1.9 g (77%).

[0049] 1H NMR (600 MHz, CDCl )3δ 2.19; 2.14; 2.12; 2.10; 2.06; 2.03; 2.01 (2CH3); 1.98; 1.93 [(3H, s) x 10];8.03-7.97 (2H, m, Ar); 7.55 (1H, t, J = 7.4 Hz, Ar); 7.43 (1H, t, J = 7.8 Hz, Ar); Glc:4.42 (1H, d, J = 7.7 Hz, H-1); 4.90 (1H, dd, J = 9.6; 7.7 Hz, H-2); 5.17-5.10 (1H, m, H-3); 3.72 (1H, dd, J = 9.5 Hz, H-4); 3.54 (1H, ddd, J = 9.9; 5.4; 2.1 Hz, H-5); 4.31-4.28 (1H, m, H-6a); 4.02-3.97 (1H, m, H-6b); Gal:4.34 (1H, d, J = 7.9 Hz, H-1); 4.82 (1H, dd, J = 9.9; 7.9 Hz, H-2); 4.09 (1H, dd, J = 9.9; 3.8 Hz, H-3); 4.95-4.93 (1H, m, H-4); 3.89-3.86 (1H, m, H-5); 4.64 (1H, dd, J = 12.1; 3.4 Hz, H-6a); 4.28-4.22 (1H, m, H-6b); Neu:2.45 (1H, dd, J = 13.9; 5.4 Hz, H-3ec); 1.78 (1H, dd, J = 13.9; 11.6 Hz, 30 H-3ax); 5.57- 5.51 (1H, m, H-4); 4.14 (1H, q, J = 10.4 Hz, H-5); 3.68 (1H, dd, J = 10.5; 1.8 Hz, H-6); 5.17-5.10 (1H, m, H-7); 5.19 (1H, ddd, J = 9.1; 6.2; 2.8 Hz, H-8); 4.28-4.22 (1H, m, H-9a); 3.92 (1H, dd, J = 12.5; 6.3 Hz, H-9b); 5.98 (1H, d, J = 10.1 Hz, 5-NH); 4.60 (1H, d, J = 15.4 Hz, H-10a); 4.28-4.22 (1H, m, H-10b); Sph:3.61 (1H, dd, J = 10.1; 4.5 Hz, H-1a); 4.02-3.97 (1H, m, H-1b); 4.50-4.45 (1H, m, H-2); 5.73 (1H, d, J = 9.2 Hz, 2-NH); 5.57-5.51 (1H, m, H-3); 5.45 (1H, ddt, J = 15.3; 7.6; 1.5 Hz, H-4); 5.86 (1H, dt, J = 15.3; 6.8 Hz, H-5); 2.00 (2H, m, H-6); 1.38-1.15 (22H m, H-7~H-17); 5 0.87 (3H, t, J = 7.0 Hz, H-18); FA:2.18-2.15 (2H, m, H-2); 1.58 (2H, hepta, J = 6.6 Hz, H-3); 1.38- 1.15 (40H m, H-4~H-23); 0.87 (3H, t, J = 7.0 Hz, H-24). MALDI-TOF MS: [M+Na] + m / z 1809.76 (calculated 1809.94).

[0050] Synthesis of GO-[5-[5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonupyranosyl-1→4-lactone]-(2→3)-O-(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl-(1→1)-(2R,3S,4E)-3-benzoyl-2-tetracos-15-enamido-4-octadecene-1,3-diol (Formula IX) (Figure 1b) [ka]

[0051] Compound IX was obtained by the procedure described above for VIII using (Z) tetracos-15-enoic acid for acylation. Yield 1.85 g (75%).

[0052] 1H NMR (600 MHz, CDCl )3δ 2.19; 2.14; 2.12; 2.10; 2.06; 2.03; 2.01 (2 CH3); 1.98; 1.93 [ (3H, s) x 10];8.03-7.97 (2H, m, Ar); 7.55 (1H, dd, J= 7.4 Hz, Ar); 7.43 (2H, t, J= 7.7 Hz, Ar); Glc:4.42 (1H, d, J= 7.8 Hz, H-1); 4.90 (1H, dd, J = 9.7; 7.7 Hz, H-2); 5.17-5.10 (1H, m, H-3); 3.72 (1H, dd, J= 9.5 Hz, H-4); 3.54 (1H, ddd, J= 9.9; 5.4; 2.1 Hz, H-5); 4.31-4.28 (1H, m, H-6a); 4.02-3.98 (1H, m, H-6b); Gal:4.33 (1H, d, J= 8.0 20 Hz, H-1); 4.82 (1H, dd, J= 9.9; 7.9 Hz, H-2); 4.09 (1H, dd, J= 9.9; 3.8 Hz, H-3); 4.94 (1H, d, J= 3.9 Hz, H-4); 3.88 (1H, dd, J= 8.6; 3.4 Hz, H-5); 4.64 (1H, dd, J= 12.1; 3.4 Hz, H-6a); 4.28-4.23 (1H, m, H-6b); Neu:2.45 (1H, dd, J= 13.9; 5.4 Hz, H-3ec); 1.78 (1H, dd, J= 13.9; 11.5 Hz, H-3ax); 5.57-5.51 (1H, m, H-4); 4.14 (1H, q, J= 10.4 Hz, H-5); 3.68 (1H, dd, J= 10.5; 1.8 Hz, H-6); 5.17-5.10 (1H, m, H-7); 5.19 (1H, ddd, J= 9.1; 25 6.2; 2.8 Hz, H-8); 4.28-4.23 (1H, m, H-9a); 3.92 (1H, dd, J= 12.5; 6.2 Hz, H-9b); 5.97 (1H, d, J= 10.2 Hz, 5-NH); 4.60 (1H, d, J= 15.4 Hz, H-10a); 4.28-4.23 (1H, m, H-10b); Sph:3.61 (1H, dd, J= 10.0; 4.5 Hz, H-1a); 4.02-3.98 (1H, m, H-1b); 4.47 (1H, ddt, J= 11.3; 7.5; 4.1 Hz, H-2); 5.73 (1H, d, J= 9.2 Hz, 2-NH); 5.57-5.51 (1H, m, H-3); 5.48-5.42 (1H, m, H-4); 5.86 (1H, dt, J= 15.2; 6.8 Hz, H-5); 2.00 (2H, m, H-6); 1.38-1.15 30 (22H m, H-7~H-17); 0.87 (3H, t, J= 6.9 Hz, H-18); FA:2.18-2.07 (2H, m, H-2); 1.63-1.52 (2H, m, J= 6.5 Hz, H-3); 5.34 (2H, t, J= 4.8 Hz, CH=CH, H-15 and H-16); 1.38-1.15 (36H m, H-4~H-14 and H-17~H-23); 0.87 (3H, t, J= 6.9 Hz, H24). MALDI-TOF MS: [M+Na] + m / z 1807.75 (calculated 1807.93).

[0053] Synthesis of HO-[3,5-didesoxy-5-hydroxyacetamido-D-glycero-α-D-galacto-2-nonupyranosyl-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranosyl-(1→1)(2R,3S,4E)-2-tetracosanamido-4-octadecene-1,3-diol (Formula X) (Figure 1b) [ka]

[0054] A suspension of VIII (1.9 g, 1.06 mmol) in dry MeOH (70 mL) was prepared, and 1.3 M NaOMe / MeOH solution (10.6 mL, 13.8 mmol) was added and stirred at room temperature for 24 h. HO (18 mL) was added, and the mixture was stirred at room temperature for an additional 24 h. Upon completion, this was purified by IR-120 (H + The solution was neutralized by stirring with CCD (R) resin, the resin was removed by filtration, washed with MeOH, and the combined filtrates were evaporated to dryness under reduced pressure. The residue was dissolved in HO (200 mL), and the resulting solution was dialyzed against HO using a 3.5 KDa membrane at 4 °C for 24 h. The dialysate was lyophilized at a constant temperature of -40 °C for 24 h to separate ganglioside X from CCD (R) resin. f 0.56, CHCl3 / MeOH / 0.25% aqueous KCl 2 5:20:5) as a homogeneous white solid. Yield 1.3 g (95%).

[0055] 1H NMR (600 MHz, DMSO-d6):Glc:4.15 (1H, d, J =7.7 Hz, H-1); 3.03 (1H,q, J= 7.6 Hz, H-2); 3.35-3.25 (3H, m, H-3, H-4, H-5); 3.80-3.71 (1H, m, H-6a); 3.64-3.55 (1H, m, H-6b); Gal:4.18 (1H, d, J= 7.7 Hz, H-1); 3.35-3.25 (2H, m, H-2, H-5); 3.98 (1H, td, J = 9.6; 8.8; 3.7 Hz, H-3); 3.69 (1H, d, J= 3.9 Hz H-4); 3.51-3.42 (2H, m, H-6); Neu:2.752.71 (1H, m, H-3ec); 1.50-1.38 (1H, m, H-3ax); 3.80-3.71 (1H, m, H-4); 3.51-3.41 (2H, m, H-5, H-6); 7.77 (1H, d, J= 7.6 Hz, 5-NH); 3.21 (1H, dd, J= 9.2; 4.4 Hz, H-7); 3.64-3.55 (2H, m, H-8, H-9a); 3.35-3.25 (1H, m, H-9b); 3.92-3.82 (2 H, m, H-10); Sph:3.99 (1H, dd, J= 10.1; 4.5 Hz, H-1a); 3.39 (1H, dd, J= 10.2; 3.6 Hz, H-1b); 3.80-3.71 (1H, m, H-2); 7.49 (1H, d, J= 9.1 Hz, 2-NH); 3.92-3.82 (1H, m, H-3); 5.30 (1H, dd, J= 12.4; 5.6 Hz, H-4); 5.53 (1H, dq, J= 13.0; 6.4 Hz, H-5); 1.93 (2H, ヘプタ, J= 7.4 Hz; H-6); 1.34- 1.18 (22H, m, H-7~H-17); 0.85 (3H, t, J= 6.5 Hz, H-18).FA:2.01 (2H, q, J=7.3 Hz, H-2);1.50-1.38(2H, m, H-3);1.34-1.18(40H, m, H-4~H-23);0.85 (3H, t, J=30 6.5 Hz, H-24). MALDI-TOF MS: [M+Na] + m / z 1304.08 (calculated 1304.66).

[0056] Synthesis of IO-[3,5-didesoxy-5-hydroxyacetamido-D-glycero-α-D-galacto-2-nonupyranosyl-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranosyl-(1→1)-(2R,3S,4E)-2-tetracosa-15-enamido-4-octadecene-1,3-diol (Formula XI) (Figure 1b). [ka]

[0057] Compound XI was obtained from intermediate IX by the procedure described above for X. Yield 1.25 g (95%).

[0058] 1H NMR (600 MHz, DMSO-d6):Glc:4.15 (1H, d, J=7.8 Hz, H-1); 3.07-3.03 (1H, m, H-2); 3.36-3.24 (3H, m, H-3, H-4, H-5); 3.80-3.71 (1H, m, H-6a); 3.65-3.54 (1H, m, H-6b); Gal:4.19 (1H, d, J= 7.7 Hz, H-1); 3.36-3.24 (2H, m, H-2, H-5); 3.96 (1H, dd, J= 9.9; 3.0 Hz, H-3); 3.69 (1H, d, J= 3.6 Hz H-4); 3.52-3.42 (2H, m, H-6); Neu:2.72 (1H,dd, J= 12.1; 4.8 Hz, H-3ec); 1.50-1.37 (1H, m, H-3ax); 3.80-3.71 (1H, m, H-4); 3.52-3.42 (2H, m, H-5, H-6); 7.85 (1H, d, J= 6.9 Hz, 5-NH); 3.23-3.19 (1H, m, H-7); 3.65-3.54 (2H, m, H-8, H-9a); 3.36-3.24 (1H, m, H-9b); 3.92-3.82 (2 H, m, H-10); Sph:4.00 (1H, dd, J=10.2;4.6 Hz、H-1a);3.39 (1H, dd, J=10.5;3.2 Hz、H-1b);3.80-3.71(1H, m, H-2);7.49 (1H, d, J=9.1Hz, 2-NH);3.92-3.82(1H, m, H-3);5.37-5.31(1H, m, H-4);5.53 (1H, dt, J=14.2;6.7 Hz, H-5);1.96-1.88(2H, m, H-6);1.34-1.18(22H, m, H-7~H-17);0.85 (3H, t, J=6.5 Hz, H-18).FA:5.31 (2H, t, J=5.2Hz, CH=CH, H-15, H-16);2.01 (2H, t, J=7.4 Hz, H-2);1.97 (2H, q, J=6.6 Hz, H-14, H-17);1.50-1.37(2H, m, H-3);1.34-1.18(32H, m, H-4~H-13, H-18~H-23);0.85 (3H, t, J=6.5 Hz, H24). MALDI-TOF MS: [M+Na]+ m / z 1301.94 (calculated 1301.81).

[0059] In NMR characterization, the terms Glc, Gal, Neu, Sph, and FA refer to molecular fragments of hydrocarbon chains provided by the compounds glucose, galactose, neuraminyl, sphingosine hydrocarbon chains, and fatty acids, respectively.

[0060] Example 2. NGcGM3 d18:1-C24:1 has antimetastatic effects in the liver, unlike NGcGM3 extracted from natural sources. Syngeneic C57BL / 6 mice were treated with 2 × 10 5 EL-4 thymoma cells were inoculated intravenously via the tail vein. Mice were randomized and distributed into groups of 7–9 animals. Experimental groups received intraperitoneal treatment with 200 μg of synthetic and natural variants of NGcGM3 or vehicle (5.6% sucrose solution, 0.75% L-histidine, and 0.5% Tween 20) on days 1, 5, and 9. Animals were sacrificed 12–13 days after tumor inoculation, and their livers were removed and weighed to determine the level of liver metastasis. Liver weights of healthy animals of the same age and batch were used as controls. Figure 2 shows the individual liver weight values ​​for animals in each group, as well as the mean and standard deviation of the mean. As can be seen, treatment with NGcGM3 d18:1-C24:1 demonstrated a statistically significant anti-metastatic effect, and no differences were observed between the liver weights of tumor-bearing animals and healthy animals treated with NGcGM3 d18:1-C24:1. Treatment with NGcGM3 extracted from natural sources did not show such antimetastatic effects (same letters p>0.05, different letters p<0.0001, ANOVA and Tukey's test).

[0061] Example 3. Treatment with NGcGM3 d18:1-C24:1 increases survival of EL-4 tumor-bearing animals. Syngeneic C57BL / 6 mice were treated with 2 × 10 5EL-4 thymoma cells (ATCC TIB-39) were inoculated intravenously via the tail vein, and the mice were randomized and distributed into groups of 7–9 animals. Experimental groups received intraperitoneal treatments with 200 μg of the ganglioside mutant NGcGM3 d18:1-C24:1, NGcGM3 from natural sources, or vehicle (5.6% sucrose solution, 0.75% L-histidine, and 0.5% Tween 20) on days 1, 5, and 9. Animals were monitored and, according to veterinary standards, were sacrificed if they showed any signs or symptoms affecting their ability to consume food and water or compromising their general health. Figure 3 shows that 13 days after tumor cell inoculation, all mice treated with vehicle solution (placebo) or natural-source NGcGM3 died, while all animals treated with NGcGM3 d18:1-C24:1 survived and remained healthy. Thus, treatment with NGcGM3 d18:1-C24:1 significantly increased the survival rate of EL-4 tumor-bearing animals (p = 0.0005, Long-Rank Mantel-Cox chi-square test).

[0062] Example 4. P3X63 mouse myeloma cells express CD1d on their surface. CD1d expression in the mouse myeloma cell line P3X63 (P3X63Ag8.653, CRL 1580) was assessed by flow cytometry using a phycoerythrin-conjugated mouse anti-CD1d monoclonal antibody (clone 1B1, eBioscience). Cells were preincubated with α-CD16 / 32 monoclonal antibody to block nonspecific binding, and a fluorophore-conjugated antibody isotype (ARL2397, BioSource) was used as a control. Cells were acquired using a Sysmex Partec flow cytometer (Sachsen, Germany) and analyzed using FlowJo 10 software (Tree Star, USA). As can be seen in Figure 4, approximately 90% of P3X63 cells express CD1d on their surface.

[0063] Example 5. Administration of NGcGM3 d18:1-C24:1 inhibits high-load P3X63 myeloma tumor growth even better than αGalCer. 1×10 6 P3X63 cells were implanted subcutaneously into syngeneic BALB / c mice. Animals were randomized and divided into 6–8 groups and received intraperitoneal treatment with 200 μg of NGcGM3 d18:1-C24:1 ganglioside, αGalCer, or vehicle (5.6% sucrose solution, 0.75% L-histidine, and 0.5% Tween 20) on days 1, 5, and 9. Tumor diameters were measured with calipers, and tumor volume (TV) was calculated using the formula TV (mm). 3 ) = π / 6 × major axis × minor axis 2 Figure 5 shows the average TV per group and the distribution of individual measurements on day 5 after tumor cell implantation. The individual tumor volume values ​​for animals in each group on day 5 of the experiment, along with the mean and standard deviation of the mean, are shown. Table 1 shows the percentage distribution of tumor volume for animals in each group.

[0064] [Table 1]

[0065] Treatment with NGcGM3 d18:1-C24:1 and αGalCer significantly inhibited the growth of subcutaneous tumors, although at different levels of statistical significance (p<0.001 for NGcGM3 d18:1-C24:1, p<0.05 for αGalCer, ANOVA and Tukey's test). In addition, 25% of tumors grew to 36 mm 3 In the NGcGM3 d18:1-C24:1 treated group, 50% of the animals had a tumor size of 36 mm compared to the αGalCer treated group, which had a tumor size of less than 36 mm. 3 In the placebo group, tumors with a TV of 36 mm 3 More than 100 tumors were present in all animals.

[0066] Example 6. NGcGM3 ganglioside d18:1-C24:1 exhibits a potent stimulatory effect on iNKT cells in vitro, superior to other synthetic variants of NGcGM3 and gangliosides purified from erythrocytes. 5×10 4 bmDCs were grown in RPMI-1640 medium containing 10% FBS for 16–24 h in the presence of αGalCer at a concentration of 100 ng / ml, and native NGcGM3 and synthetic mutants NGcGM3 d18:1-C24:1, NGcGM3 d18:1-C24:0, NGcGM3 d18:1-C18:0, NGcGM3 d18:1-C18:1, and NGcGM3 d18:1-C18:2 at concentrations of 0.1, 1, and 10 μg / ml, or vehicle (0.1% DMSO). 5 FF13 hybridoma cells were added and co-cultured with bmDCs for 24 hours. Culture supernatants were collected and evaluated for IL-2 content by ELISA. Figure 6 shows that, in contrast to NGcGM3 ganglioside extracted from natural sources, which did not stimulate IL-2 secretion by iNKT cells at any of the concentrations tested, a synthetic variant of NGcGM3 at the highest concentration tested (10 μg / ml) was able to activate iNKT cell hybridomas. Differences were observed among the NGcGM3 variants in terms of antigenicity to iNKT cells based on the length and structure of the fatty acid in the ceramide. The d18:1-C24:1 NGcGM3 variant showed potent activation of FF13 hybridoma cells and was the only variant able to activate iNKT cells at the same concentration of αGalCer (100 ng / ml) and surpass the effect of αGalCer at a concentration of 10 μg / ml (p<0.05, ANOVA and Tukey's test).

[0067] Example 7. Administration of NGcGM3 d18:1-C24:1 activates iNKT cells in vivo, whereas NGcGM3 d18:1-C18:0 and NGcGM3 extracted from erythrocytes are inactive. C57BL / 6 mice were intraperitoneally injected with 200 μg of natural NGcGM3 ganglioside, NGcGM3 d18:1-C24:1, NGcGM3 d18:1-C18:0, and 2 μg of αGalCer or vehicle (5.6% sucrose, 0.75% L-histidine, and 0.5% Tween-20), and blood was collected 16 hours after administration. Serum IFNγ levels were assessed by ELISA as an indirect measure of iNKT cell activation (Mouse IFNγ ELISA Ready-SET-Go!, eBioscience). Figure 7 shows that IFNγ levels were higher in mice receiving NGcGM3 d18:1-C24:1 than in the remaining experimental groups and statistically higher than in animals in the placebo group (p<0.05) (Kruskal-Wallis, Dunn, NC). This result suggests in vivo activation of iNKT cells mediated by NGcGM3 d18:1-C24:1.

[0068] Example 8. Synthetic variants of ganglioside NGcGM3 have a greater stimulatory effect on iNKT cells than synthetic variants of ganglioside NAcGM3 with the same ceramide structure. 5×10 4 bmDCs were cultured in RPMI-1640 medium containing 10% FBS for 16-24 hours in the presence of 10 μg / ml and 1 μg / ml NGcGM3 d18:1-C24:1 ganglioside, GM3 d18:1-C24:1, or vehicle (0.1% DMSO). 5 FF13 hybridoma cells were added and co-cultured with bmDCs for 24 hours. Culture supernatants were collected, and IL-2 content was quantified by ELISA. Figure 8 shows that the synthetic ganglioside mutants NGcGM3 and GM3, which have the same ceramide structure, differ in their ability to stimulate iNKT cells. The NGcGM3 mutant exhibits a stronger effect on iNKT cell activation than the GM3 mutant (ANOVA, Tukey's test).

[0069] Example 9. Nanoparticles of NGcGM3 d18:1-C24:1 and N. meningitidis OPMC exhibit potent antimetastatic effects in the liver, in contrast to nanoparticles formed with NGcGM3 d18:1-C18:0 or NGcGM3 extracted from red blood cells. Syngeneic C57BL / 6 mice were treated with 2 × 10 5 EL-4 thymoma cells were inoculated intravenously via the tail vein, and the mice were randomized and distributed into groups of 7–9 animals. Experimental groups received intraperitoneal treatments with 10–30 μg of nanoparticles of NGcGM3 d18:1-C24:1, NGcGM3 d18:1-C18:0, or native NGcGM3 with N. meningitidis OMPC, or with Tris / HCl solution (placebo) on days 1, 5, and 9. Animals were sacrificed 12–13 days after tumor inoculation. To determine the level of liver metastatic spread, their livers were excised and weighed. Figure 9 shows the individual liver weight values ​​for animals in each group, as well as the mean and standard deviation of the mean. Notably, treatment with nanoparticles containing NGcGM3 d18:1-C24:1 ganglioside showed a statistically significant anti-metastatic effect compared to the placebo group, whereas groups treated with nanoparticles containing NGcGM3 d18:1-C18:0 ganglioside or gangliosides obtained from natural sources did not show such an effect (same letters p>0.05, different letters p=0.0015, ANOVA and Tukey's test).

[0070] Example 10. Administration of NGcGM3 d18:1-C24:1 incorporated into nanoparticles containing N. meningitidis OMPC inhibits the growth of high tumor burden P3X63 murine myeloma even better than αGalCer and systemic NGcGM3 d18:1-C24:1. 1×10 6P3X63 myeloma cells were implanted subcutaneously into syngeneic BALB / c mice. Animals were randomized and divided into groups of 6–8 and treated on days 1, 5, and 9 with vehicle solution (5.6% sucrose, 0.75% L-histidine, and 0.5% Tween-20), αGalCer, NGcGM3 d18:1-C24:1, or OMPC and NGcGM3 d18:1-C24:1 nanoparticles (GlycoVax 24:1). Subcutaneous tumor diameters were measured every 2–3 days with a vernier caliper and calculated as TV (mm). 3 ) = π / 6 × major axis × minor axis 2 TV was calculated according to the formula (1). Figure 10 shows the TV for individual animals in each group, as well as the mean and standard deviation of each case, on day 5 after tumor cell implantation, at which point all animals in the placebo group had measurable tumors. In contrast to treatment with αGalCer, which did not demonstrate a statistically significant antitumor effect, treatment with NGcGM3 d18:1-C24:1 and GlycoVax 24:1 demonstrated significant inhibition of tumor growth compared to the control group (p = 0.0031 and p = 0.0004, respectively, nonparametric ANOVA, Kruskal-Wallis, Dunn's multiple comparison test).

[0071] Table 2 shows the percentage distribution of tumor volume in animals in each group.

[0072] [Table 2]

[0073] Treatment with GlycoVax 24:1 demonstrated the strongest antitumor effect, with 75% of animals achieving tumor size of 36 mm 3 In the group treated with systemic NGcGM3 d18:1-C24:1 ganglioside, 50% of the animals had tumors of 36 mm 3 Treatment with αGalCer reduced tumor volume to 36 mm in 25% of animals. 3 was reduced to less than

[0074] Example 11. Nanoparticles of NGcGM3 d18:1-C24:1 and N. meningitidis OMPC activate iNKT cells, unlike nanoparticles obtained from the incorporation of NGcGM3 d18:1-C18:0 or NGcGM3 extracted from natural sources. 5×10 4 bmDCs were cultured in RPMI-1640 medium containing 10% FBS for 16-24 hours in the presence of nanoparticles at concentrations of 100 ng / ml, 1 μg / ml, or 10 μg / ml, αGalCer, or vehicle (0.1% DMSO). 5 The nanoparticles were co-cultured with FF13 hybridoma cells for 24 hours. The culture supernatants were collected, and the IL-2 content was quantified by ELISA. Figure 11 shows that nanoparticles containing NGcGM3 d18:1-C24:1 ganglioside (GlycoVax 24:1) activated iNKT cell hybridomas similarly to αGalCer at concentrations of 10 μg / ml and 1 μg / ml, and such an effect was maintained at a concentration of 100 ng / ml. In the case of nanoparticles obtained using NGcGM3 ganglioside extracted from natural sources, no activation of iNKT cell hybridomas was observed at any of the concentrations tested, and only a moderate effect was observed with nanoparticles containing NGcGM3 d18:1-C18:0 ganglioside at the highest concentration evaluated (10 μg / ml).

[0075] Example 12. Adoptive transfer of dendritic cells incubated with NGcGM3 d18:1C24:1 produces a potent anti-metastatic effect in the liver, whereas transfer of dendritic cells incubated with NGcGM3 extracted from erythrocytes has no effect. Syngeneic C57BL / 6 mice were treated with 2 × 10 5 Mice were inoculated intravenously via the tail vein with EL-4 thymoma cells, randomized, and divided into groups of 7–9 animals. The day after tumor inoculation, 6 × 10 cells were cultured with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 ganglioside (10 μg / ml), or NGcGM3 extracted from natural sources (10 μg / ml) for 16 h. 5bmDCs were administered intravenously in a single dose. Cells were thoroughly washed with serum-free RPMI-1640 medium to remove excess lipids before transfer. 12–13 days after tumor inoculation, animals were sacrificed, and livers were harvested and weighed to determine the level of liver metastasis. Livers from healthy mice of the same age and batch were used as a reference. Figure 12 shows the individual liver weights of animals in each group, as well as the mean and standard deviation of the mean. Clearly, transfer of bmDCs pulsed with NGcGM3 d18:1-C24:1 induced a significant reduction in EL-4 liver metastasis compared to the placebo group transferred with empty dendritic cells. Similarly, transfer of bmDCs incubated with gangliosides extracted from natural sources had no effect (same letters p > 0.05, different letters p < 0.05, ANOVA and Tukey's test).

[0076] Example 13. Adoptive transfer of dendritic cells incubated with NGcGM3 d18:1C24:1 increases the survival rate of tumor-bearing animals, in contrast to the transfer of dendritic cells incubated with NGcGM3 extracted from erythrocytes. Syngeneic C57BL / 6 mice were treated with 2 × 10 5 Mice were inoculated intravenously via the tail vein with EL-4 thymoma cells, randomized, and divided into groups of 7–9 animals. The day after tumor inoculation, 6 × 10 cells were cultured for 16 h with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 ganglioside (10 μg / ml), or ganglioside extracted from natural sources (10 μg / ml). 5bmDCs were administered intravenously in a single dose. Cells were thoroughly washed with serum-free RPMI-1640 medium to remove excess lipids before transfer. Mice were monitored according to veterinary standards for signs or symptoms affecting their ability to consume food and water or general health. Figure 13 shows that 13 days after tumor cell inoculation, all animals in the placebo group receiving empty dendritic cells and all animals receiving cells pulsed with naturally occurring NGcGM3 died. In contrast, all animals (100%) in the group receiving bmDCs exposed to NGcGM3 d18:1-C24:1 were alive and healthy. Statistically distinct survival curves indicate a significant increase in survival (p<0.0001) for animals treated with bmDCs precultured in the presence of NGcGM3 d18:1-C24:1 (Long-Rank Mantel-Cox, chi-square test).

[0077] Example 14. Adoptive transfer of dendritic cells incubated with NGcGM3 d18:1-C24:1, in contrast to transfer of dendritic cells incubated with NGcGM3 extracted from red blood cells, reduces tumor growth in P3X63 murine myeloma-bearing animals. 1×10 6 P3X63 cells were implanted subcutaneously into syngeneic BALB / c mice. Animals were randomized and divided into groups of 6–8. The day after implantation, 6 × 10 cells were implanted subcutaneously into syngeneic BALB / c mice. The cells were then pre-incubated for 16 h with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 ganglioside (10 μg / ml), or ganglioside extracted from natural sources (10 μg / ml). 5 The cells were given a single intravenous injection of 1000 bmDCs. Cells were thoroughly washed with serum-free RPMI-1640 medium to remove excess lipids before transfer. After tumor growth, tumor diameter was measured with a caliper and calculated using the formula TV (mm 3 ) = π / 6 × major axis × minor axis 2The TV was determined according to the method described above. Figure 14 shows that the transfer of bmDCs pulsed with NGcGM3 d18:1C24:1 induced an antitumor effect by significantly (p<0.0001) reducing tumor growth compared to the group treated with empty bmDCs. When bmDCs incubated with gangliosides extracted from natural sources were transferred, no antitumor effect was observed (ANOVA and Tukey's test).

[0078] Example 15. Transfer of dendritic cells incubated with NGcGM3 d18:1-C24:1, as opposed to dendritic cells incubated with NGcGM3 extracted from red blood cells, results in the activation of NKT cells in vivo in BALB / c mice. 1×10 6 P3X63 cells were implanted subcutaneously into syngeneic BALB / c mice. Animals were randomized and divided into groups of 6–8. The day after implantation, 6 × 10 cells were implanted into the subcutaneous tissue of the mice, which had been pre-incubated for 16 h with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 (10 μg / ml), or gangliosides extracted from natural sources (10 μg / ml). 5 Animals received a single intravenous injection of bmDCs. Cells were thoroughly washed with serum-free RPMI-1640 medium to remove excess lipids before transfer. 16 hours after transfer, blood was collected from 2–3 animals in each group, and serum IFNγ levels were assessed by ELISA. Figure 15 shows that both empty bmDC transfer and bmDC transfer pulsed with NGcGM3 extracted from red blood cells failed to stimulate early IFNγ secretion. However, in animals transferred with bmDCs precultured in the presence of NGcGM3 d18:1-C24:1, significant levels of IFNγ were detected 16 hours after treatment. This early IFNγ secretion is associated with iNKT cell activation.

[0079] Example 16. Transfer of dendritic cells incubated with NGcGM3 d18:1-C24:1, as opposed to dendritic cells incubated with NGcGM3 extracted from red blood cells, results in the activation of NKT cells in vivo in C57BL / 6 mice. Syngeneic C57BL / 6 mice were treated with 2 × 10 5 Mice were inoculated intravenously via the tail vein with EL-4 thymoma cells, randomized, and divided into groups of 7–9 animals. The day after tumor inoculation, 6 × 10 cells were cultured with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 (10 μg / ml), or gangliosides extracted from natural sources (10 μg / ml) for 16 h. 5 bmDCs were administered intravenously in a single dose. Cells were thoroughly washed with serum-free RPMI-1640 medium to remove excess lipids before transfer. 16 hours after transfer, blood was collected from 2–3 animals in each group, and serum IFNγ levels were assessed by ELISA. Figure 16 shows that transfer of vehicle-pulsed bmDCs and bmDCs pulsed with natural source NGcGM3 did not activate iNKT cells, whereas transfer of bmDCs pre-pulsed with NGcGM3 d18:1-C24:1 induced iNKT cell activation with early IFNγ secretion.

[0080] Example 17. NGcGM3 d18:1-C24:1 incorporated into nanoparticles bearing N. meningitidis OMPC induces strong specific antibody responses when administered to mice, unlike NGcGM3 d18:1-C18:0 and NGcGM3 extracted from red blood cells. Syngeneic C57BL / 6 mice were treated with 2 × 10 5EL-4 thymoma cells were inoculated intravenously via the tail vein, and the mice were randomized and distributed into groups of 7–9 animals. Experimental groups received intraperitoneal treatments with 10–30 μg of nanoparticles containing N. meningitidis OMPC containing NGcGM3 d18:1-C24:1, NGcGM3 d18:1-C18:0, or NGcGM3 extracted from natural sources, or with a placebo in Tris / HCl solution on days 1, 5, and 9. On day 13, blood was collected from 3–4 animals per group, and specific IgM and IgG antibody responses to NGcGM3 were assessed by ELISA. For this purpose, a single serum pool per group was prepared from the sera of each individual animal. For detection of NGcGM3-specific antibodies and titration by ELISA, NUNC PolySorp 96-well plates were coated with 0.16 nmol / well of NGcGM3 (natural source) dissolved in methanol. The solvent was evaporated for 1–2 h at 37°C, and a 16–24 h blocking step was performed using 1% (m / v) lipid-free bovine serum albumin fraction V in phosphate-buffered saline (PBS). After addition of serum at the dilution range to be evaluated and washing with PBS-Tween 20, 0.05% (v / v) biotinylated goat anti-IgM mouse IgG antibody (Sigma) was added at a 1 / 5000 dilution in blocking solution. After 1 h of incubation at 37°C, alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch) was added at a 1 / 2000 dilution in blocking solution. After 1 h at 37°C, the enzymatic reaction was visualized using 1 mg / ml p-nitrophenyl phosphate (PNPP) dissolved in 1 M diethanolamine buffer (pH 9.6) and 1 mM MgCl2. Absorbance was read at 405 nm. To eliminate the influence of nonspecific signals, serum was also analyzed in empty wells containing only methanol. The absorbance at each serum dilution was corrected by subtracting the value from the empty wells. The serum titer was defined as the reciprocal of the highest dilution that resulted in a final absorbance value greater than 0.1. Figure 17 shows that anti-NGcGM3 antibody responses were present only in animals injected with nanoparticles containing NGcGM3 d18:1-C24:1.The resulting IgM and IgG antibody titers of 1 / 320 specific for NGcGM3 may have been considered extraordinary due to the lipid nature of this antigen and the short time frame of only 13 days. Nanoparticles formulated with gangliosides extracted from natural sources or NGcGM3 d18:1-C18:0 did not generate an anti-NGcGM3 antibody response.

[0081] Example 18. The antitumor activity of NGcGM3 d18:1-C24:1 ganglioside is NKT cell dependent. Syngeneic C57BL / 6 mice were treated intraperitoneally with 1 mg / 200 μL PBS of anti-NK1.1 depleting antibody (clone PK136, ATCC) or 20 μL / 200 μL PBS of polyclonal anti-asialoGM1 antibody (OriGene Technologies). Treatment with anti-NK1.1 antibody eliminated over 90% of the NK and NKT cell populations, while anti-asialoGM1 antibody was used to selectively deplete the NK cell population. 48 hours after antibody treatment, all animals, including the two groups of mice that received 200 μL of PBS intraperitoneally as a control, received 2 × 10 5 EL-4 thymoma cells were inoculated intravenously. On days 1, 5, and 9 after tumor inoculation, the experimental groups received intraperitoneal treatment with 200 μg of NGcGM3 d18:1-C24:1 or vehicle (placebo group) according to the following experimental group distribution: Group 1: Placebo + PBS Group 2 NGcGM3 d18:1-C24:1, 200μg / 200μL+PBS Group 3: Placebo + αNK1.1 Group 4 NGcGM3 d18:1-C24:1, 200μg / 200μL+αNK1.1 Group 5: Placebo + α-asialo GM1 Group 6 NGcGM3 d18:1-C24:1, 200μg / 200μL+α-SialoGM1

[0082] Equal doses of depleting antibodies were administered on days 4 and 11 after tumor inoculation to maintain depletion of the relevant populations throughout the experimental period. Animals were sacrificed 12–13 days after tumor inoculation, and livers were removed and weighed to determine the level of liver metastasis. Livers from healthy mice of the same age and batch were used as controls. Figure 18a shows the effect of depletion of NKT and NK cell populations on the antitumor effect of NGcGM3 d18:1-C24:1 treatment. The anti-metastatic effect of NGcGM3 d18:1-C24:1 treatment was present in mice in which NK and NKT cell populations were unaffected, whereas the absence of these populations in animals treated with anti-NK1.1 Mab eliminated the aforementioned antitumor effect (same letters p > 0.05, different letters p < 0.0001, ANOVA and Tukey's test). These results suggest that the antitumor effect of NGcGM3 d18:1-C24:1 treatment is dependent on NK or NKT cells. However, when we evaluated the antitumor effect of NGcGM3 d18:1-C24:1 treatment in animals depleted of only the NK cell population (Figure 18b), we observed that the antitumor effect was maintained despite population depletion (same letter p>0.05, different letters p<0.0001, ANOVA and Tukey's test), indicating that this population is not directly responsible for the observed antimetastatic effect. Collectively, these results suggest that the antitumor effect of NGcGM3 d18:1-C24:1 is primarily dependent on NKT cell activity.

Claims

1. formula [Equation 1] (Wherein R is -C ２３ H ４７ and -C ２３ H ４５ and corresponding to the anomeric α-trisaccharide.

2. A pharmaceutical composition comprising the ganglioside of claim 1 as an active substance and a pharmaceutically acceptable vehicle.

3. The composition of claim 2 containing another immunomodulatory agent.

4. A composition according to any one of claims 2 to 3, comprising an antigen.

5. The composition of claim 2 in the form of nanoparticles or liposomes.

6. 6. The composition of claim 5, wherein the nanoparticles are formed by hydrophobic insertion of one or more gangliosides according to any one of claims 1 to 2 into a hydrophobic outer membrane protein of a gram-negative bacterium.

7. 7. The composition of claim 6, wherein the hydrophobic outer membrane protein is derived from the bacterium Neisseria meningitidis.

8. 10. Use of the ganglioside of claim 1 in the manufacture of a medicament for the treatment of cancer and its metastasis.

9. 10. Use of a pharmaceutical composition according to any one of claims 2 to 7 in the manufacture of a medicament for the treatment of cancer and its metastases.

10. A method for treating a mammal suffering from cancer, comprising administering to the mammal a therapeutically effective amount of the ganglioside of claim 1, which has activity against the malignant tumor and its metastasis.

11. The method of claim 10, wherein the tumor is CD1d positive or CD1d negative.

12. 11. The method of claim 10, wherein the metastasis is to the liver.

13. 13. The method of any one of claims 10 to 12, wherein the compound is provided as a nanoparticle or a liposome.

14. 13. The method of any one of claims 10 to 12, wherein the mammal is a human.

15. 10. An in vitro method for preparing dendritic cells loaded with gangliosides according to claim 1, comprising: a) incubating dendritic cells obtained from a mammal with the ganglioside, b) using the resulting cells in adoptive cell transfer therapy A method comprising:

16. 10. An in vitro method for preparing dendritic cells loaded with the pharmaceutical composition of any one of claims 2 to 6, comprising: a) incubating dendritic cells obtained from a mammal with the composition, b) using the cells obtained in a) in adoptive cell transfer therapy. A method comprising:

17. 17. The method of any one of claims 15 to 16, wherein the mammal is a human.

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