Synthetic variants of ganglioside NGcGM3 and their use in treatment of cancer

Abstract

The invention relates to the field of biotechnology, in particular in the aspect of cancer immunotherapy. Described herein are synthetic variants of GM3 ganglioside (NGcGM3) containing glycoacylated sialic acid and compositions comprising said derivatives and methods for producing the same. The compounds are useful for the treatment of malignancies and metastases thereof. According to the invention, the anti-tumor effect of these derivatives is at least partially attributed to their ability to stimulate iNKT cells.

Description

Technical Field

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

[0002] In the prior art of natural or synthetic compounds derived from α-galactosylceramide (αGalCer), the presence of glycosphingolipids with anti-tumor activity and as stimulators of invariant NKT (iNKT) cells has been convincingly described (U.S. Patent No. 5,936,076).

[0003] In contrast, for other types of glycolipids, particularly gangliosides, the evidence for them being iNKT cell-activating compounds is conflicting and poorly understood, and to our knowledge, there is no evidence of their anti-tumor activity. Existing experimental results have shown that ganglioside GD3 is a tumor-associated glycolipid that can induce CD1d-restricted iNKT cell responses (Park et al., Immunology, 2008, 123:145-155), while ganglioside GM3 seems to have an inhibitory effect on this response, as co-immunization of mice with antigen-presenting cells (APCs) pulsed with this glycolipid and GD3 prevented iNKT cell activation.

[0004] Conversely, results have shown how GD3 inhibits the innate immune response in ovarian cancer by inhibiting the activation of iNKT cells (Webb et al., Can. Res., 2012, 72:3744-3752), indicating how these tumors avoid the anti-tumor effects of iNKT cells through this ganglioside, thus providing an early escape mechanism.

[0005] Paget et al. also found the same contradiction, namely that gangliosides GM3 and GD3 may be endogenous activators of iNKT cells, depending on the structural changes that occur to ceramides within dendritic cells (DCs) stimulated by Toll-like receptor (TLR) agonists (Paget et al., PLoS Biology, 2019, 17(3):e3000169). They found that synthetic variants of GM3 and GD3 with acetylated sialic acid (NAcGM3 and NAcGD3) and with d18:1-C24:1 ceramide were both able to activate iNKT cells in a CD1d-dependent manner. However, Paget et al. did not teach that synthetic variants of GM3 containing glycolyl-acetylated sialic acid (NGcGM3) have any iNKT cell activation ability, nor did they show evidence of anti-tumor activity for NAcGM3 or NAcGD3 d18:1-C24:1.

[0006] On the other hand, it has been demonstrated that NGcGM3 is an evolutionarily fixed tumor neoantigen and thus of interest for cancer immunotherapy, which forms the basis of the different strategies described (Labrada et al., Semin Oncol, 2018, 45:41-51). Based on the following experimental facts, the prior art supports the identification of NGcGM3 as a tumor antigen: reducing the expression of this ganglioside in tumor cell lines decreased the growth and metastatic spread of solid tumors in mice. Of particular significance for the present invention is the observation of a reduction in subcutaneous tumors generated by implanting P3X63 murine myeloma cells (highly expressing NGcGM3) pretreated with a glucosylceramide synthase inhibitor, thus demonstrating how this ganglioside acts as a stimulator of tumor progression (deLeon et al., Cancer Immunol Immunother, 2006, 55:443-450).

[0007] Based on this evidence, no one has described the direct anti-tumor application of the NGcGM3 ganglioside; although the immunotherapy methods described to date have identified NGcGM3 as an antigen. Among them, the one closest to the present invention is the GlycoVaxGM3 vaccine, which is a nanoparticulated product obtained by combining NGcGM3 with the outer membrane protein complex of Neisseria meningitidis. This vaccine induces specific antibodies against the ganglioside in both experimental animals and cancer patients (Labrada et al., Semin Oncol, 2018, 45:41-51). However, the prior art shows 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] Previously, there was evidence that NGcGM3 could bind to CD1d (Gentilini et al., Cancer Immunol Immunother, 2016, 65:551 - 562), which was consistent with the findings of the authors of the present invention. However, this study did not show evidence that this ganglioside could directly activate iNKT cells, as it started with a population of CD3 cells purified from human peripheral monocytes, which were activated by αGalCer and IL - 2 in culture. This procedure increased the number of iNKT cells, demonstrating their ability to bind to a CD1d - IgG1 fusion protein containing NGcGM3. These experiments were conducted using a natural mixture of NGcGM3, so it was not possible to determine whether any specific molecular species was responsible for the described effect, and they also did not teach any anti - tumor effect on the iNKT NGcGM3 population. + cell population, which were activated by αGalCer and IL - 2 in culture. This procedure increased the number of iNKT cells, demonstrating their ability to bind to a CD1d - IgG1 fusion protein containing NGcGM3. These experiments were conducted using a natural mixture of NGcGM3, so it was not possible to determine whether any specific molecular species was responsible for the described effect, and they also did not teach any anti - tumor effect on the iNKT NGcGM3 + population. Summary of the Invention

[0010] One object of the present invention is a synthetic ganglioside of NGcGM3 represented by formula A, which has anti - tumor and anti - metastatic activities.

[0011] Formula A

[0012]

[0013] wherein R is selected from the group consisting of:

[0014] C 23 H 47 and

[0015] C23H45

[0016] In addition, the present invention relates to pharmaceutical compositions comprising the ganglioside shown in A as an active ingredient and a pharmaceutically acceptable vehicle. In particular, these compositions may comprise another immunomodulator. Additionally, they may have antigens. Such compositions can be in the form of nanoparticles or liposomes. Specifically, the nanoparticles are formed by hydrophobically inserting one or more of the gangliosides described herein into the hydrophobic outer membrane protein of Gram - negative bacteria, especially Neisseria meningitidis.

[0017] Another embodiment of the present invention is the use of the ganglioside represented by formula A or a pharmaceutical composition containing the same in the preparation of a drug for treating cancer and its metastases.

[0018] In yet another embodiment, the present invention comprises administering a therapeutically effective amount of a ganglioside as described herein or a pharmaceutical composition comprising the same in the form of nanoparticles or liposomes to a mammal, particularly a human, suffering from cancer. The tumor to be treated can be CD1d positive or CD1d negative. In particular, liver metastases are treated.

[0019] In another embodiment, the present invention describes an in vitro method for preparing dendritic cells loaded with a ganglioside of formula A or a pharmaceutical composition comprising the same, and the method is based on: incubating dendritic cells obtained from a mammal, particularly a human, with the ganglioside, and using the obtained cells for adoptive cell transfer therapy. DETAILED DESCRIPTION OF THE INVENTION

[0021] The synthetic variants of interest of the ganglioside NGcGM3 according to the present invention and as described above are represented by formula A (i.e., formula X and XI). The ceramide composition of the two compounds has a common sphingosine (an amino alcohol having 18 carbon atoms and an unsaturated hydrocarbon chain); while they differ in the fatty acid, which is tetracosanoic acid in compound X and nervonic acid in the case of compound XI.

[0022] SYNTHETIC METHOD

[0023] For the synthesis of the ganglioside NGcGM3 represented by formula A, a protocol developed by the present inventors was used. First, sialylation of the hexa-benzylated lactose acceptor was carried out with a phenylthio-type N-glycolylneuraminic acid donor. After isolation of the target α-anomeric trisaccharide by formation of its 1→4 lactone (the anomeric trisaccharide β does not undergo lactonization), it still plays a protective role in subsequent series of synthetic steps. The synthetic process used includes the following steps:

[0024] a) Debenzylation by hydrogenolysis

[0025] b) Complete O-acetylation of the resulting derivative

[0026] c) Selective removal of the acetyl group attached to the anomeric carbon of the trisaccharide glucose unit

[0027] d) Preparation of trichloroacetimidate trisaccharide donor

[0028] e) Glycosylation of azidosphingosine benzoate

[0029] f) Reduction of the azido group of the synthesized glycoside

[0030] g) Acylation of the formed amino functional group using a second lipid chain carrier reagent and final removal of the protecting groups (including the lactone) from the resulting product

[0031] By means of Figure 1aWith respect to a and b, the synthetic steps of the compound represented by Formula A above can be better understood. In these figures, the following abbreviations are used:

[0032] Abbreviations of chemical structures: Ac: acetyl; Bn: benzyl; Bz: benzoyl; Me: methyl; SPh: phenylthio.

[0033] Abbreviations of 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; NH4OAc: ammonium acetate; NIS: N-iodosuccinimide; TfOH: trifluoromethanesulfonic acid.

[0034] Source and preparation of the lipid antigens used

[0035] According to the method described in (Svennerholm L. Biochem Biophys Acta 1957, 24: 604-11), the gangliosides used were quantified by a colorimetric resorcinol assay to detect lipid-bound sialic acid. The αGalCer (KRN7000) used as a reference was purchased from Enzo Life Science (Farmingdale, NY, USA) or Avanti Polar lipids (Alabaster, AL, USA). Initially, all lipids were dissolved in a 2:1 chloroform:methanol mixture and divided into aliquots suitable for daily use. The solvent was evaporated and the dried aliquots were stored at -20 °C for use. For in vitro experiments, a stock solution of glycolipid was prepared in anhydrous dimethyl sulfoxide (DMSO) and sonicated, while for in vivo experiments, the lipids were dissolved in a vehicle solution containing 5.6% sucrose, 0.75% L-histidine, and 0.5% Tween-20.

[0036] Hybridomas of iNKT cells

[0037] The FF13 mouse iNKT cell hybridomas previously described by Schumann J. (Schumann J. Eur J Immunol. 2007, 37:1431 - 41) were provided by Dr. Lucia Mori (University Hospital Basel, Switzerland) and cultured in RPMI - 1640 medium containing GlutaMAX - I and 25 mM HEPES, which was supplemented with 10% heat - inactivated fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin, and 55 μM 2 - mercaptoethanol.

[0038] Obtaining mature dendritic cells

[0039] Dendritic cells derived from mature bone marrow precursors (bmDC) were used as APCs for in vitro iNKT cell activation assays and in vivo adoptive transfer experiments. bmDC were differentiated from bone marrow progenitors in the presence of granulocyte / macrophage colony - stimulating factor (GM - CSF) according to a previously described protocol (Inaba K. J Exp Med. 1992, 176:1693 - 1702) with some modifications. Briefly, bone marrow progenitors 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). On day 3, the cultures were replenished with fresh medium, and the maturation of bmDC was induced with 1 μg / ml LPS. On day 7, the cells were harvested and used for iNKT cell activation assays or adoptive transfer.

[0040] Obtaining mixed nanoparticles of NGcGM3 d18:1 - C24:1 or NGcGM3 d18:1 - C24:0 with bacterial outer membrane protein complex (OMPC)

[0041] Disperse the OMPC of Gram-negative bacteria from the group consisting of Neisseria meningitidis (N. meningitidis), Salmonella typhi, Salmonella enteritis, Haemophilus influenzae, Bordetella pertussis or Escherichia coli (E. coli) in a 0.01 M Tris-HCL buffer solution at pH 8.5 at a final concentration of 0.5 to 3 mg / ml, place it in an oscillating reactor for 12 hours, where sodium deoxycholate (DOC) is 10 to 15 mM, and sodium dodecyl sulfate (SDS) is 0.25 to 5 mM. Next, add synthetic ganglioside NGcGM3 18:1-24:1 or NGcGM3 18:1-24:0 equivalent to the added mass of OPMC, and continue to oscillate for 3 to 10 hours. Then use a tangential filtration system with a 10-100 kDa membrane to remove the detergent. Ultracentrifuge the ultrafiltration solution at 100,000 g for 0.5 to 2 hours. Concentrate the supernatant to adjust its concentration to the required dose, and sterilize it by filtering through a capsule sterilizing filter with a pore size of 0.2 μm.

[0042] The dose of the nanoparticles used for treating a subject suffering from cancer or its metastasis is 10 μg to 2 mg per injection, preferably 30 μg to 1 mg.

[0043] Obtain mixed nanoparticles of NGcGM3 d18:1-C24:1 or NGcGM3 d18:1-C24:0 and bacterial outer membrane protein complex (OMPC), which may also contain immunomodulators and / or tumor antigens

[0044] In another embodiment of the present invention, mixed nanoparticles as described above are prepared, and these mixed nanoparticles additionally contain an immunomodulator selected from Toll-like receptor (TLR) agonists 3, 7 or 9.

[0045] Mixed nanoparticles as described above can also be obtained, which include tumor antigens, preferably neoantigens from public mutations and private mutations.

[0046] The compounds of the present invention represented by formula A (i.e., formula X and XI) have anti-tumor, anti-metastatic and immunostimulatory activities and can be used to treat tumors and their metastases.

[0047] (1) Anti-tumor activity

[0048] As shown in Examples 5, 10, and 14, when murine myeloma P3X63 Ag 8.653 (ATCC NCRL 1580) (X63) was inoculated into mice in the SQ manner, the compounds of Formulas X and XI, the formulations with nanoparticles obtained by inserting these gangliosides into a mixture of hydrophobic lipids or proteins from microorganisms as mediators, and the preparations of dendritic cells pulsed with an effective amount of the ganglioside represented by Formula A exhibited anti-tumor activity against these cells.

[0049] (2) Anti-metastatic activity

[0050] As shown in Examples 2, 3, 9, 12, 13, and 18, when cells of murine EL4 thymoma were inoculated via the tail vein of mice, the compounds of Formulas X and XI, the formulations with nanoparticles obtained by inserting these gangliosides into a mixture of lipids or hydrophobic proteins from microorganisms as mediators, and the preparations of dendritic cells pulsed with an effective amount of the ganglioside represented by Formula A had liver anti-metastatic activity against murine EL4 thymoma.

[0051] (3) Efficacy against human tumors expressing CD1d

[0052] The compounds of Formulas X and XI, the formulations with nanoparticles obtained by inserting these gangliosides into a mixture of lipids or hydrophobic proteins from microorganisms as mediators, and the preparations of dendritic cells pulsed with an effective amount of the ganglioside represented by Formula A can be successfully used against CD1d-positive hematological malignancies such as multiple myeloma, and can also be used against human solid tumors expressing CD1d and their metastases. These include lung cancer, head and neck cancer, prostate cancer, neuroblastoma and other brain tumors, melanoma, colorectal cancer, and renal cancer (Ingram, Z. Cells 2021, 10, 1329). Similarly, these treatments can also be effective against CD1d-negative tumors and metastases. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1. Schematic diagram of overall ganglioside synthesis: a) NGcGM3 d18:1-C24:1, b) NGcGM3 d18:1C24:0.

[0054] Figure 2 . Effect of treatment with NGcGM3 extracted from natural sources and synthetic NGcGM3 d18:1-C24:1 on the hepatic metastatic spread of EL-4 tumor cells.

[0055] Figure 3. Effects of treatment with NGcGM3 extracted from natural sources and synthetic NGcGM3 d18:1-C24:1 on the survival of C57BL / 6 mice bearing EL-4 tumor cells.

[0056] Figure 4 . Expression of CD1d molecules on the cell surface of P3X63 mouse myeloma cells measured by flow cytometry.

[0057] Figure 5 . Antitumor effect of NGcGM3 d18:1-C24:1 treatment on subcutaneously implanted P3X63 myeloma cells.

[0058] Figure 6 . Effects of different molecular species of NGcGM3 on in vitro iNKT cell activation.

[0059] Figure 7 . Effects of different molecular species of NGcGM3 on in vivo iNKT cell activation measured by serum levels of IFNγ.

[0060] Figure 8 . Comparison of the effects of different molecular species of NGcGM3 and GM3 with the same ceramide structure on in vitro iNKT cell activation measured by IL-2 secretion in the culture.

[0061] Figure 9 . Effects of treatment with NGcGM3 molecular species and nanoparticles of Neisseria meningitidis OMPC on the hepatic metastatic spread of EL-4 tumor cells.

[0062] Figure 10 . Antitumor effect of treatment with NGcGM3 d18:1-C24:1 or nanoparticles of NGcGM3 d18:1-C24:1 and Neisseria meningitidis OMPC on subcutaneously implanted P3X63 myeloma cells.

[0063] Figure 11 . Effects of NGcGM3 molecular species and nanoparticles of Neisseria meningitidis OMPC on in vitro iNKT cell activation measured by IL-2 secretion in the culture.

[0064] Figure 12 . Effects of adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3 on the hepatic metastatic spread of EL-4 tumor cells.

[0065] Figure 13 . Effects of adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3 on the survival of C57BL / 6 mice in the EL-4 tumor model.

[0066] Figure 14 . Effect of adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3 on tumor growth of P3X63 cells.

[0067] Figure 15 . IFNγ levels in the sera of P3X63 tumor-bearing mice after adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3.

[0068] Figure 16 . IFNγ levels in the sera of EL-4 tumor-bearing mice after adoptive transfer of dendritic cells incubated with different molecular species of NGcGM3.

[0069] Figure 17 . Comparison of specific antibody responses against NGcGM3 induced by injection of nanoparticles containing NGcGM3 molecular species and Neisseria meningitidis OPMC in EL-4 tumor-bearing mice.

[0070] Figure 18a . Effect of dual depletion of NK and NKT cells on the action of NGcGM3 d18:1-C24:1 in an EL-4 liver metastatic spread model.

[0071] Figure 18b . Effect of NK cell depletion on the action of NGcGM3 d18:1C24:1 in an EL-4 cell liver metastatic spread model. Examples

[0072] The present invention will be described in detail below with reference to experimental examples, but the present invention should not be considered limited to these examples.

[0073] Example 1. Chemical synthesis of NGcGM3 variants

[0074] The method for synthesizing the compounds of the present invention and their chemical-physical properties are shown below (see Figure 1a and b).

[0075] A. Synthesis of benzyl O-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosyl)-1→4-lactone)-(2→3)-O-(2,6-di-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-α / β-D-glucopyranoside (Formula III)( Figure 1a ).

[0076]

[0077] Prepare a solution of sialic acid donor methyl (I) 5-acetoxyacetamido-4,7,8,9,10-tetra-O-acetyl-3,5-dideoxy-2-phenylthio-D-glycero-α-D-galacto-2-nonulopyranosilonate (I) (7.25 g; 11.3 mmol) and 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 anhydrous CH3CN (56 mL), add powdered molecular sieve (8.7 g) and stir for 10 minutes at room temperature. Then cool the mixture to -30 °C, add NIS (3.48 g; 15.5 mmol), then add TfOH (67 μL; 0.76 mmol), and stir at the same temperature for 2 hours. Monitor the reaction by TLC, and when the reaction is complete, add CH2Cl2 (120 mL), and filter through a Celite 545 layer. Wash the filtrate with saturated aqueous Na2S2O until the faint red color disappears, and after separating the organic phase, neutralize it with Et3N, dry it over anhydrous Na2SO4, and then evaporate the solvent under reduced pressure. The residue obtained contains unreacted acceptor II, the α- and β-isomers of the formed trisaccharide, and the removal products of donor I.

[0078] Dissolve the product mixture obtained in the above reaction in anhydrous CH2Cl2 (180 mL), cool the solution to 0 °C, add DBU (2.16 mL; 14.47 mmol) and stir at the same temperature for 2 hours. After neutralizing with glacial AcOH diluted in CH2Cl2 (1:4 v / v), evaporate the solvent to dryness under reduced pressure, and purify the residue by column chromatography (toluene / acetone 15:1) to recover the remaining unreacted acceptor II, followed by α[1→4]-lactone (III). After evaporating the solvent, compound III is obtained as an amorphous white solid, homogeneous by TLC (TLC) (R f : 0.56; toluene / acetone 2:1). Yield: 5.6 g (62%).

[0079] 1H NMR (600 MHz, CDCl3): δ 2.15; 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).

[0080] MALDI-TOF MS: [M+Na] + m / z 1404.37 (calcd 1404.52).

[0081] B. Synthesis of Acetyl O-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-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 )。

[0082]

[0083] Dissolve α[1→4]-lactone (III) (5.6 g; 4.05 mmol) in 230 mL of a MeOH / AcOH mixture (9:1 v / v), add 10% Pd / C (0.95 g), evacuate the gas from the balloon using an Ar(g) stream, then inject H2(g) and stir at room temperature for 24 h. After that, filter off the catalyst through a Celite 545 layer and evaporate the solvent to dryness under reduced pressure. Suspend the obtained solid in Ac2O (5 mL), stir the mixture at 0 °C for 10 min, add pre-distilled BF3OEt2 (0.8 mL; 6.3 mmol) and stir at the same temperature for 1 h. After completion of the reaction, neutralize the resulting solution with saturated aqueous NaHCO3, add CH2Cl2, and separate the phases. After separating the organic phase, wash the aqueous phase with CH2Cl2 twice, combine the organic extracts, dry over anhydrous Na2SO4, and evaporate off the solvent under reduced pressure to give Compound IV as a white amorphous solid, which is homogeneous by CCD (R f : 0.73; toluene / acetone 1:1). Yield: 4.1 g (93%). A mixture of α / β anomers (7.6:3.4) was obtained. The NMR data refer to the α-anomer, which is the major anomer.

[0084] 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).

[0085] MALDI-TOF MS: [M+Na]+ m / z 1116.28 (calcd 1116.30).

[0086] C. Synthesis of 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5'-dideoxy-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-glucopyranose (Formula V)( Figure 1a )

[0087]

[0088] Prepare a solution of IV (4.1 g; 3.74 mmol) in anhydrous DMF (20 mL), add NH4OAc (0.47 g; 6.19 mmol) and stir the mixture at room temperature for 24 h. After completion of the reaction, add AcOEt (100 mL) and wash with saturated aqueous NaCl solution (5 x 18 mL). Separate the organic phase, dry over anhydrous Na2SO4 and remove the solvent under reduced pressure to dryness. Compound V is obtained as an amorphous white solid and is homogeneous by CCD (R f : 0.59; toluene / acetone 1:1). Yield: 3.55 g (90%). A mixture of α / β anomers (7:3) is obtained. The NMR data refer to the α-anomer, which is the major anomer.

[0089] 1H NMR (600 MHz, CDCl3): δ 2.19; 2.12 (3 CH3); 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).

[0090] MALDI-TOF MS: [M+Na] +m / z 1074.11 (calcd 1074.29).

[0091] D. Synthesis of O-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-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-glucopyranoside (Formula VI)( Figure 1a )

[0092]

[0093] A solution of V (3.55 g; 3.37 mmol) in anhydrous CH2Cl2 (40 mL) was prepared, trichloroacetonitrile (10.9 mL; 109 mmol) was added, DBU (0.52 mL; 3.46 mmol) was added dropwise, and the mixture was stirred at room temperature for 2 h. After completion of the reaction, the mixture was evaporated to dryness under reduced pressure and the resulting residue was purified by column chromatography (CH2Cl2 / acetone 6:1) to give the compound VI as an amorphous white solid, which was homogeneous by CCD (Rf 0.61; toluene / acetone 1:1). Yield: 2.95 g (73%).

[0094] 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); 4.11 (1H, m, 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).

[0095] MALDI-TOF MS: The product decomposed during the analysis and only the peak corresponding to the starting compound V ([M+Na] + m / z 1074.19) was observed in the spectrum.

[0096] E. Synthesis of O-(5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-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)-(2S,3R,4E)-2-azido-3-benzoyl-4-octadecene-1,3-diol (Formula VII)( Figure 1b ).

[0097]

[0098] Prepare a solution of VI (2.95 g; 2.47 mmol) and azido-sphingosine benzoate (2.11 g; 4.94 mmol) dissolved in anhydrous CH2Cl2 (25 mL), add powdered molecular sieve (5 g) and stir at room temperature for 30 minutes. Cool the mixture to 0 °C, add freshly distilled BF3-OEt2 (0.65 mL; 5.17 mmol) and stir at the same temperature for 2 hours. After completion of the reaction, filter the mixture through a Celite 545 layer, neutralize the filtrate with Et3N, and evaporate to dryness under reduced pressure. Purify the residue by column chromatography (CH2Cl2 / acetone 15:1) to recover the remaining unreacted azido-sphingosine benzoate, and then purify by CH2Cl2 / acetone 10:1 to obtain pure glycoside VII, which is an amorphous white solid and is homogeneous after CCD (toluene / acetone 2:1, R f : 0.58). Yield: 2.02 g (56%).

[0099] 1H NMR (600 MHz, CDCl3): δ 2.19; 2.13; 2.12; 2.10; 2.08; 2.06; 2.03 (2 CH3); 2.01; 1.98 [(3H, s, CH3) x 10]; 8.05–8.00 (2H, m, ortho 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 to H-17); 0.86 (3H, t, J = 7.0 Hz, H-18).

[0100] MALDI-TOF MS: [M+Na]+ m / z 1485.93 (calcd 1485.58).

[0101] F. Synthesis of O-[5-[5-Acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-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 )。

[0102]

[0103] A solution of glycoside VII (2.02 g; 1.38 mmol) was prepared in a 70 mL mixture of pyridine / H2O / Et3N (10:1:0.3 v / v / v), cooled to 0 °C, and a stream of H2S(g) was bubbled through for 1 h. The reactor was sealed, the mixture was stirred at the same temperature, and after 6 h, H2S(g) was bubbled into the mixture for an additional 1 h. After sealing the reactor, the mixture was stirred at 0 °C for 16 h. The progress of the reaction was monitored by TLC, and after completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was co-evaporated with toluene several times to remove the H2O and pyridine residues present. The resulting solid was dissolved in CH2Cl2 (100 mL), 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 x 20 mL), the organic phase was dried over anhydrous Na2SO4, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography successively using mixtures of CH2Cl2 / acetone in different ratios (15:1 → 10:1) to give VIII as an amorphous white solid, which was homogeneous by TLC (toluene / acetone 2:1, R f : 0.58). Yield: 1.9 g (77%).

[0104] 1H NMR (600 MHz, CDCl₃) δ 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, 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, 30H-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 to 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, hept, J = 6.6 Hz, H-3); 1.38 - 1.15 (40H, m, H-4 to H-23); 0.87 (3H, t, J = 7.0 Hz, H-24).

[0105] MALDI-TOF MS: [M + Na] + m / z 1809.76 (calcd 1809.94).

[0106] G. Synthesis of O-[5-[5-Acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-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-tetracosan-15-enamido-4-octadecene-1,3-diol (Formula IX)( Figure 1b ).

[0107]

[0108] By the procedure for VIII above, acylation with (Z)-tetracos-15-enoic acid gave compound IX. Yield: 1.85 g (75%).

[0109] 1H NMR (600 MHz, CDCl₃) δ 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 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; 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 to 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 to H-14 and H-17 to H-23); 0.87 (3H, t, J = 6.9 Hz, H24).

[0110] MALDI-TOF MS: [M+Na] + m / z 1807.75 (calcd 1807.93).

[0111] H. Synthesis of O-[3,5-dideoxy-5-hydroxyacetamido-D-glycero-α-D-galacto-2-nonulopyranosyl-(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 )

[0112]

[0113] A suspension of Formulation VIII (1.9 g; 1.06 mmol) in anhydrous MeOH (70 mL) was treated with 1.3 M NaOMe / MeOH solution (10.6 mL; 13.8 mmol) and stirred at room temperature for 24 h. H2O (18 mL) was added and the mixture was stirred at room temperature for an additional 24 h. Upon completion, it was neutralized by stirring with IR-120(H + ) resin, the resin was filtered off, washed with MeOH, and the combined filtrates were evaporated to dryness under reduced pressure. The residue was dissolved in H2O (200 mL) and the resulting solution was dialyzed against H2O for 24 h at 4 °C using a 3.5 kDa membrane. The dialyzed solution was lyophilized for 24 h at a constant temperature of -40 °C to give the ganglioside X as a white solid, which was homogeneous by CCD(R f : 0.56; CHCl3 / MeOH / 0.25% aqueous KCl 25:20:5). Yield: 1.3 g (95%).

[0114] 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: 2.75 2.71 (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 (2H, 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, septet, J = 7.4 Hz; H-6); 1.34 - 1.18 (22H, m, H-7 to 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 to H-23); 0.85 (3H, t, J = 6.5 Hz, H-24).

[0115] MALDI-TOF MS: [M+Na] + m / z 1304.08 (calcd 1304.66).

[0116] I. Synthesis of O-[3,5-dideoxy-5-hydroxyacetamido-D-glycero-α-D-galacto-2-nonulopyranosyl-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranosyl-(1→1)-(2R,3S,4E)-2-tetracosan-15-enamido-4-octadecene-1,3-diol (Formula XI)( Figure 1b ).

[0117]

[0118] Compound XI was obtained from Intermediate IX through the above procedure for X. Yield: 1.25 g (95%).

[0119] 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 (2H, 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 = 9.1 Hz, 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 to H-17); 0.85 (3H, t, J = 6.5 Hz, H-18). FA: 5.31 (2H, t, J = 5.2 Hz, 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 to H-13, H-18 to H-23); 0.85 (3H, t, J = 6.5 Hz, H24).

[0120] MALDI-TOF MS: [M+Na] + m / z 1301.94 (calcd 1301.81).

[0121] In NMR characterization, the terms Glc, Gal, Neu, Sph, and FA refer to the molecular fragments of the compounds glucose, galactose, neuraminyl, sphingosine hydrocarbon chain, and hydrocarbon chain provided by fatty acids, respectively.

[0122] Example 2. Different from NGcGM3 extracted from natural sources, NGcGM3 d18:1-C24:1 has an anti-metastatic effect in the liver

[0123] Intravenously inoculate 2x10 5 EL-4 thymoma cells into syngeneic C57BL / 6 mice via the tail vein. Randomize the mice and divide them into groups of 7 - 9 animals. The 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. The animals were sacrificed at 12 - 13 days after tumor inoculation, and the livers were removed and weighed to determine the level of liver metastatic spread. The liver weights of healthy animals of the same age and batch were used as controls. Figure 2 The individual liver weight values, as well as the mean and standard deviation of the mean, of the animals in each group are shown. It can be seen that treatment with NGcGM3 d18:1-C24:1 showed a statistically significant anti-metastatic effect, such that no difference was observed between the liver weights of tumor-bearing animals treated with NGcGM3 d18:1-C24:1 and healthy animals. Treatment with NGcGM3 extracted from natural sources did not show such an anti-metastatic effect (same letters indicate p > 0.05; different letters indicate p < 0.0001; ANOVA and Tukey tests).

[0124] Example 3. Treatment with NGcGM3 d18:1-C24:1 improves the survival rate of EL-4 tumor-bearing animals

[0125] Intravenously inoculate 2x10 5 EL-4 thymoma cells (ATCC TIB-39) into syngeneic C57BL / 6 mice via the tail vein; randomize the mice and divide them into groups of 7 - 9 animals. The experimental groups received intraperitoneal treatment with 200 μg of the ganglioside variant NGcGM3 d18:1-C24:1 and 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. The animals were monitored according to veterinary standards, and if the animals showed any signs or symptoms that affected their ability to ingest food and water or impaired their general health, they were sacrificed. Figure 3On day 13 after inoculation of tumor cells, all mice in the group receiving the vehicle solution (placebo) or treated with NGcGM3 from natural sources had died, while all animals treated with NGcGM3 d18:1-C24:1 survived and remained healthy. Thus, it was observed that treatment with NGcGM3 d18:1-C24:1 significantly increased the survival rate of EL-4 tumor-bearing animals (p = 0.0005, log-rank Mantel-Cox, chi-square test).

[0126] Example 4. P3X63 murine myeloma cells express CD1d on the surface

[0127] CD1d expression in the murine myeloma cell line P3X63 (P3X63Ag8.653, CRL 1580) was evaluated by flow cytometry using a mouse anti-CD1d monoclonal antibody conjugated to phycoerythrin (clone 1B1, eBioscience). Cells were pre-incubated with an α-CD16 / 32 monoclonal antibody to block non-specific binding, and the fluorophore-conjugated antibody isotype (ARL2397; BioSource) was used as a control. Cells were acquired on a Sysmex Partec flow cytometer (Sachsen, Germany) and analyzed in FlowJo 10 software (Tree Star, USA). As Figure 4 can be seen, approximately 90% of P3X63 cells express CD1d on their surface.

[0128] Example 5. Administration of NGcGM3 d18:1-C24:1 inhibits tumor growth with a high P3X63 myeloma burden, even superior to αGalCer

[0129] 1x10 6 P3X63 cells were implanted subcutaneously into syngeneic BALB / c mice. Animals were randomized into groups of 6 - 8 animals 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 0.5%) on days 1, 5, and 9. Tumor diameters were measured with calipers, and the tumor volume (TV) was calculated according to the following formula: TV (mm 3 ) = π / 6 × long diameter × (short diameter) 2 . Figure 5 The distribution of the mean TV and individual measurements for each group on day 5 after tumor cell implantation is shown. The individual tumor volume values for each group of animals on day 5 of the experiment, as well as the mean and standard deviation of the mean in each case, are shown. Table 1 shows the percentage distribution of the tumor volumes of the animals in each group.

[0130] Table 1. Percentage distribution of tumor volume of animals in each group

[0131] Placebo αGalCer NGcGM3 24:1 <![CDATA[TV>36mm 3 > 100％ 75％ 50％ <![CDATA[TV<36mm 3 > - 25％ 50％

[0132] Treatment with NGcGM3 d18:1-C24:1 and αGalCer significantly inhibited the growth of subcutaneous tumors, but with different levels of statistical significance (p < 0.001 for NGcGM3 d18:1-C24:1 and p < 0.05 for αGalCer, ANOVA and Tukey tests). Additionally, in the group treated with NGcGM3 d18:1-C24:1, 50% of the animals had tumors with a TV less than 36 mm 3 compared with 25% of the tumors being less than 36 mm in the group treated with αGalCer 3 . In the placebo group, all animals had tumors with a TV greater than 36 mm 3 .

[0133] Example 6. NGcGM3 ganglioside d18:1-C24:1 showed a strong stimulatory effect on iNKT cells in vitro, superior to other synthetic variants of NGcGM3 and gangliosides purified from erythrocytes

[0134] In the presence of 100 ng / mL of αGalCer and natural NGcGM3 and synthetic variants 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 x 10 4 bmDCs were grown in RPMI-1640 medium containing 10% FBS for 16 - 24 hours. Then, 10 5 FF13 hybridoma cells were added and co-cultured with bmDCs for 24 hours. The culture supernatant was collected and the IL-2 content was evaluated by ELISA Figure 6It was shown that at the highest concentration tested (10 μg / ml), the synthetic variant of NGcGM3 was able to activate iNKT cell hybridomas, in contrast to the NGcGM3 ganglioside extracted from natural sources, which did not stimulate IL-2 secretion of iNKT cells at any concentration tested. Based on the length and structure of the fatty acids in the ceramide, differences among NGcGM3 variants were observed in terms of the antigenicity of NGcGM3 variants towards iNKT cells. The D18:1-C24:1 NGcGM3 variant showed strong activation of the FF13 hybridoma cells, which was the only variant capable of activating iNKT cells at the same concentration of αGalCer (100 ng / ml) and exceeding the effect of αGalCer at a concentration of 10 μg / ml (p<0.05, ANOVA and Tukey tests).

[0135] Example 7. Administration of NGcGM3 d18:1-C24:1 activates iNKT cells in vivo, while NGcGM3 d18:1-C 18:0 and NGcGM3 extracted from red blood cells are inactive

[0136] C57BL / 6 mice were intraperitoneally injected with 200 μg of natural NGcGM3 ganglioside, NGcGM3 d18:1-C24:1, NGcGM3 d18:1-C 18:0 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 evaluated by ELISA as an indirect measure of iNKT cell activation (Mouse IFNγ ELISA Ready-SET-Go!, eBioscience). Figure 7 It was shown that in mice receiving NGcGM3 d18:1-C24:1, IFNγ levels were higher than those in the remaining experimental groups and were statistically higher (p<0.05) than the animals in the placebo group (Kruskal-Wallis, Dunn). This result indicates that NGcGM3 d18:1-C24:1 mediates iNKT cell activation in vivo.

[0137] Example 8. Synthetic variants of the ganglioside NGcGM3 have a greater stimulatory effect on iNKT cells than synthetic variants of the ganglioside NAcGM3 with the same ceramide structure

[0138] In the presence of 10 μg / ml and 1 μg / ml of the NGcGM3 d18:1-C24:1 ganglioside, GM3 d18:1-C24:1 or vehicle (0.1% DMSO), 5x10 4bmDCs were cultured in RPMI-1640 medium containing 10% FBS for 16 - 24 hours. Next, 10 5 FF13 hybridoma cells were added and co-cultured with bmDCs for 24 hours. The culture supernatant was collected, and the IL-2 content was quantified by ELISA. As can be seen from Figure 8 the synthetic ganglioside variants NGcGM3 and GM3 with the same ceramide structure differed in their ability to stimulate iNKT cells. The NGcGM3 variant showed a stronger effect on iNKT cell activation than the GM3 variant (ANOVA, Tukey test).

[0139] Example 9. Compared with nanoparticles formed with NGcGM3 d18:1-C18:0 or NGcGM3 extracted from red blood cells, nanoparticles of NGcGM3 d18:1-C24:1 and OMPC of Neisseria meningitidis showed potent anti-metastatic effects in the liver

[0140] 2 x 10 5 EL-4 thymoma cells were intravenously inoculated into syngeneic C57BL / 6 mice via the tail vein; the mice were randomized and divided into groups of 7 - 9 animals. The experimental groups received intraperitoneal treatment with 10 - 30 μg of nanoparticles of NGcGM3 d18:1-C24:1, NGcGM3 d18:1-C18:0 and native NGcGM3 with OMPC of Neisseria meningitidis or Tris / HCl solution (placebo) on days 1, 5, and 9. The animals were sacrificed 12 - 13 days after tumor inoculation. To determine the level of liver metastatic spread, their livers were removed and weighed. Figure 9 The individual liver weight values of the animals in each group, as well as the mean and standard deviation of the mean, are shown. Notably, treatment with nanoparticles containing the NGcGM3 d18:1-C24:1 ganglioside showed a statistically significant anti-metastatic effect compared to the placebo group, while the groups treated with nanoparticles containing the NGcGM3 d18:1-C18:0 ganglioside or gangliosides obtained from natural sources did not show such an effect (the same letters indicate p > 0.05; different letters indicate p = 0.0015; ANOVA and Tukey test).

[0141] Example 10. Administration of NGcGM3 d18:1-C24:1 integrated into nanoparticles together with OMPC of Neisseria meningitidis inhibited the growth of P3X63 murine myeloma with high tumor burden, even better than αGalCer and systemic NGcGM3 d18:1-C24:1

[0142] 1 x 10 6P3X63 myeloma cells were subcutaneously implanted into syngeneic BALB / c mice. The animals were randomized into groups of 6 - 8 animals and treated with vehicle solution (5.6% sucrose, 0.75% L - histidine, and 0.5% Tween - 20), αGalCer, NGcGM3 d18:1 - C24:1, or nanoparticles of OMPC and NGcGM3 d18:1 - C24:1 (GlycoVax 24:1) on days 1, 5, and 9. The subcutaneous tumor diameter was measured with calipers every 2 - 3 days, and the TV was calculated according to the following formula: TV (mm 3 ) = π / 6 × long diameter × (short diameter) 2 . Figure 10 Shows the TV of individual animals in each group and the mean and standard deviation of the mean at day 5 after tumor cell implantation, at which time all animals in the placebo group had measurable tumors. The αGalCer treatment did not show a statistically significant anti - tumor effect. In contrast, treatment with NGcGM3 d18:1 - C24:1 and GlycoVax 24:1 showed significant tumor growth inhibition relative to the control group (p = 0.0031 and p = 0.0004, respectively, non - parametric ANOVA, Kruskal - Wallis, Dunn's multiple comparison test).

[0143] Table 2 shows the percentage distribution of the tumor volume of animals in each group.

[0144] Table 2. Percentage distribution of tumor volume of animals in each group

[0145]

[0146] Treatment with GlycoVax 24:1 showed the strongest anti - tumor effect, with 75% of the animals having tumors smaller than 36 mm 3 . For the group treated with systemic NGcGM3 d18:1 - C24:1 ganglioside, 50% of the animals were observed to have tumors smaller than 36 mm 3 . Treatment with αGalCer reduced the tumor volume to less than 36 mm in 25% of the animals 3 .

[0147] Example 11. Nanoparticles of NGcGM3 d18:1 - C24:1 and Neisseria meningitidis OMPC activate iNKT cells, which is different from the nanoparticles obtained by incorporating NGcGM3 d18:1 - C18:0 or NGcGM3 extracted from natural sources

[0148] In the presence of nanoparticles, αGalCer or vehicle (0.1% DMSO) at a concentration of 100 ng / ml, 1 μg / ml or 10 μg / ml, 5 x 10 4 bmDCs were cultured in RPMI-1640 medium containing 10% FBS for 16 - 24 hours. Then the bmDCs were co-cultured with 10 5 FF13 hybridoma cells for 24 hours. The culture supernatant was collected and the IL-2 content was quantified by ELISA. As Figure 11 can be seen, nanoparticles containing NGcGM3 d18:1-C24:1 ganglioside (GlycoVax24:1) activated iNKT cell hybridomas at concentrations of 10 μg / ml and 1 μg / ml similar to αGalCer, and this effect was still maintained at a concentration of 100 ng / mL. In the case of nanoparticles obtained with NGcGM3 ganglioside extracted from natural sources, no activation of iNKT cell hybridomas was observed at any test concentration, while in the case of nanoparticles containing NGcGM3 d18:1-C18:0 ganglioside, a moderate effect was only observed at the highest evaluated concentration (10 μg / ml).

[0149] Example 12. Adoptive transfer of dendritic cells incubated with NGcGM3 d18:1C24:1 produces a strong anti-metastatic effect in the liver, while transfer of dendritic cells incubated with NGcGM3 extracted from red blood cells is ineffective

[0150] 2 x 10 5 EL-4 thymoma cells were intravenously inoculated into syngeneic C57BL / 6 mice via the tail vein; the mice were randomized and divided into groups of 7 - 9 animals. On the day after tumor inoculation, 6 x 10 5 bmDCs that had been pre-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 hours were intravenously administered as a single dose. The cells were thoroughly washed with serum-free RPMI-1640 medium before transfer to remove excess lipids. The animals were sacrificed 12 - 13 days after tumor inoculation, the livers were collected and the level of liver metastatic spread was determined by weighing. The livers of healthy mice of the same age and batch were used as a reference. Figure 12Shows the individual liver weight values of the animals in each group, as well as the mean and standard deviation of the mean. Clearly, compared to the placebo group that received transferred empty dendritic cells, the EL-4 liver metastases induced by transferring bmDC pulsed with NGcGM3 d18:1-C24:1 were significantly reduced. Similarly, transferring bmDC incubated with gangliosides extracted from natural sources was ineffective (the same letters indicate p>0.05, different letters indicate p<0.05, ANOVA and Tukey tests).

[0151] Example 13. Contrary to transferring dendritic cells incubated with NGcGM3 extracted from red blood cells, adoptive transfer of dendritic cells incubated with NGcGM3 d18:1C24:1 increased the survival of tumor-bearing animals

[0152] Intravenously inoculate 2x10 5 EL-4 thymoma cells into syngeneic C57BL / 6 mice via the tail vein; randomize the mice and divide them into groups of 7-9 animals. On the day after tumor inoculation, 6x10 5 bmDCs that had been pre-cultured with vehicle (0.1% DMSO), NGcGM3 d18:1-C24:1 ganglioside (10 μg / ml), or gangliosides extracted from natural sources (10 μg / mL) for 16 hours were intravenously administered as a single dose. Wash the cells thoroughly with serum-free RPMI-1640 medium before transfer to remove excess lipids. Monitor the mice for signs or symptoms that affect their ability to eat and drink or impair their overall health status according to veterinary standards. As can be seen from Figure 13 At 13 days after tumor cell inoculation, all animals in the placebo group that received empty dendritic cells and all animals that received cells pulsed with NGcGM3 from natural sources had died. In contrast, in the group that received bmDC exposed to NGcGM3 d18:1-C24:1, all animals (100%) survived and remained healthy. The statistically different survival curves indicate that the survival rate of animals treated with bmDC previously cultured in the presence of NGcGM3 d18:1-C24:1 was significantly increased (p<0.0001) (log-rank Mantel-Cox, chi-square test).

[0153] Example 14. Compared to transferring dendritic cells incubated with NGcGM3 extracted from red blood cells, adoptive transfer of dendritic cells incubated with NGcGM3 d18:1-C24:1 reduced tumor growth in P3X63 murine myeloma tumor-bearing animals

[0154] Inject 1x10 6Individual P3X63 cells were subcutaneously implanted into syngeneic BALB / c mice. The animals were randomized into groups of 6 - 8 animals and received a single intravenous dose of 6x10 5 bmDCs on the day after implantation. These bmDCs were pre-cultured with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 ganglioside (10 μg / ml), or ganglioside extracted from natural sources (10 μg / ml) for 16 hours. The cells were thoroughly washed with serum-free RPMI-1640 medium before transfer to remove excess lipids. Tumor growth was followed by measuring the tumor diameter with calipers, and the TV was determined according to the following formula: TV (mm 3 ) = π / 6 × long diameter × (short diameter) 2 . Figure 14 showed that transfer of bmDCs pulsed with NGcGM3 d18:1C24:1 induced an anti-tumor effect by significantly (p < 0.0001) reducing tumor growth compared to the group receiving empty bmDC treatment. No anti-tumor effect was observed when transferring bmDCs incubated with ganglioside extracted from natural sources (ANOVA and Tukey test).

[0155] Example 15. Activation of NKT cells in vivo in BALB / c mice occurs when transferring dendritic cells incubated with NGcGM3d18:1-C24:1, but not when transferring dendritic cells incubated with NGcGM3 extracted from erythrocytes

[0156] 1x10 6 Individual P3X63 cells were subcutaneously implanted into syngeneic BALB / c mice. The animals were randomized into groups of 6 - 8 animals and received a single intravenous dose of 6x10 5 bmDCs on the day after implantation. These bmDCs were pre-cultured with vehicle (DMSO 0.1%), NGcGM3 d18:1-C24:1 (10 μg / ml), or ganglioside extracted from natural sources (10 μg / ml) for 16 hours. The cells were thoroughly washed with serum-free RPMI-1640 medium before transfer to remove excess lipids. Sixteen hours after transfer, blood was collected from 2 - 3 animals in each group, and serum IFNγ levels were evaluated by ELISA. Figure 15 showed that neither transfer of empty bmDCs nor transfer of bmDCs pulsed with NGcGM3 extracted from erythrocytes was able to stimulate early IFNγ secretion. However, significant levels of IFNγ were detected at 16 hours post-treatment in animals that had received transfer of bmDCs previously cultured in the presence of NGcGM3 d18:1-C24:1. This early secretion of IFNγ is associated with the activation of iNKT cells.

[0157] Example 16. In vivo activation of NKT cells in C57BL / 6 mice occurred upon transfer of dendritic cells incubated with NGcGM3d18:1-C24:1, but not upon transfer of dendritic cells incubated with NGcGM3 extracted from erythrocytes.

[0158] Syngeneic C57BL / 6 mice were intravenously inoculated via the tail vein with 2 x 10 5 EL-4 thymoma cells; the mice were randomized and divided into groups of 7 - 9 animals. On the day after tumor inoculation, 6 x 10 5 bmDCs that had been pre-cultured for 16 hours with vehicle (0.1% DMSO), NGcGM3d18:1-C24:1 (10 μg / ml), or ganglioside extracted from natural sources (10 μg / mL) were intravenously administered as a single dose. Cells were thoroughly washed with serum-free RPMI-1640 medium prior to transfer to remove excess lipid. Sixteen hours after transfer, blood was collected from 2 - 3 animals per group, and serum IFNγ levels were evaluated by ELISA. In Figure 16 , transfer of vehicle-pulsed bmDCs and transfer of bmDCs pulsed with natural-source NGcGM3 failed to activate iNKT cells, whereas transfer of bmDCs previously pulsed with NGcGM3 d18:1-C24:1 induced activation of iNKT cells, as manifested by early IFNγ secretion.

[0159] Example 17. When administered to mice, NGcGM3 d18:1-C24:1 incorporated into nanoparticles together with Neisseria meningitidis OMPC induced a strong specific antibody response, which was different from that when incorporating NGcGM3d18:1-C18:0 and NGcGM3 extracted from erythrocytes.

[0160] Syngeneic C57BL / 6 mice were intravenously inoculated via the tail vein with 2 x 10 5EL-4 thymoma cells were used, and these mice were randomized and divided into groups of 7 - 9 animals. The experimental groups received an intraperitoneal treatment with 10 - 30 μg of nanoparticles with Neisseria meningitidis OMPC or Tris / HCl solution (placebo) on days 1, 5, and 9. These nanoparticles contained NGcGM3d18:1 - C24:1, NGcGM3 d18:1 - C18:0, or NGcGM3 extracted from natural sources, respectively. On day 13, blood was collected from 3 - 4 animals in each group, and the specific IgM and IgG antibody responses against NGcGM3 were evaluated by ELISA. For this purpose, a single serum pool for each group was prepared from the sera of individual animals in each case. For the detection and titration of NGcGM3 - specific antibodies by ELISA, NUNC PolySorp 96 - well plates were coated with 0.16 nmol / well of NGcGM3 (from natural sources) dissolved in methanol. The solvent was evaporated at 37 °C for 1 - 2 hours, and a blocking step was carried out with 1% (m / v) fat - free bovine serum albumin fraction V in phosphate - buffered saline (PBS) for 16 - 24 hours. Subsequently, sera were added within the dilution range to be evaluated, and after washing with PBS - Tween 20 0.05% (v / v), biotinylated goat anti - IgM + mouse IgG antibody (Sigma) diluted 1 / 5000 in the blocking solution was added. After incubation at 37 °C for 1 hour, streptavidin conjugated with alkaline phosphatase (Jackson Immnoresearch) diluted 1 / 2000 in the blocking solution was added. After 1 hour at 37 °C, the enzymatic reaction was developed with 1 mg / ml p - nitrophenyl phosphate (PNPP) dissolved in 1 M diethanolamine buffer at pH 9.6 and containing 1 mM MgCl2. The absorbance was read at 405 nm. To eliminate the influence of non - specific signals, sera were also analyzed in empty wells containing only methanol. The absorbance at each serum dilution was corrected by subtracting the value of the empty well. The serum titer was defined as the reciprocal of the highest dilution that produced a final absorbance value greater than 0.1. Figure 17 The anti - NGcGM3 antibody response was shown to be present only in animals injected with nanoparticles containing NGcGM3 d18:1 - C24:1. The obtained IgM and IgG antibody titers specific for NGcGM3 were 1 / 320, which can be said to be very excellent considering the lipid nature of this antigen and the relatively short time frame of only 13 days. Nanoparticles formulated with gangliosides extracted from natural sources or NGcGM3 d18:1 - C18:0 did not produce an anti - NGcGM3 antibody response.

[0161] Example 18. The anti - tumor activity of NGcGM3 d18:1 - C24:1 ganglioside is NKT - cell - dependent

[0162] Allogeneic C57BL / 6 mice were intraperitoneally treated with anti-NK1.1 depleting antibody (clone PK136, ATCC) at 1 mg / 200 μl PBS or polyclonal anti-asialoGM1 antibody (OriGene Technologies) at 20 μL / 200 μL PBS. Treatment with anti-NK1.1 antibody eliminated more than 90% of the NK and NKT cell populations, while anti-asialoGM1 antibody was used to selectively deplete the NK cell population. Forty-eight hours after antibody treatment, 2 x 10 5 EL-4 thymoma cells were intravenously inoculated into all animals, including two groups of mice that had received 200 μL PBS intraperitoneally as a control. On days 1, 5, and 9 after tumor inoculation, according to the allocation of the following experimental groups, the experimental groups received intraperitoneal treatment with 200 μg NGcGM3 d18:1-C24:1 or vehicle (placebo group):

[0163] Group 1: Placebo + PBS

[0164] Group 2: NGcGM3 d18:1-C 24:1, 200 μg / 200 μL + PBS

[0165] Group 3: Placebo + αNK1.1

[0166] Group 4: NGcGM3 d18:1-C24:1, 200 μg / 200 μL + αNK1.1

[0167] Group 5: Placebo + α-asialoGM1

[0168] Group 6: NGcGM3 d18:1-C24:1, 200 μg / 200 μL + α-sialoGM1

[0169] To maintain depletion of the target population throughout the experiment, the same dose of depleting antibody was administered on days 4 and 11 after tumor inoculation. The animals were sacrificed on days 12 - 13 after tumor inoculation, the livers were removed and the level of liver metastatic spread was determined by weighing. The livers from healthy mice of the same age and batch were used as a reference. Figure 18aShow the effect of depletion of NKT and NK cell populations on the anti-tumor effect of NGcGM3d18:1-C24:1 treatment. Although the anti-metastatic effect of NGcGM3 d18:1-C24:1 treatment was present in mice in which the NK and NKT cell populations were not affected, these cell populations were absent in animals treated with anti-NK1.1 Mab, thus abolishing the above anti-tumor effect (same letters indicate p > 0.05; different letters indicate p < 0.0001; ANOVA and Tukey tests). These results suggest that the anti-tumor effect of NGcGM3 d18:1-C24:1 administration is dependent on NK or NKT cells. However, when the anti-tumor effect of NGcGM3 d18:1-C24:1 administration was evaluated in animals in which only the NK cell population was depleted ( Figure 18b ), the anti-tumor effect was observed to be maintained despite cell population depletion (same letters indicate p > 0.05; different letters indicate p < 0.0001; ANOVA and Tukey tests), suggesting that this cell population is not directly responsible for the observed anti-metastatic effect. Collectively, these results suggest that the anti-tumor effect of NGcGM3d18:1-C24:1 is mainly dependent on NKT cell activity.

Claims

1. A synthetic NGcGM3 ganglioside represented by the following formula: which corresponds to the anomeric α-trisaccharide, wherein R is selected from the group consisting of the following: -C 23 H 47 and -C 23 H 45 。 2. A pharmaceutical composition comprising the ganglioside according to claim 1 as an active substance and a pharmaceutically acceptable vehicle.

3. The composition according to claim 2, which contains another immunomodulator.

4. The composition according to any one of claims 2-3, which contains an antigen.

5. The composition according to claim 2, which is in the form of nanoparticles or liposomes.

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

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

8. Use of the ganglioside according to claim 1 in the preparation of a medicament for the treatment of cancer and its metastases.

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

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

11. The method according to claim 10, wherein the tumor is CD1d-positive or CD1d-negative.

12. The method according to claim 10, wherein the metastasis is a liver metastasis.

13. The method according to any one of claims 10-12, wherein the compound is provided in the form of nanoparticles or liposomes.

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

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

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

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

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