Development and application of novel glycolipid Toll-like Receptor 2 agonist

Abstract

The invention belongs to the field of medicinal chemistry and pharmacology, and relates to application of a novel glycolipid TLR2 agonist 13-C in preparation of vaccine adjuvants and anti-tumor treatment. The compound is excellent in human TLR2 agonist activity, EC50 reaches 2.2 nM, and the compound is simple in structure, easy to prepare and excellent in solubility and has industrial application potential. In a B16-OVA tumor model, the antibody induction capacity of the compound is superior to that of a positive control drug Diprovocim, and the anti-tumor activity of the compound is more remarkable; in addition, PD-L1 expression of tumor tissue can be effectively down-regulated, and a synergistic anti-tumor effect is generated with a PD-L1 monoclonal antibody Atezolizumab. Besides, 13-C and a vaccine adjuvant QS-21 are combined for use, so that a synergistic enhancement effect is achieved, the antibody titer induced by a vaccine can be remarkably improved, antibody subtypes are enriched, meanwhile, cellular immunity and humoral immunity response of an organism are stimulated, and the effect of dual immune activation is achieved.

Description

Technical Field

[0001] This invention relates to the fields of medicinal chemistry and pharmacology, specifically to the application of a novel glycolipid Toll-like Receptor 2 agonist in vaccine adjuvants and tumor immunotherapy. Background Technology

[0002] Vaccines play a vital role in preventing and controlling infectious diseases and combating tumors, but the lack of suitable adjuvants has been a bottleneck in the development of novel vaccines. Adjuvants can not only enhance antibody-mediated immune responses but also strengthen and increase the specificity of B-lymphocyte and T-lymphocyte-mediated immune responses to vaccine antigens. Furthermore, adjuvants can overcome competition between antigens in combined vaccines, thereby enhancing immune responses in young children and the elderly. However, to date, only a limited number of adjuvants have been approved for use due to significant regulatory hurdles.

[0003] Small molecules or biomolecules synthesized targeting pattern recognition receptors (PRRs) are next-generation vaccine adjuvants, belonging to the category of immune enhancers. Recent evidence suggests that PRRs recognize not only pathogen-associated molecular patterns (PAMPs) but also endogenous molecules (DAMPs) released by damaged cells. The field of vaccine adjuvant development has been revolutionized since the discovery of these innate immune receptors responsible for detecting PAMPs and activating downstream signaling. Several ligands targeting different PRRs have been identified as vaccine adjuvants. However, each PRR has its own specific tissue localization and downstream gene signaling pathways, providing opportunities to tailor adjuvants with desired properties.

[0004] TLRs (Toll-like Receptors) are the most characteristic members of the PRR family, responsible for sensing invading pathogens in extracellular and intracellular endosomes and lysosomes. Different TLRs recognize different microbial molecules and their own components. Each TLR has its specific tissue localization and downstream signaling pathways. TLR2 is expressed on the surface of various cells, including monocytes, macrophages, endothelial cells, epithelial cells, natural killer cells, dendritic cells, myelosuppressive cells, platelets, and mast cells. It can detect components of bacteria, mycoplasma, fungi, and viruses, including bacterial and mycoplasma lipoproteins. Furthermore, because TLR2 can form heterodimers with TLR1 and TLR6 on the cell surface, its library of pathogen-associated molecular patterns (PAMPs) is very extensive.

[0005] TLR agonists are commonly used as immunotherapies or vaccine adjuvants for treating cancer, allergies, and infectious diseases. Some TLR agonists are currently in clinical trials or have been approved by the FDA. For example, the TLR2 and TLR4 agonist BCG is used to prevent tuberculosis and treat bladder cancer; the TLR4 agonist monophosphorylated lipid A is used as an adjuvant for the hepatitis B vaccine Fendrix and the HPV-associated cervical cancer vaccine Cervarix; the TLR5 agonist entolimo has been used to treat tumors, infections, and digestive system diseases; and the TLR7 agonist imiquimod has been widely used to treat malignant skin cancer. Unlike other members of the TLR family, TLR2 can form heterodimers with TLR1 or TLR6, making the structures of TLR2 agonists more diverse. These promising potential applications motivate us to explore and discover new TLR2 agonists.

[0006] Clardy's team discovered CaLGL-1, a lipid-based small molecule agonist with a suitable molecular weight, from Collinsella aerofaciens. Knockout experiments further demonstrated that CaLGL-1 is a TLR2-dependent agonist (EC2-dependent agonist) in mouse bone marrow-derived dendritic cells (BMDCs). 50 = 3.2 μM). Although CaLGL-1 has poor agonistic activity against human TLR2, it still provides a promising lead compound for the development of an effective, specific human TLR2 receptor agonist.

[0007] The structural formula of CalGL-1:

[0008] The purpose of this invention is to develop a novel glycolipid Toll-like Receptor 2 agonist for use in vaccine adjuvants and anti-tumor applications, providing new ideas for subsequent research on TLR-2 agonists and related drugs. Summary of the Invention

[0009] This invention discloses the application of a novel glycolipid Toll-like Receptor 2 agonist in vaccine adjuvants and antitumor applications.

[0010] The purpose of this invention is to provide a novel glycolipid toll-like receptor 2 compound, the specific structural formula of which is shown in (I) below: Where: R 1 For CO(CH2)nCH3, R 2 For hydrogen, CO(CH2)mCH3, R 3The formula is CO(CH2)mCH3, where X is a heteroatom such as oxygen or sulfur, Y is a heteroatom such as oxygen or nitrogen, and n and m are fatty chains with 8-18 methylene groups. Specifically, X is O or S, Y is O or NH, and n and m are fatty chains with 8-18 methylene groups. Preferably, X is O or S, Y is O or NH, and when X is S, Y is not NH, and n and m are fatty chains with 8-18 methylene groups. More preferably, when X is O, Y is O, and n and m are fatty chains with 8-18 methylene groups.

[0011] The novel glycolipid Toll-like Receptor 2 agonists disclosed in this invention include, but are not limited to, the following structures: .

[0012] Another object of the present invention is to provide a method for synthesizing novel glycolipid TLR2 agonists, wherein the compounds involved in the present invention can be synthesized by the following method:

[0013] Method 1: Step a) Using inexpensive and readily available D-galactose as the starting material, compound (II) was obtained by TBS protection. Step b), compound II reacts with acetone glycerol under the action of TMSI to undergo a glycosylation reaction to give compound III-a; Step c), compound III-a is selectively de-propylene under acidic conditions to give diol IV-a; Step d), compound IV-a is esterified under EDCI conditions to give lipid compound Va; Step e), compound Va is deprotected by the TBS protecting group to obtain VI-a; Step f), compound VI-a is esterified under EDCI conditions to give compound Ia.

[0014] Method 2: Step b1), compound II reacts with (S)-glycidol under the action of TMSI to undergo a glycosylation reaction to give compound III-b; Step c1), compound III-b undergoes a Staudinger reaction with NaN3 and PPh3 to give compound IV-b; In step d1), compound IV-b undergoes esterification under EDCI conditions to yield the lipid compound Vb; Step e1), compound Vb is deprotected by the TBS protecting group to obtain compound VI-b; Step f1), compound VI-b was esterified under EDCI conditions to give compound Ib. Specifically, the detailed steps for Method 1 are as follows: Step a) Using commercially available D-galactose as the starting material, anhydrous N,N-dimethylformamide as the solvent, and imidazole as the base, compound (II) was obtained by TBS protection at room temperature. Step b), compound II was reacted in anhydrous dichloromethane as solvent, N-ethyldiisopropylamine as base, (S)-(2,2-dimethyl-1,3-dioxolane-4-yl) alcohol and activated 4Å molecular sieve as additives, under an argon atmosphere, at -60°C, and at room temperature to obtain compound III-a. Step c), compound III-a is reacted in a trifluoroacetic acid:water:dichloromethane ratio of 2.5:2.5:95 from 0°C to room temperature for 30 minutes to obtain the large-scale product IV-a; In step d), compound IV-a is reacted in tetrahydrofuran (or toluene) as solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as condensing agent, the corresponding acid as reactant, and a catalytic amount of 4-dimethylaminopyridine as additive, at room temperature for 12 hours (or heated to 90°C) to obtain the large-scale product Va. Step e) Compound Va was reacted with anhydrous tetrahydrofuran as solvent, and pyridine hydrogen fluoride was slowly added dropwise at 0°C. The reaction was then carried out at room temperature for 2 hours. After the reaction was detected to be complete by thin-layer chromatography, the mixture was placed in an ice bath and the reaction was quenched by slow addition of saturated sodium bicarbonate solution. The mixture was then extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and finally obtained by column chromatography to yield the large-scale product VI-a. Step f), compound VI-a is reacted in tetrahydrofuran (or toluene) as solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as condensing agent, the corresponding fatty acid as reactant, and a catalytic amount of 4-dimethylaminopyridine as additive. The reaction is carried out at room temperature for 12 hours (or heated to 90°C). After the reaction is detected by thin-layer chromatography to ensure complete reaction, the organic phase is concentrated under reduced pressure, and finally column chromatography is used to obtain the large-scale product compound Ia.

[0015] Another object of the present invention is to provide a composition of a novel glycolipid toll-like receptor 2 agonist and its use in vaccine adjuvants and antitumor agents.

[0016] The novel glycolipid Toll-like Receptor 2 agonist disclosed in this invention and its pharmaceutically acceptable carrier or diluent can be used to form a composition.

[0017] The present invention also discloses the use of the above composition in the preparation of a drug, which can activate the TLR2 receptor and activate the signaling pathway mediated by it, and can be used as a vaccine additive to improve the vaccine protection efficiency; or for tumor immunotherapy to treat or improve diseases or conditions; or for conditional immunity to treat or improve inflammation; or for promoting intestinal peristalsis to treat or improve intestinal diseases.

[0018] The novel glycolipid toll-like receptor 2 agonist I disclosed in this invention can also be used in combination with other drugs for tumor immunotherapy and tumor vaccines; preferably, compound I or its composition can be used in combination with antibodies; more preferably, compound I or its composition can be used in combination with anti-PD-L1 monoclonal antibodies.

[0019] This invention has the advantages of safe operation, environmental friendliness, mild reaction conditions, and high yield. The raw materials and additives used in this invention are inexpensive and readily available, resulting in minimal environmental impact.

[0020] This invention meets the requirements of green synthesis and has significant practical application value. Attached Figure Description

[0021] Figure 1 The expression of TNF-α in mouse-derived cells (BMDC) for all candidate compounds.

[0022] Figure 2 The expression of TNF-α in human cells (THP-1) for all candidate compounds.

[0023] Figure 3 The serum antibody titer of compound 13-C in animals.

[0024] Figure 4 The antitumor activity of the 13-C compound.

[0025] Figure 5 It provides adjuvant activity for 13-C used alone and in combination with QS-21 in OVA antigens.

[0026] Figure 6 It provides adjuvant activity for 13-C used alone and in combination with QS-21 in gE antigen. Detailed Implementation

[0027] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following specific embodiments are described to further illustrate the above-mentioned content of the present invention. However, this should not be construed as limiting the scope of implementation of the present invention. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0029] In the experimental methods described in the following examples, all solvents and reagents used were analytically pure or chemically pure; anhydrous solvents were processed according to standard methods; column chromatography and silica gel plates were all standard models. Unless otherwise specified, anhydrous sodium sulfate was used as the drying agent for the extracted organic phase. LC-MS was performed using an Agilent 1260 high-performance liquid chromatography-ion trap mass spectrometer (ESI source) with a diode array detector (DAD) at detection wavelengths of 210 nm and 254 nm. Unless otherwise specified, all system models are standard methods; all reagents and materials, unless otherwise specified, are commercially available.

[0030] Example 1: A method for synthesizing a novel glycolipid TLR2 agonist 13-C (m=11). 13-C (m=11)

[0031] Step 1: Synthesis of Compound II II

[0032] Commercially available D-galactose (30 mmol) was added to a 250 mL round-bottom flask equipped with a magnetic stirrup, and anhydrous N,N-dimethylformamide (DMF, 150 mL) was added to the system and the flask was placed at 0 °C. Imidazole (6.6 equivalents) and tert-butyldimethylchlorosilane (TBSCl, 7.25 equivalents) were then slowly added in portions to the system. The reaction was then allowed to proceed at room temperature for 48 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the reaction system was slowly poured into ice water in portions. The aqueous phase was then extracted with dichloromethane (100 mL × 3), and the organic phases were combined. The organic phase was then washed successively with 5% hydrochloric acid, saturated sodium bicarbonate, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then anhydrous methanol (20 mL) was added. A white solid precipitated, which was filtered to give product (II) in 80% yield. 1H NMR (400 MHz, CDCl3) δ5.15 (d, J = 2.4 Hz, 1H), 4.09 (t, J = 3.8 Hz, 1H), 4.00 (t, J = 4.1 Hz, 1H), 3.92 (t, J = 2.8 Hz, 1H), 3.78 – 3.70 (m, [M+ Na] + = 773.7.

[0033] Step 2: Synthesis of compound III-a1 III-a1

[0034] The product (II) (10 mmol) obtained in the first step and the activated 4Å molecular sieve were added to a 250 mL round-bottom flask equipped with a magnetic stir bar and protected with argon. Anhydrous dichloromethane (DCM, 100 mL) was then added, and the mixture was stirred at room temperature for 30 minutes. The flask was then placed at -40 °C, and trimethyliodosilane (TMSI, 1.2 equivalents) was added. After reacting for 30 minutes, N-ethyldiisopropylamine (DIPEA, 2.0 equivalents) and (S)-(2,2-dimethyl-1,3-dioxolane-4-yl)methanol ((S)-(+)-1,2-Isopropylideneglycerol, 1.2 equivalents) were slowly added. (Equivalent), then reacted at room temperature for 2 hours. After the reaction was detected to be complete by thin-layer chromatography, the liquid obtained after filtering off the molecular sieve was washed with saturated sodium bicarbonate solution and saturated brine. The organic phase was dried with anhydrous sodium sulfate and concentrated under reduced pressure. The residue was separated and purified by column chromatography using 100:1 petroleum ether / ethyl acetate (PE / EtOAc) as the eluent to obtain product (III-a1) in 74% yield. 1H NMR(400 MHz, CDCl3) δ 4.81 (d, J = 2.2 Hz, 1H), 4.28 (m, 1H) 4.11 (dd, J = 5.8,3.3 Hz, 1H), 4.05 (m, 1H), 4.01 (m, 1H), 3.93 (dd, J = 5.8, 3.0 Hz, 1H), 3.81(dd, J = 8.3, 6.2 Hz, 1H), 3.77 (dd, J = 10.0 Hz, 4.7 Hz, 1H), 3.75 (m, 1H), 3.65 (dd, J = 9.9, 6.7 Hz, 1H), 3.57 (dd, J = 9.8, 5.8 Hz, 1H), 3.41 (dd, J =10.2, 6.7 Hz, 1H), 1.39 (s, 3H), 1.34 (s, 3H), 0.87 (m, 36H), 0.10 – 0.03 (m,24H). [M + Na] + = 773.6.

[0035] Step 3: Synthesis of compound IV-a1 IV-a1

[0036] The product (III-a1) (8.5 mmol) obtained in step 2 was added to a 500 mL round-bottom flask equipped with a magnetic stirrup. 400 mL of dichloromethane (DCM) was added, and the flask was placed in an ice bath. Trifluoroacetic acid (TFA, 10.6 mL) and water (H2O, 10.6 mL) were then added. The mixture was allowed to react at room temperature for 30 minutes. After the reaction was confirmed to be complete by thin-layer chromatography, the flask was placed in an ice bath, and the reaction was quenched by slow addition of saturated sodium bicarbonate solution. The mixture was then extracted three times with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was eluted with 100:1 petroleum ether / ethyl acetate (PE / EtOAc) and then subjected to column chromatography to give product (IV-a1) in 83% yield. 1H NMR (600 MHz, CDCl3) δ 4.83 (d, J =2.3 Hz, 1H), 4.12 (dd, J = 4.7, 3.0 Hz, 1H), 4.02 – 3.96 (m, 2H), 3.82 (q, J= 4.6 Hz, 1H), 3.74 (td, J = 6.1, 3.9 Hz, 1H), 3.67 (d, J = 4.8 Hz, 2H), 3.63 (dd, J = 10.2, 6.6 Hz, 1H), 3.58 (m, 2H), 3.33 (d, J = 4.3 Hz, 1H), 2.41 (t,J = 6.1 Hz, 1H), 0.90 – 0.85 (m, 36H), 0.12 – 0.03 (m, 24H). [M + Na] + =733.6.

[0037] Step 4: Synthesis of compound 13-CVa (m=11) 13-CVa (m=11)

[0038] The product (IV-a1) (5 mmol) obtained in step 3 was added to a 100 mL round-bottom flask equipped with a magnetic flask. Anhydrous tetrahydrofuran (THF, 60 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 2.0 equivalence), 4-dimethylaminopyridine (DMAP, 0.1 equivalence), and tridecanoic acid (2.0 equivalence) were added to the flask. The mixture was then reacted at room temperature for 12 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the mixture was moved to room temperature and ethyl acetate (100 mL) was added. The organic phase was washed with saturated sodium bicarbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the product (13-CVa (m=11)).

[0039] Step 5: Synthesis of compound 13-C-VI-a (m=11) 13-C-VI-a (m=11)

[0040] The product obtained in step four (13-CVa (m=11)) (5 ​​mmol) was added to a 100 mL plastic reaction flask equipped with a magnetic flask. Anhydrous tetrahydrofuran (THF) was added to the flask, and pyridine hydrogen fluoride (6.0 equivalent) was slowly added dropwise at 0 °C. The reaction was then carried out at room temperature for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the flask was placed in an ice bath, and the reaction was quenched by slowly adding saturated sodium bicarbonate solution. The flask was then extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was then concentrated under reduced pressure to obtain the product (13-C-VI-a (m=11)).

[0041] Step 6: Synthesis of compound 13-C (m=11) 13-C (m=11)

[0042] The product obtained in step 5 (13-C-VI-a (m=11)) (0.2 mmol) was added to a 50 mL round-bottom flask equipped with a magnetic stirrup. Anhydrous tetrahydrofuran (THF, 10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 2.0 equivalent), 4-dimethylaminopyridine (DMAP, 0.1 equivalent), and tridecanoic acid (2.0 equivalent) were added to the flask. The mixture was then reacted at room temperature for 12 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the mixture was transferred to room temperature, and ethyl acetate (100 mL) was added. The organic phase was washed with saturated sodium bicarbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography using dichloromethane / methanol (DCM / MeOH) 20:1 eluent to obtain the product (13-C (m=11)) in 92% yield. 1 H NMR (400 MHz, CDCl3) δ 5.03 (s, 1H), 4.35 – 4.25 (m, 1H), 4.23 – 4.14 (m, 2H), 4.13 – 3.97 (m, 5H), 3.92 – 3.60 (m, 4H), 3.57(dt, J = 10.5, 3.5 Hz, 1H), 2.39 – 2.30 (m, 4H), 2.00 (m, 1H), 1.62 (m, 4H), 1.33 (s, 1H), 1.27 (m, 36H), 0.87 (t, J = 6.7 Hz, 6H). MS (ESI) calcd forC 35 H 66 O 10 , m / z 646.5; found 669.6 [M + Na] +

[0043] Example 2: A method for synthesizing a novel glycolipid TLR2 agonist 13-A (n=11). 13-A (n=11)

[0044] Step 1: Synthesis of compound 13-AVa 13-AVa

[0045] (IV-a1) (5 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stirrup. Anhydrous tetrahydrofuran (THF, 60 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 2.0 equivalent), 4-dimethylaminopyridine (DMAP, 0.1 equivalent), and lauric acid (2.0 equivalent) were added to the flask. The mixture was then reacted at room temperature for 12 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the mixture was moved to room temperature, and ethyl acetate (100 mL) was added. The organic phase was washed with saturated sodium bicarbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the product (13-AVa).

[0046] Step 2: Synthesis of compound 13-A-VI-a 13-A-VI-a

[0047] The product (13-AVa) (5 mmol) obtained in the first step was added to a 100 mL plastic reaction flask equipped with a magnetic flask. Anhydrous tetrahydrofuran (THF) was added to the flask, and pyridine hydrogen fluoride (6.0 equivalent) was slowly added dropwise at 0 °C. The reaction was then carried out at room temperature for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the flask was placed in an ice bath, and the reaction was quenched by slowly adding saturated sodium bicarbonate solution. The flask was then extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was then concentrated under reduced pressure to obtain the product (13-A-VI-a).

[0048] Step 3: Synthesis of compound 13-A (n=11) 13-A (n=11)

[0049] The product obtained in the second step (13-A-VI-a) (0.2 mmol) was added to a 50 mL round-bottom flask equipped with a magnetic stirrup. Anhydrous tetrahydrofuran (THF, 10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 2.0 equivalent), 4-dimethylaminopyridine (DMAP, 0.1 equivalent), and lauric acid (2.0 equivalent) were added. The mixture was then reacted at room temperature for 12 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the mixture was transferred to room temperature, and ethyl acetate (100 mL) was added. The organic phase was washed with saturated sodium bicarbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography using dichloromethane / methanol (DCM / MeOH) 20:1 eluent to obtain the product (13-A (n=11)) in 92% yield. 1 H NMR (600 MHz, CDCl3) δ 5.00 (s, 1H), 4.58 (s, 1H), 4.25 (dd, J= 11.6, 7.7 Hz, 1H), 4.16 (m, 1H), 4.10 (m, 4H), 4.04 (d, J = 15.8 Hz, 2H), 4.00 – 3.97 (m, 1H), 3.75 – 3.65 (m, 2H), 3.56 (dt, J = 10.5, 5.2 Hz, 1H), 2.33 (q, J = 7.1 Hz, 6H), 1.60 (qt, J = 7.7, 3.4 Hz, 6H), 1.46 – 1.38 (m,4H), 1.26 (d, J = 23.3 Hz, 54H), 0.87 (t, J = 6.9 Hz, 9H). MS (ESI) calcd.for C 34 H 64 O 10 , m / z 632.5; found 655.6 [M + Na] +

[0050] Example 3: A method for synthesizing a novel glycolipid TLR2 agonist, 14-AS (n=12). 14-AS (n=12)

[0051] Step 1: Synthesis of compound 14-AS-III-a 14-AS-III-a

[0052] Compound (II) (750 mg, 1 mmol) and 4 Å molecular sieve (1.0 g) were added to a 50 mL round-bottom flask equipped with a magnetic stir bar. Anhydrous dichloromethane (CH₂Cl₂, 10 mL) was added to the reaction flask under argon atmosphere, and the mixture was stirred at room temperature for 3 h. The reaction mixture was then cooled to -40 °C, and trimethyliodosilane (TMSI, 170 μL, 1.2 mmol, 1.2 eq.) was added. The reaction was continued at -40 °C for 30 min, and thin-layer chromatography confirmed that compound II was completely converted to two glycosyl iodides. Subsequently, (R)-(2,2-dimethyl-1,3-dioxy)thiol (1.5 mmol, 1.5 eq.) and N,N-diisopropylethylamine (DIPEA, 348 μL, 2.0 mmol, 2.0 eq.) were added. The reaction mixture was then stirred at room temperature for another 3 h until the iodide was observed to be completely converted to a product with a slightly higher TLC Rf value. After the raw material was confirmed to have completely disappeared by thin-layer chromatography, the mixture was filtered through diatomaceous earth to remove the 4 Å molecular sieve and washed with dichloromethane. The combined organic phases were washed successively with saturated sodium bicarbonate (NaHCO3) solution, 10% sodium thiosulfate solution (Na2S2O3), and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a 100:1 petroleum ether / ethyl acetate (PE / EtOAc) eluent to give the colorless oily target compound (14-AS-III-a) in 76% yield. 1 H NMR (400 MHz, CDCl3) δ 5.25 (d, J = 3.3 Hz, 1H), 4.22 (m, 1H), 4.14 – 4.07 (m, 2H), 3.94 (m, 1H), 3.82 (m, 1H), 3.76 – 3.70(m, 2H), 3.68 – 3.63 (m, 1H), 3.61 – 3.52 (m, 1H), 2.98 (dd, J = 13.4, 5.5Hz, 1H), 2.66 (dd, J = 13.4, 7.8 Hz, 1H), 1.39 (s, 3H), 1.33 (s, 3H), 0.90(s, 9H), 0.89 (s, 9H), 0.88 (s, 9H), 0.85 (s, 9H), 0.13 (s, 6H), 0.09 (s,6H), 0.07 (s, 6H), 0.04 (s, 6H). MS (ESI) calcd. for C 36 H 78O7Si4, m / z 766.5;found 789.6 [M + Na] +

[0053] Step 2: Synthesis of compound 14-AS-IV-a 14-AS-IV-a

[0054] The first step (14-AS-III-a) (635 mg, 0.85 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA), water, and dichloromethane (CH2Cl2) in a volume ratio of 2.5:2.5:95, yielding a total volume of 42 mL. The solution was stirred at room temperature for 30 min. After the reaction was confirmed by thin-layer chromatography to be completely dissolved, saturated sodium bicarbonate (NaHCO3) solution was added to quench the reaction. The mixture was then extracted with dichloromethane, and the combined organic layers were washed successively with saturated sodium bicarbonate solution and saturated brine. Finally, the organic layers were dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a 10:1 petroleum ether / ethyl acetate (PE / EtOAc) eluent to obtain the desired target compound (14-AS-IV-a) in 80% yield as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.13 (d, J= 3.6 Hz, 1H), 4.13 (m, 1H), 4.00 (m, 1H), 3.87 (m, 1H), 3.80 (m, 2H), 3.66(m, 2H), 3.59 (m, 2H), 3.53 (m, 1H), 2.78 (m, 1H), 2.48 (m, 1H), 0.90 (s,18H), 0.86 (s, 18H), 0.13 (s, 6H), 0.09 (s, 6H), 0.07 (s, 6H), 0.04 (s, 6H).MS (ESI) calcd. for C 33 H 74 O7Si4, m / z 726.4; found 749.5 [M + Na] +

[0055] Step 3: Synthesis of compound 14-ASVa 14-ASVa

[0056] The second-step compound (14-AS-IV-a) (355 mg, 0.5 mmol) and lauric acid (120 mg, 0.6 mmol) were added to a 50 mL round-bottom flask equipped with a magnetic stir bar. Anhydrous tetrahydrofuran (10 mL) was then added to the reaction flask, followed by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 192 mg, 1 mmol) and 4-dimethylaminopyridine (DMAP, 12.2 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and then the organic phase was washed sequentially with saturated sodium bicarbonate (NaHCO3) solution and saturated brine. Finally, the organic layer was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography with a 30:1 petroleum ether / ethyl acetate (PE / EtOAc) eluent to obtain the desired target compound (14-ASVa) in 92% yield as a colorless oil. 1 H NMR(600 MHz, CDCl3) δ 5.12 (d, J = 3.5 Hz, 1H), 4.19 – 4.10 (m, 3H), 4.02 (m,1H), 3.99 (m, 1H), 3.82 (m, 1H), 3.79 (m, 1H), 3.67 (dd, J = 10.6, 4.8 Hz, 1H), 3.58 (dd, J = 10.7, 4.8 Hz, 1H), 3.48 (d, J = 6.6 Hz, 1H), 2.86 (dd, J =14.3, 3.6 Hz, 1H), 2.77 (dd, J = 14.4, 6.7 Hz, 1H), 2.30 (t, J = 7.6 Hz, 2H),1.60 (m, 2H), 1.26 (m, 16H), 0.91 (s, 9H), 0.90 (s, 9H), 0.88 (s, 9H), 0.85(s, 9H), 0.14 (s, 6H), 0.10 (s, 6H), 0.08 (s, 6H), 0.04 (s, 6H). MS (ESI)calcd. for C 45 H 96 O8Si4, m / z 908.6; found 931.7 [M + Na] +

[0057] Step 4: Synthesis of compound 14-AS-VI-a 14-AS-VI-a

[0058] The compound from step three (14-ASVa) (268 mg, 0.3 mmol) was added to a 10 mL plastic flask equipped with a magnetic stirrup. Anhydrous tetrahydrofuran (THF, 2 mL) was added, followed by pyridine hydrogen fluoride (HF·py, 0.5 mL). The reaction mixture was stirred at room temperature for 2 h. After the starting material was confirmed to have completely disappeared by thin-layer chromatography, saturated sodium bicarbonate aqueous solution (NaHCO3) was added to quench the reaction. The mixture was then extracted with ethyl acetate (EtOAc), and the combined organic phases were washed successively with 0.1 N dilute hydrochloric acid (HCl) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane / methanol (DCM / MeOH) 20:1 eluent to give the desired target compound (14-AS-VI-a) in a white foamy form in 84% yield. 1 H NMR (400 MHz, CD3OD) δ 5.34 (d, J = 4.5 Hz, 1H), 4.20 – 4.07 (m, 4H), 4.06 – 3.98 (m, 1H), 3.83 (m, 1H), 3.77 (m, 1H), 3.67 – 3.54 (m, for C 21 H 40 O8S, m / z 452.2; found 475.3 [M + Na] +

[0059] Step 5: Synthesis of compound 14-AS (n=12) 14-AS (n=12)

[0060] Compound (14-AS-VI-a) (87.25 mg, 0.2 mmol) and tetradecanoic acid (0.21 mmol) from step four were added to 20 mL round-bottom flasks equipped with magnetic stirrups. Anhydrous tetrahydrofuran (5 mL) was added to the reaction flasks, followed by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 58 mg, 0.3 mmol) and 4-dimethylaminopyridine (DMAP, 5.0 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and then the organic phase was washed sequentially with saturated sodium bicarbonate solution (NaHCO3) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography with a 30:1 dichloromethane / methanol (DCM / MeOH) eluent to obtain the desired target compound (14-AS) in a colorless oil in 76% yield. 1 H NMR(600 MHz, CDCl3) δ 5.37 (d, J = 4.7 Hz, 1H), 4.79 – 4.68 (m, 1H), 4.30 (m,3H), 4.26 – 4.23 (m, 2H), 4.11 (m, 4H), 4.07 (s, 1H), 4.03 – 3.97 (m, 1H),3.88 (m, 1H), 2.90 (dd, J = 14.2, 2.8 Hz, 1H), 2.78 (dd, J = 14.3, 7.2 Hz,1H), 2.33 (t, J = 7.6 Hz, 4H), 1.60 (m, 4H), 1.26 (m, 36H), 0.87 (t, J = 6.9Hz, 6H). MS (ESI) calcd. for C 35 H 66 O9S, m / z 662.4; found 685.5 [M + Na] +

[0061] Example 4: A method for synthesizing a novel glycolipid TLR2 agonist, 14-AN (n=12). 14-AN (n=12)

[0062] Step 1: Synthesis of compound 14-AN-III-a 14-AN-III-a

[0063] Compound (II) (750 mg, 1 mmol) and 4 Å molecular sieve (1.0 g) were added to a 50 mL round-bottom flask equipped with a magnetic stir bar. Anhydrous dichloromethane (CH2Cl2, 10 mL) was added to the reaction flask under argon atmosphere, and the mixture was stirred at room temperature for 3 h. The reaction mixture was then cooled to -40 °C, and trimethyliodosilane (TMSI, 170 μL, 1.2 mmol, 1.2 eq.) was added. The reaction was stirred at -40 °C for another 30 min. Thin-layer chromatography confirmed that compound II was completely converted to two glycosyl iodides. Subsequently, (S)-epoxyethanol (1.5 mmol, 1.5 eq.) and N,N-diisopropylethylamine (DIPEA, 348 μL, 2.0 mmol, 2.0 eq.) were added to the reaction flask, and the reaction mixture was stirred at room temperature for another 3 h until the iodides were observed to be completely converted to the product with a slightly higher TLC Rf value. After the raw material was completely eliminated by thin-layer chromatography, the 4Å molecular sieve was removed by diatomaceous earth filtration and the mixture was washed with dichloromethane. The combined organic phases were washed successively with saturated sodium bicarbonate solution (NaHCO3), 10% sodium thiosulfate solution (Na2S2O3), and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography using a 100:1 petroleum ether / ethyl acetate (PE / EtOAc) eluent to obtain the desired target compound (14-AN-III-a) in a colorless oily form with a yield of 72%. 1 H NMR (400 MHz, CDCl3) δ 4.83 (d, J = 2.2 Hz, 1H), 4.12 (dd, J= 5.8, 3.3 Hz, 1H), 4.01 (t, J = 2.7 Hz, 1H), 3.95 (dd, J = 5.9, 2.9 Hz, 1H), 3.76 (dt, J = 12.4, 6.1 Hz, 2H), 3.65 (dd, J = 9.9, 6.7 Hz, 1H), 3.59 – 3.51(m, 2H), 3.15 – 3.08 (m, 1H), 2.76 – 2.70 (m, 1H), 2.64 (dd, J = 5.3, 2.6 Hz, 1H), 0.91 – 0.78 (m, 39H), 0.09 – 0.01 (m, 25H). MS (ESI) calcd. forC 33 H 72O7Si4, m / z 692.4; found 715.5 [M + Na] +

[0064] Step 2: Synthesis of compound 14-AN-IV-a 14-AN-IV-a

[0065] The first step (14-AN-III-a) was added to a 50 mL round-bottom flask equipped with a magnetic stir bar and dissolved in methanol:water (8:1). Then, NaN3 (4.25 mmol, 5.0 eq.) and NH4Cl (1.87 mmol, 2.2 eq.) were added to the reaction flask, and the mixture was reacted at 60 °C for 12 h. After the starting material was completely eliminated as confirmed by thin-layer chromatography, the reaction system was moved to room temperature, and a saturated NaHCO3 solution was added to the mixture. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were dried over anhydrous Na2SO4, filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate 40:1) to obtain the desired target compound (14-AN-IV-a) in 80% yield. 1 H NMR (600 MHz, CDCl3) δ4.84 (d, J = 2.4 Hz, 1H), 4.13 (m, 1H), 4.00 – 3.98 (m, 2H), 3.92 (m, 1H), 3.76 – 3.72 (m, 1H), 3.65 – 3.61 (m, for C 33 H 73 N3O7Si4, m / z 735.5; found 758.6 [M + Na] +

[0066] Step 3: Synthesis of compound 14-ANVa 14-ANVa

[0067] The compound from step two (14-AN-IV-a) (588 mg, 0.8 mmol) was added to a 50 mL round-bottom flask equipped with a magnetic stir bar and dissolved in tetrahydrofuran:water (10:1). Then, PPh3 (1.6 mmol, 2.0 eq.) was added to the reaction flask, and the reaction mixture was stirred overnight at room temperature. After the starting material was completely eliminated by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate 30:1) to give the desired target compound (14-ANVa) as a colorless oil in 70% yield. 1 H NMR (600 MHz, CDCl3) δ 4.81 (d, J = 2.4 Hz, 1H), 4.10 (dd, J = 5.0, 3.1 Hz, 1H), 3.97 (t, J = 2.7 Hz, 1H), 3.95 (dd, J =5.0, 3.6 Hz, 1H), 3.72 (m, 2H), 3.62 (m, 1H), 3.58 – 3.52 (m, 3H), 2.79 (dd,J = 12.8, 4.1 Hz, 1H), 2.71 (dd,J = 12.8, 6.9 Hz, 1H), 2.57 (s, 3H), 0.89 –0.83 (m, 36H), 0.14 – -0.05 (m, 24H). MS (ESI) calcd. for C 33 H 75 NO7Si4, m / z709.5; found 732.6 [M + Na] +

[0068] Step 4: Synthesis of compound 14-AN-VI-a 14-AN-VI-a

[0069] The compound from step three (14-ANVa) (355 mg, 0.5 mmol) and lauric acid (120 mg, 0.6 mmol) were added to a 50 mL round-bottom flask equipped with a magnetic flask and dissolved in anhydrous tetrahydrofuran (10 mL). Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 192 mg, 1 mmol) and 4-dimethylaminopyridine (DMAP, 12.2 mg, 0.1 mmol) were added to the system, and the reaction mixture was stirred at room temperature for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and the organic phase was washed sequentially with saturated sodium bicarbonate (NaHCO3) solution and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography using a 30:1 petroleum ether / ethyl acetate (PE / EtOAc) eluent to obtain the desired target compound (14-4-N-VI-a) in 92% yield as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 6.18 (t, J = 5.9 Hz, 1H), 4.76 (d, J = 2.4 Hz, 1H), 4.06 (dd, J =4.9, 3.2 Hz, 1H), 3.94 – 3.90 (m, 2H), 3.84 (d, J = 2.9 Hz, 1H), 3.79 (m,1H), 3.67 (m, 1H), 3.56 (m, 1H), 3.53 (m, 2H), 3.45 (dd, J = 10.7, 7.1 Hz,1H), 3.36 (m, 1H), 3.18 (m, 1H), 2.09 (t, J = 7.6 Hz, 2H), 1.54 (m, 2H), 1.19(m, 16H), 0.84 – 0.76 (m, 39H), 0.05 – -0.07 (m, 24H). MS (ESI) calcd. forC 45 H 97 NO8Si4, m / z 891.6; found 914.7 [M + Na] +

[0070] Step 5: Synthesis of compound 14-AN-VII-a 14-AN-VII-a

[0071] The compound from step four (14-AN-VI-a) (268 mg, 0.3 mmol) was added to a 10 mL plastic flask equipped with a magnetic stir bar and dissolved in anhydrous tetrahydrofuran (THF, 3 mL). Then, pyridine hydrogen fluoride (HF·Py, 0.5 mL) was added to this solution, and the reaction mixture was stirred at room temperature for 2 h. After the starting material was confirmed to have completely disappeared by thin-layer chromatography, the reaction was quenched with saturated sodium bicarbonate solution (NaHCO3), and the product was extracted with ethyl acetate (EtOAc). The organic phase was washed sequentially with 0.1 N dilute hydrochloric acid (HCl) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a dichloromethane / methanol (DCM / MeOH) 20:1 eluent to obtain the desired target compound (14-AN-VII-a) in a white foamy form with a yield of 84%. 1 H NMR (400MHz, CD3OD) δ 4.93 (s, 1H), 4.03 (m, 3H), 3.88 (m, 1H), 3.78 (m, 1H), 3.68(m, 3H), 3.48 (dd, J = 10.4, 4.3 Hz, 1H), 3.44 – MS (ESI) calcd. for C 21 H 40 O8S, m / z 452.2; found 475.3[M + Na] +

[0072] Step 6: Synthesis of compound 14-AN (n=12) 14-AN (n=12)

[0073] The fifth-step compound (14-AN-VII-a) (90 mg, 0.2 mmol) and tetradecanoic acid (0.21 mmol) were added separately to 50 mL round-bottom flasks equipped with magnetic flasks and dissolved in anhydrous tetrahydrofuran (4 mL). Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 58 mg, 0.3 mmol) and 4-dimethylaminopyridine (DMAP, 5.0 mg, 0.04 mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and the organic phase was washed sequentially with saturated sodium bicarbonate solution (NaHCO3) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol 20:1) to obtain the desired target compound (14-AN) in 80% yield as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.01 (d, J =2.3 Hz, 1H), 4.26 (m, 1H), 4.17 (m, 1H), 4.14 – 4.07 (m, 4H), 4.04 (m, 2H), 4.01 – 3.95 (m, 1H), 3.82 – 3.65 (m, 1H), 3.52 (m, 1H), 2.46 (s, 1H), 2.33(t, J = 7.6 Hz, 4H), 1.60 (m, 4H), 1.26 (m, 36H), 0.87 (t, J = 6.8 Hz, 6H).MS (ESI) calcd. for C 35 H 67 O9N, m / z 645.5; found 668.6 [M + Na] +

[0074] Example 5: A method for synthesizing a novel glycolipid TLR2 agonist 13-B (n=11). 13-B (n=11)

[0075] Step 1: Synthesis of compound 13-BVa 13-BVa

[0076] (IV-a) (3.55 g, 5.0 mmol) and lauric acid (2.20 g, 11 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic flask and dissolved in anhydrous toluene (40 mL). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.92 g, 10 mmol) and 4-dimethylaminopyridine (DMAP, 122.2 mg, 1 mmol) were then added to the solution. The reaction mixture was stirred at 80 °C for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and the organic phase was washed sequentially with saturated sodium bicarbonate solution (NaHCO3) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography using a 30:1 petroleum ether / ethyl acetate (PE / EtOAc) eluent to obtain the desired target compound (13-BVa) in a colorless oily form with a yield of 83%. 1 H NMR (400 MHz, CDCl3) δ 5.18 (m, 1H), 4.78 (d, J = 2.0 Hz, 1H), 4.33 (dd, J = 11.9, 3.5 Hz, 1H), 4.17 (dd, J =11.9, 6.5 Hz, 1H), 4.11 (dd, J = 5.7, 3.2 Hz, 1H), 3.98 (t, J = 2.6 Hz, 1H), 3.91 (dd, J = 5.7, 3.2 Hz, 1H), 3.76 (m, 2H), 3.64 – 3.62 (m, 1H), 3.54 (m,2H), 2.27 (m, 4H), 1.58 (m, 4H), 1.25 (m, 32H), 0.86 (m, 42H), 0.14 – 0.01(m, 24H). MS (ESI) calcd. for C 57 H 118 NO 10 Si4, m / z 1074.7; found 1097.8 [M + Na] +

[0077] Step 2: Synthesis of compound 13-B-VI-a 13-B-VI-a

[0078] The product obtained in the first step (13-BVa) (3.22 g, 3.0 mmol) was added to a 50 mL plastic flask equipped with a magnetic stir bar and dissolved in anhydrous tetrahydrofuran (THF, 30 mL). Then, pyridine hydrogen fluoride (HF·Py, 5 mL) was added to the reaction flask, and the reaction mixture was stirred at room temperature for 2 h. After the starting material was confirmed to have completely disappeared by thin-layer chromatography, the reaction was quenched with saturated sodium bicarbonate solution (NaHCO3), and the product was extracted with ethyl acetate (EtOAc). The organic phase was washed sequentially with 0.1 N dilute hydrochloric acid (HCl) and saturated brine; finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with dichloromethane / methanol (DCM / MeOH) 20:1 eluent to give the desired target compound (13-B-VI-a) in a white foamy form in 82% yield. 1 H NMR (600 MHz, CDCl3) δ 5.19 (m, 1H), 4.93 (m, 2H), 4.64 (m, 1H), 4.33 (dd, J = 12.1, 3.2Hz, 1H), 4.22 (d, J = 7.1 Hz, 1H), 4.13 – 4.03 (m, 3H), 4.00 (d, J = 5.9 Hz,1H), 3.96 (m, 1H), 3.88 – 3.82 (m, 1H), 3.76 – 3.63 (m, 3H), 3.58 (dd, J =11.0, 5.7 Hz, 1H), 2.28 (q, J = 8.3 Hz, 4H), 1.56 (m, 4H), 1.24 (m, 32H),0.85 (t, J = 6.9 Hz, 6H). MS (ESI) calcd. for C 33 H 62 O 10 , m / z 618.4; found 641.5[M + Na] +

[0079] Step 3: Synthesis of compound 13-B (n=11) 13-B (n=11)

[0080] The product obtained in the second step (13-B-VI-a) (124 mg, 0.2 mmol) and tridecanoic acid (0.21 mmol) were added separately to a 50 mL round-bottom flask equipped with a magnetic flask and dissolved in anhydrous tetrahydrofuran (5 mL). Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 58 mg, 0.3 mmol) and 4-dimethylaminopyridine (DMAP, 5.0 mg, 0.04 mmol) were added to the system, and the reaction mixture was stirred at room temperature for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and the organic phase was washed sequentially with saturated sodium bicarbonate solution (NaHCO3) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (dichloromethane / methanol 20:1) to obtain the desired target compound (13-B) in a colorless oil with a yield of 78%. 1 H NMR (600 MHz, CDCl3) δ 5.19 (m, 1H), 5.00(s, 1H), 4.36 – 4.27 (m, 2H), 4.19 (dd, J = 11.8, 3.7 Hz, 1H), 4.14 – 4.10(m, 1H), 4.09 (m, 1H), 4.06 (d, J = 11.8 Hz, 2H), 4.00 (s, 1H), 3.80 (dd, J =11.0, 6.0 Hz, 1H), 3.60 (dd, J = 11.0, 4.7 Hz, 1H), 3.47 (s, 1H), 3.01 – 2.83(m, 1H), 2.37 – 2.29 (m, 6H), 1.79 (s, 1H), 1.67 – 1.57 (m, 6H), 1.34 – 1.23(m, 50H), 0.87 (t, J = 7.0 Hz, 9H). MS (ESI) calcd. for C 46 H 86 O 11 , m / z 814.6;found 837.7 [M + Na] +

[0081] Example 6: A method for synthesizing a novel glycolipid TLR2 agonist, 16-D (m=14). 16-D (m=14)

[0082] Step 1: Synthesis of compound 16-DVa 16-DVa

[0083] (IV-a) (3.55 g, 5.0 mmol) and hexadecanoic acid (11 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirrup and dissolved in anhydrous toluene (40 mL). Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.92 g, 10 mmol) and 4-dimethylaminopyridine (DMAP, 122.2 mg, 1 mmol) were added to this solution. The reaction mixture was stirred at 80 °C for 12 h. After the starting material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and the organic phase was washed successively with saturated sodium bicarbonate solution (NaHCO3) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure to obtain a colorless oily target compound (16-DVa).

[0084] Step 2: Synthesis of compound 16-D-VI-a 16-D-VI-a

[0085] The product obtained in the first step (16-DVa) (3.0 mmol) was added to a 50 mL plastic flask equipped with a magnetic stir bar and dissolved in anhydrous tetrahydrofuran (THF, 30 mL). Then, pyridine hydrogen fluoride (HF·Py, 5 mL) was added to the reaction flask, and the reaction mixture was stirred at room temperature for 2 h. After the starting material was confirmed to have completely disappeared by thin-layer chromatography, the reaction was quenched with saturated sodium bicarbonate solution (NaHCO3), and the product was extracted with ethyl acetate (EtOAc). The organic phase was washed successively with 0.1 N dilute hydrochloric acid (HCl) and saturated brine; finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure to obtain the target compound (16-D-VI-a).

[0086] Step 3: Synthesis of compound 16-D (m=14) 16-D (m=14)

[0087] The product obtained in the second step (16-D-VI-a) (124 mg, 0.2 mmol) and tridecanoic acid (0.21 mmol) were added separately to 50 mL round-bottom flasks equipped with magnetic inlets and dissolved in anhydrous tetrahydrofuran (5 mL). Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 58 mg, 0.3 mmol) and 4-dimethylaminopyridine (DMAP, 5.0 mg, 0.04 mmol) were added to the system, and the reaction mixture was stirred at room temperature for 12 h. After the raw material was completely eliminated by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate (EtOAc), and the organic phase was washed sequentially with saturated sodium bicarbonate solution (NaHCO3) and saturated brine. Finally, the organic phase was dried with anhydrous sodium sulfate (Na2SO4), filtered through defatted cotton, and the organic solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol 20:1) to obtain the desired target compound (16-D(m=14)) in 78% yield as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 5.19 (m, 1H),5.00 (s, 1H), 4.37 – 4.27 (m, 2H), 4.19 (dd, J = 11.8, 3.7 Hz, 1H), 4.13 –4.03 (m, 5H), 4.00 (s, 1H), 3.80 (dd, J = 11.0, 6.0 Hz, 1H), 3.60 (dd, J =11.0, 4.7 Hz, 1H), 3.48 (s, 1H), 2.91 (d, J = 10.8 Hz, 1H), 2.40 – 2.26 (m,6H), 1.64 – 1.56 (m, 6H), 1.32 – 1.21 (m, 66H), 0.87 (t, J = 6.9 Hz, 9H). MS(ESI) calcd. for C 54 H 102 O 11 , m / z 926.7; found 949.8 [M + Na] +

[0088] Example 7: Synthesis of other compounds

[0089] Other compounds disclosed in this invention can be synthesized according to the methods of Examples 1-6. Specific compounds are shown in the table below: serial number structural <![CDATA[ 1 H NMR or MS<!-- 19 --> ]]> 12-AN (n=10) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 6.64 – 6.46 (m,1H), 5.13 (d, J = 6.7 Hz, 1H), 5.03 (p, J =5.2 Hz, 1H), 4.93 (s, 1H), 4.73 (dd, J =10.6, 5.8 Hz, 2H), 4.42 – 4.30 (m, 1H), 4.09(d, J = 5.8 Hz, 1H), 4.05 – 3.99 (m, 2H),3.86 (q, J = 9.0, 4.8 Hz, 1H), 3.74 (dd, J =11.1, 4.6 Hz, 1H), 3.68 (d, J = 6.6 Hz, 2H),3.61 (dt, J = 14.3, 4.9 Hz, 1H), 3.53 (dd, J= 11.1, 5.2 Hz, 1H), 3.35 (dq, J = 13.7, 7.0,6.4 Hz, 1H), 2.30 (t, J = 7.6 Hz, 2H), 2.14(t, J = 7.6 Hz, 2H), 1.56 (dp, J = 14.2, 7.1Hz, 4H), 1.25 (dd, J = 13.3, 5.3 Hz, 32H),0.86 (t, J = 7.0 Hz, 6H). MS (ESI) calcd forC 33 H 63 O9N, m / z 617.5; found 640.6 [M + Na] + ]]> 12-AS (n=10) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.37 (d, J = 4.7Hz, 1H), 4.72 (m, 1H), 4.32 – 4.27 (m, 3H),4.25 (m, 2H), 4.13 (m, 2H), 4.11 – 4.08 (m,2H), 4.07 (s, 1H), 4.00 (m, 1H), 3.88 (m,1H), 2.90 (dd, J = 14.4, 2.9 Hz, 1H), 2.78(dd, J = 14.3, 7.3 Hz, 1H), 2.33 (t, J = 7.6Hz, 4H), 1.60 (m, 4H), 1.27 (m, 32H), 0.87(t, J = 7.0 Hz, 6H). MS (ESI) calcd forC 33 H 62 O9S, m / z 634.4; found 657.5 [M + Na] + ]]> 10-A (n=8) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H), 4.77– 4.66 (m, 1H), 4.35 – 4.29 (m, 1H), 4.25 (m,1H), 4.15 (m, 1H), 4.07 (m, 7H), 4.01 – 3.95(m, 2H), 3.68 (dd, J = 10.6, 6.2 Hz, 1H),3.54 (dd, J = 10.6, 3.1 Hz, 1H), 2.32 (m,4H), 1.66 – 1.54 (m, 4H), 1.25 (m, 28H), 0.86(t, J = 6.7 Hz, 6H). MS (ESI) calcd forC 31 H 58 O 10 , m / z 590.4; found 613.4 [M + Na] + ]]> 12-A (n=10) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H), 4.66(m, 1H), 4.26 (m, 2H), 4.16 (m, 1H), 4.18 –4.13 (m, 9H)3.68 (dd, J = 10.7, 5.6 Hz, 1H),3.55 (dd, J = 10.6, 2.4 Hz, 1H), 2.33 (m,4H), 1.59 (m, 4H), 1.26 (m, 34H), 0.86 (t, J= 6.7 Hz, 6H). MS (ESI) calcd for C 33 H 62 O 10 , m / z 618.4; found 641.4 [M + Na] + ]]> 14-A (n=12) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 5.01 (s, 1H), 4.26(m, 1H), 4.20 – 3.95 (m, 9H), 3.80 (m, 1H),3.75 – 3.64 (m, 1H), 3.52 (m, 1H), 2.46 (s,1H), 2.33 (t, J = 7.6 Hz, 4H), 2.26 (s, 1H),1.60 (m, 4H), 1.24 (s, 36H), 0.87 (t, J = 6.8Hz, 6H). MS (ESI) calcd for C 35 H 66 O 10 , m / z646.5; found 669.6 [M + Na] + <!-- 20 -->]]> 16-A (n=14) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H), 4.68(m, 1H), 4.35 – 4.21 (m, 2H), 4.16 (m, 1H),4.12 – 3.95 (m, 9H), 3.68 (m, 1H), 3.55 (m,1H), 2.33 (t, J = 7.6 Hz, 4H), 1.60 (m, 4H),1.25 (m, 40H), 0.86 (t, J = 6.8 Hz, 6H). MS(ESI) calcd for C 37 H 70 O 10 , m / z 674.5; found697.6 [M + Na] + ]]> 18-A (n=16) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H), 4.77(m, 1H), 4.42 (m, 1H), 4.32 – 4.19 (m, 2H),4.18 – 4.06 (m, 6H), 4.01 (m, 3H), 3.67 (m,1H), 3.54 (m, 1H), 2.32 (t, J = 7.6Hz, 4H),1.58 (m, 4H), 1.25 (m, 44H), 0.86 (t, J = 6.6Hz, 6H). MS (ESI) calcd for C 39 H 74 O 10 , m / z702.5; found 725.6 [M + Na] + ]]> 10-B (n=8) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.19 (p, J = 5.2Hz, 1H), 4.99 (s, 1H), 4.34 – 4.25 (m, 2H),4.17 (m, 1H), 4.12 – 4.09 (m, 1H), 4.08 (m,1H), 4.06 – 4.02 (m, 2H), 4.00 (s, 1H), 3.79(dd, J = 11.0, 5.9 Hz, 1H), 3.59 (dd, J =10.9, 4.8 Hz, 1H), 2.99 (d, J = 10.3 Hz, 1H),2.37 – 2.27 (m, 6H), 1.65 – 1.55 (m, 6H),1.34 – 1.22 (m, 444H), 0.86 (t, J = 6.9 Hz,9H). MS (ESI) calcd for C 43 H 80 O 11 , m / z 772.6;found 795.7 [M + Na] + ]]> 12-B (n=10) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.19 (p, J = 5.3Hz, 1H), 4.99 (s, 1H), 4.36 – 4.25 (m, 2H),4.17 (dd, J = 11.6, 4.3 Hz, 2H), 4.10 (dd, J= 12.0, 6.0 Hz, 1H), 4.08 (d, J = 1.9 Hz,1H), 4.06 – 4.02 (m, 2H), 3.99 (d, J = 7.6Hz, 1H), 3.79 (dd, J = 11.0, 5.9 Hz, 1H),3.66 – 3.62 (m, 1H), 3.59 (dd, J = 11.0, 4.8Hz, 1H), 3.01 (d, J = 10.5 Hz, 1H), 2.40 –2.25 (m, 6H), 1.68 – 1.54 (m, 7H), 1.26 (d, J= 22.4 Hz, 50H), 0.86 (t, J = 7.0 Hz, 9H). MS(ESI) calcd for C 45 H 84 O 11 , m / z 800.6; found823.7 [M + Na] + ]]> 14-B (n=12) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 5.23 – 5.16 (m,1H), 5.01 (s, 1H), 4.38 – 4.28 (m, 2H), 4.20(m, 1H), 4.13 – 4.04 (m, 5H), 4.00 (d, J =9.7 Hz, 1H), 3.81 (dd, J = 11.0, 5.9 Hz, 1H),3.60 (dd, J = 10.9, 4.6 Hz, 1H), 3.46 – 3.35(m, 1H), 2.87 (d, J = 11.2 Hz, 1H), 2.40 –2.26 (m, 6H), 1.60 (m, 6H), 1.26 (m, 52H),0.87 (t, J = 6.7 Hz, 9H). MS (ESI) calcd forC 47 H 88 O 11 , m / z 828.6; found 851.7 [M + Na] + <!-- 21 -->]]> 16-B (n=14) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 5.23 – 5.16 (m,1H), 4.99 (s, 1H), 4.35 – 4.25 (m, 2H), 4.17(dd, J = 11.6, 4.0 Hz, 1H), 4.08 (m, 4H),4.01 – 3.98 (m, 1H), 3.79 (dd, J = 11.0, 5.9Hz, 1H), 3.60 (m, 2H), 3.00 (d, J = 10.5 Hz,1H), 2.38 – 2.26 (m, 6H), 1.59 (m, 6H), 1.30– 1.22 (m, 56H), 0.86 (t, J = 6.8 Hz, 9H). MS(ESI) calcd for C 49 H 92 O 11 , m / z 856.7; found879.8 [M + Na] + ]]> 18-B (n=16) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 5.19 (m, 1H), 5.00(s, 1H), 4.35 – 4.25 (m, 2H), 4.15 (m, 2H),4.09 – 4.06 (m, 2H), 4.04 (s, 1H), 4.00 (d, J= 6.8 Hz, 1H), 3.79 (dd, J = 11.0, 5.9 Hz,1H), 3.59 (m, 2H), 2.97 (d, J = 10.6 Hz, 1H),2.47 – 2.21 (m, 6H), 1.60 (m, 6H), 1.26 (m,60H), 0.87 (t, J = 6.7 Hz, 9H). MS (ESI)calcd for C 51 H 96 O 11 , m / z 884.7; found 907.8 [M+ Na] + ]]> 10-C (m=8) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H), 4.70(s, 1H), 4.37 – 4.20 (m, 2H), 4.16 (m, 2H),4.07 (m, 6H), 3.99 (m, 2H), 3.68 (dd, J =10.5, 6.2 Hz, 1H), 3.55 (dd, J = 10.6, 2.9Hz, 1H), 2.33 (td, J = 7.7, 3.6 Hz, 4H), 1.60(m, 4H), 1.25 (m, 30H), 0.86 (t, J = 6.7 Hz,6H). MS (ESI) calcd for C 32 H 60 O 10 , m / z 604.4;found 427.5 [M + Na] + ]]> 14-C (m=12) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.02 (s, 1H), 4.39(s, 1H), 4.28 (dd, J = 11.6, 7.8 Hz, 1H),4.22 – 4.12 (m, 2H), 4.12 – 4.04 (m, 4H),3.99 (m, 1H), 3.89 – 3.72 (m, 2H), 3.69 (dd,J = 10.6, 6.6 Hz, 1H), 3.56 (m, 2H), 2.34 (q,J = 7.2 Hz, 4H), 2.16 – 1.93 (m, 1H), 1.61(m, 4H), 1.32 – 1.22 (m, 38H), 0.87 (t, J =7.0 Hz, 6H). MS (ESI) calcd for C 36 H 68 O 10 , m / z660.5; found 683.6 [M + Na] + ]]> 16-C (m=14) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 5.03 (s, 1H), 4.28(dd, J = 11.7, 7.9 Hz, 1H), 4.22 – 4.13 (m,2H), 4.13 – 4.02 (m, 5H), 4.01 – 3.96 (m,1H), 3.70 (dd, J = 10.6, 6.4 Hz, 2H), 3.56(dd, J = 10.5, 3.5 Hz, 1H), 3.47 (m, 1H),2.34 (td, J = 7.6, 4.4 Hz, 4H), 1.95 (s, 1H),1.68 – 1.56 (m, 4H), 1.41 (s, 1H), 1.31 –1.22 (m, 42H), 0.87 (t, J = 6.7 Hz, 6H). MS(ESI) calcd for C 38 H 72 O 10 , m / z 688.5; found711.6 [M + Na] + <!-- 22 -->]]> 18-C (m=16) <![CDATA[ 1 H NMR (400 MHz, CDCl3) δ 5.02 (s, 1H), 4.56– 4.21 (m, 2H), 4.21 – 4.12 (m, 2H), 4.11 –4.02 (m, 4H), 3.99 (s, 1H), 3.93 – 3.64 (m,3H), 3.56 (dd, J = 10.5, 3.4 Hz, 1H), 2.34(td, J = 7.6, 4.3 Hz, 4H), 2.07 (s, 1H), 1.62(m, 4H), 1.41 (m, 1H), 1.26 (m, 46H), 0.87(t, J = 6.6 Hz, 6H). MS (ESI) calcd forC 40 H 76 O 10 , m / z 716.5; found 739.6 [M + Na] + ]]> 20-C (m=18) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.01 (s, 1H), 4.26(dd, J = 11.6, 7.8 Hz, 1H), 4.21 – 3.95 (m,11H), 3.69 (dd, J = 10.6, 6.5 Hz, 1H), 3.56(dd, J = 10.6, 3.4 Hz, 1H), 2.51 (s, 1H),2.33 (q, J = 7.0 Hz, 4H), 1.61 (m, 4H), 1.26(m, 50H), 0.87 (t, J = 6.9 Hz, 6H). MS (ESI)calcd for C 42 H 80 O 10 , m / z 744.6; found 767.7 [M+ Na] + ]]> 10-D (m=8) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.19 (m, 1H), 5.01(s, 1H), 4.36 – 4.28 (m, 2H), 4.19 (dd, J =11.8, 3.6 Hz, 1H), 4.13 – 4.03 (m, 5H), 4.00(s, 1H), 3.80 (dd, J = 11.0, 5.9 Hz, 1H),3.60 (dd, J = 11.0, 4.7 Hz, 1H), 3.44 (s,1H), 2.89 (d, J = 10.9 Hz, 1H), 2.37 – 2.29(m, 6H), 1.61 (m, 6H), 1.31 – 1.23 (m, 42H),0.87 (t, J = 6.9 Hz, 9H). MS (ESI) calcd forC 42 H 78 O 11 , m / z 758.6; found 781.7 [M + Na] + ]]> 13-D (m=11) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.19 (m, 1H), 4.99(s, 1H), 4.32 (dd, J = 11.9, 4.1 Hz, 1H),4.28 (dd, J = 11.7, 7.9 Hz, 1H), 4.17 (dd, J= 11.7, 4.1 Hz, 1H), 4.10 (dd, J = 11.9, 6.0Hz, 1H), 4.08 (d, J = 2.1 Hz, 1H), 4.05 (qd,J = 4.2, 1.8 Hz, 2H), 4.00 (s, 1H), 3.79 (dd,J = 11.0, 5.9 Hz, 1H), 3.59 (dd, J = 11.0,4.8 Hz, 1H), 3.03 (s, 1H), 2.39 – 2.25 (m,6H), 1.67 – 1.54 (m, 6H), 1.26 (m, 54H), 0.86(t, J = 7.0 Hz, 9H). MS (ESI) calcd forC 48 H 90 O 11 , m / z 842.7; found 865.8 [M + Na] + <!-- 23 -->]]> 14-D (m=12) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.20 (m, 1H), 4.99(s, 1H), 4.32 (dd, J = 11.9, 4.1 Hz, 1H),4.28 (dd, J = 11.7, 7.9 Hz, 1H), 4.17 (dd, J= 11.7, 4.1 Hz, 2H), 4.10 (dd, J = 11.9, 6.0Hz, 1H), 4.08 (m, 1H), 4.07 – 4.03 (m, 2H),4.00 (s, 1H), 3.79 (dd, J = 11.0, 5.9 Hz,1H), 3.68 – 3.57 (m, 2H), 3.01 (d, J = 10.4Hz, 1H), 2.36 – 2.27 (m, 6H), 1.59 (m, 6H),1.26 (m, 58H), 0.87 (t, J = 7.0 Hz, 9H). MS(ESI) calcd for C 50 H 94 O 11 , m / z 870.7; found893.8 [M + Na] + ]]> 18-D (m=16) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.19 (m, 1H), 5.01(s, 1H), 4.35 – 4.27 (m, 2H), 4.19 (dd, J =11.8, 3.6 Hz, 1H), 4.13 – 4.02 (m, 5H), 4.00(s, 1H), 3.80 (dd, J = 11.0, 5.9 Hz, 1H),3.60 (dd, J = 11.0, 4.7 Hz, 1H), 3.53 – 3.35(m, 1H), 2.88 (d, J = 10.9 Hz, 1H), 2.39 –2.26 (m, 6H), 1.60 (m, 6H), 1.25 (m, 74H),0.87 (t, J = 7.0 Hz, 9H). MS (ESI) calcd forC 58 H 110 O 11 , m / z 982.8; found 1005.9 [M + Na] + ]]> 20-D (m=18) <![CDATA[ 1 H NMR (600 MHz, CDCl3) δ 5.20 (m, 1H), 5.01(s, 1H), 4.34 – 4.28 (m, 2H), 4.19 (dd, J =11.8, 3.7 Hz, 1H), 4.08 (m, 5H), 4.00 (s,1H), 3.80 (dd, J = 11.0, 5.9 Hz, 1H), 3.60(dd, J = 10.9, 4.7 Hz, 1H), 3.47 (s, 1H),2.91 (d, J = 10.9 Hz, 1H), 2.39 – 2.27 (m,6H), 1.60 (m, 6H), 1.33 – 1.19 (m, 82H), 0.87(t, J = 6.9 Hz, 9H). MS (ESI) calcd forC 62 H 118 O 11 , m / z 1038.9; found 1061.9 [M + Na] + ]]>

[0090] Example 8: Bioactivity evaluation of a novel glycolipid TLR2 agonist

[0091] (i) Expression of TNF-α in mouse-derived cells (BMDC) for all candidate compounds:

[0092] A) Isolation and culture of mouse bone marrow-derived dendritic cells (BMDCs). Femurs and tibias were isolated from 6-8 week old female C57BL / 6 mice after cervical dislocation. Attached muscle tissue was removed, and the bones were disinfected by immersion in 70% alcohol for 3 min, followed by rinsing with RPMI 1640 basal medium to remove residual alcohol. The ends of the bones were cut off, and the bone marrow cavity was repeatedly rinsed with RPMI 1640 basal medium. Bone marrow was collected, centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. Red blood cell lysis buffer was added and incubated at 4°C for 5 min. The cells were centrifuged again at 1200 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with RPMI 1640 basal medium, and the collected cells were filtered through a 300-mesh nylon mesh to obtain purified mouse bone marrow leukocytes. The collected cells were counted, and the cell concentration was adjusted to 1 x 10⁻⁶ cells / mL with RPMI 1640 complete medium containing 10% fetal bovine serum. 6 Cells / mL were seeded into 6-well cell culture plates, and recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) were added to each well at a concentration of 10 ng / mL. The culture plates were placed in a 37°C incubator with 5% CO2 and saturated humidity. On day 6 of induction, non-adhesive cells and loosely adherent cells were harvested, which are immature bone marrow-derived dendritic cells (BMDCs) for subsequent experiments.

[0093] B) TNF-α expression of all candidate compounds in murine BMDCs. All candidate compounds were dissolved in DMSO to prepare a 10 mM stock solution. LPS, Pam3CSK4, and Diprovocim served as positive controls (all stock solutions were 10 mM, dissolved in DMSO), DMSO served as a negative control, and Ctrl served as a blank control. All candidate compounds and control stock solutions were diluted to 10 μM with serum-free RPMI 1640 medium to prepare working solutions. BMDCs cultured to day 6 were subjected to a 5 × 10⁻⁶ ppm induction culture. 5The cells were seeded at a density of [number] wells in 24-well plates and cultured overnight. The following day, the culture medium of the BMDC-inoculated plates was changed, and all candidate compounds and working solutions for each control group were added and cultured in a cell culture incubator. After 24 hours, the supernatant was collected and analyzed using a mouse TNF-α ELISA kit (Jianglai JL10484). The results are as follows: Figure 1 As shown.

[0094] (II) Expression of TNF-α in human cells (THP-1) for all candidate compounds:

[0095] Human monocytic leukemia cells (THP-1) were purchased from the American Type Culture Collection and cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.05 mM β-mercaptoethanol at 37°C under 5% CO2 and saturated humidity. THP-1 cells were collected by centrifugation and the cell concentration was adjusted to 1 x 10⁻⁶ cells / mL with serum-containing RPMI 1640 complete medium. 5 Cells / mL were seeded into 24-well cell culture plates, and 50 nM PMA was added to each well for induction. After 24 h, the culture plates seeded with THP-1 cells were replaced with the working solutions of all candidate compounds and control groups, and cultured in a cell culture incubator (the working solution grouping and concentration were as described previously). After 24 h, the supernatant was collected and analyzed using a human TNF-α ELISA kit (Jianglai JL10208). The results are as follows. Figure 2 As shown.

[0096] (III) Serum antibody titers of compound 13-C in animals:

[0097] A) Prepare a vaccine using ovalbumin (OVA) antigen and compound 13-C. Dilute the OVA antigen and compound 13-C with PBST (PBS + 5% Tween-80) to prepare the vaccine. Set up the following OVA model vaccines: blank control Ctrl (PBST), negative control OVA (5 mg / kg), positive control OVA (5 mg / kg) + Diprovocim (10 mg / kg), dose 1 OVA (5 mg / kg) + 13-C (10 mg / kg), dose 2 OVA (5 mg / kg) + 13-C (20 mg / kg).

[0098] B) Mouse Immunization Experiment. Four 6-8 week old female C57BL / 6 mice were used in each group. Each mouse was subcutaneously injected with the OVA model vaccine described above in the right hind limb groin. The vaccines were injected on days 0, 14, and 28. Blood was collected from the orbital sinus on days 28 and 42. 100-200 μL of whole blood was collected from each mouse. The blood was allowed to stand at 4°C for 1 hour, then centrifuged (1000 g, 4°C, 20 min). The supernatant serum was collected and stored at -80°C.

[0099] C) ELISA Immunoassay. Prepare OVA antigen solution (10 μg / mL) by dissolving in 0.1M carbonate buffer (pH 9.6); coat each well of a 96-well ELISA plate with 100 μL of the solution, seal, and incubate overnight at 4°C; the next day, incubate at 37°C for 1 hour, discard the solution, and wash the plate three times (300 μL / well / wash) with PBST (PBS + 0.05% Tween-20); add 100 μL of 5% BSA to each well, incubate at 37°C for 1 hour, discard the solution, and wash the plate three times with PBST; dilute the serum with PBS to 400, 800, 1600, 3200, 6400, 12800, 25600, and 51200 times, respectively, and add the diluted serum to the sealed 96-well plates (100 μL / well). In each well, three sub-wells were prepared for each dilution gradient. The plates were sealed and incubated at 37°C for 2 hours, then the solution was discarded and the plates were washed five times. HRP (horseradish peroxidase)-labeled Goat anti-mouse IgG was diluted with 5% BSA (1:5000). The diluted secondary antibody was added to the corresponding wells (100 μL / well) and incubated at 37°C for 1 hour, then the solution was discarded and the plates were washed five times. Then, 100 μL of TMB solution was added to each well, and the plates were incubated at 37°C in the dark for 10 minutes. The color development was terminated by adding 50 μL of 0.5 M H₂SO₄ solution. The absorbance (OD) at 450 nm was measured using a microplate reader. The OD values ​​of each group's corresponding serum dilution were compared with the Ctrl group. The highest dilution with a ratio greater than 1 was recorded as the serum antibody titer for that group. The results are as follows: Figure 3 As shown.

[0100] (iv) Antitumor activity of 13-C compound:

[0101] A) Culture and animal tumor bearing of B16F10-OVA tumor cells. Mouse melanoma-transfected OVA cells (B16F10-OVA) were purchased from Shanghai Jinyuan Biotechnology and cultured in RPMI 1640 medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C under 5% CO2 and saturated humidity. Puromycin (1 μg / mL) was used for maintenance during passage. B16F10-OVA cells were expanded to a larger culture size 40 days after mouse immunization. At 43 days, B16F10-OVA cells were collected by digestion and centrifugation, washed twice with sterile PBS, resuspended in PBS, and counted. Mice were shaved beforehand and subcutaneously injected with B16F10-OVA cells (4 x 10⁻⁶ cells / mL). 5 B16F10-OVA tumor animal model was constructed using cells / 100μL / animal.

[0102] B) Combination therapy with anti-PD-L1 monoclonal antibody and tumor growth and survival curves in mice. Mice were intraperitoneally injected with anti-PD-L1 monoclonal antibody (200 μg / 100 μL / mouse) on days 3, 6, and 9 after tumor implantation. Tumor volume was measured daily thereafter, and the longest diameter (l) and the maximum transverse diameter (w) in the vertical direction were recorded using calipers. The tumor volume was calculated using the formula V = l * w. 2 / 2. Calculate tumor volume and plot tumor growth curves in mice. The tumor volume in mice reaches 2000 mm². 3 Mice were sacrificed, and survival curves were plotted. The results are as follows: Figure 4 As shown.

[0103] (v) Adjuvant activity of compound 13-C alone and in combination with QS-21 in OVA / gE antigen

[0104] A) Preparation of single-component and two-component nanoliposomes of 13-C and QS-21. Weigh 1 mg of dioleoyl lecithin, 0.25 mg of cholesterol, and 50 μg of 13-C compound or 50 μg of QS-21, dissolve them in 2 mL of ethanol, mix thoroughly, and evaporate the organic solvent by rotary evaporation in a 25 mL round-bottom flask at 40 °C and 0.08 MPa vacuum. Then, vacuum dry the solution to form a uniform film on the inner wall of the flask. Add 0.5 mL of PBS solution, and rotate the solution in a 55 °C water bath to detach the film and disperse it in the PBS. Sonicate on ice for 10 min (650 W, 2 s sonication, 3 s interval) to obtain the liposome solution. Store in a sealed container at 4 °C.

[0105] B) Prepare liposomal vaccines containing chicken ovalbumin (OVA) antigen or recombinant human varicella-zoster virus nucleocapsid protein E (gE) + 13-C and QS-21 in single-component and two-component formulations. Dilute the OVA antigen or gE antigen and compound 13-C with QS-21 in PBS to prepare single-component and two-component liposomal formulations. Set up a blank control (Ctrl (PBS), a negative control (OVA (20 μg / animal) or gE (5 μg / animal), and a positive control (OVA (20 μg / animal) or gE (5 μg / animal) + AS01). B (50 uL / each, AS01) B Developed for GSK the Shingrix vaccine adjuvant component, including single 13-C liposome OVA (20 ug / vial) or gE (5 ug / vial) + 13-C liposome (50 uL / vial), single QS-21 liposome OVA (20 ug / vial) or gE (5 ug / vial) + QS-21 liposome (50 uL / vial), and 13-C plus QS-21 two-component liposome OVA (20 ug / vial) or gE (5 ug / vial) + 13-C liposome (50 uL / vial) + QS-21 liposome (50 uL / vial) and other OVA or gE model vaccines.

[0106] C) Mouse Immunization Experiment. Four 6-8 week old female C57BL / 6 mice were used in each group. Each mouse was subcutaneously injected with the OVA or gE model vaccine mentioned above in the right hind limb groin. The vaccines were injected on days 0, 14, and 28. Blood was collected from the orbital sinus on days 14, 28, and 42. 100-200 μL of whole blood was collected from each mouse. The blood was allowed to stand at 4°C for 1 hour, then centrifuged (1000 g, 4°C, 20 min). The supernatant serum was collected and stored at -80°C.

[0107] D) ELISA Immunoassay. Prepare OVA antigen solution (10 μg / mL) or gE antigen solution (2 μg / mL) by dissolving in 0.1M carbonate buffer (pH 9.6); coat each well of a 96-well ELISA plate with 100 μL of the solution, seal, and incubate overnight at 4°C; the next day, incubate at 37°C for 1 h, discard the solution, and wash the plate three times with PBST (PBS + 0.05% Tween-20) (300 μL / well / wash); add 100 μL of 5% BSA to each well, incubate at 37°C for 1 h, discard the solution, and wash the plate three times with PBST; dilute the serum with PBS to 400, 800, 1600, 3200, 6400, 12800, 25600, and 51200 times, respectively, and add the diluted serum to the sealed 96-well plates (100 μL / well). In each well, three copies of each dilution gradient were prepared, sealed, and incubated at 37°C for 2 h. The solution was then discarded and the plate was washed 5 times. HRP (horseradish peroxidase)-labeled Goat anti mouse IgG, Goat anti mouse IgG1, and Goat anti mouse IgG2c were diluted with 5% BSA (1:5000). The diluted secondary antibody was added to the corresponding well (100 μL / well) and incubated at 37°C for 1 h. The solution was then discarded and the plate was washed 5 times. Then, 100 μL of TMB solution was added to each well, and the plate was incubated at 37°C in the dark for 10 min. The color development was terminated by adding 0.5 M H2SO4 solution (50 μL / well). The absorbance (OD) at 450 nm was measured using a microplate reader. The OD values ​​of each group's corresponding serum dilution were compared with those of the Ctrl group. The highest dilution with a ratio greater than 1 was recorded as the serum antibody titer for that group. The results are as follows: Figure 5 , Figure 6 As shown.

[0108] E) Flow cytometry detection of mouse spleen-specific T lymphocytes. Mice were euthanized by cervical dislocation, and the spleen was aseptically dissected and isolated in a laminar flow hood after being disinfected by immersion in 75% ethanol. The cells were ground in sterile culture dishes, filtered through a 70 μm screen, and collected. Lymphocytes were isolated and collected stepwise according to the manufacturer's instructions using a mouse spleen lymphocyte isolation kit (Solomon P8860). The cell concentration was adjusted to 5 × 10⁶ cells / mL using serum-free medium. 6Cells were cultured at 1000 cells / mL. OVA or gE peptide pools were added to stimulate cell culture. At 20 h, prepared BFA was added to block Golgi transport. Cells were harvested after 24 h, and surface staining antibody prepared in FACS buffer was added. After mixing by pipetting, the cells were incubated at 37 °C in the dark for 30 min. After incubation, cells were washed once with 1 mL of FACS buffer, centrifuged at 500 g for 5 min, and the supernatant was discarded using a vacuum pump. Cells were gently vortexed, and 300 µL of fixative was added to each tube. The cells were gently vortexed and incubated at room temperature in the dark for 20 min. 1 mL of Perm / Wash Buffer was added, and the cells were incubated at room temperature for 30 min to fix and perforate the membrane. The cells were centrifuged at 800 g for 5 min, and the supernatant was discarded. Another 1 mL of Perm / Wash Buffer was added, but not mixed. The cells were centrifuged at 800 g for 5 min, and the supernatant was discarded. The pre-prepared intracellular staining antibody was added, and the cells were mixed and incubated overnight at 4 °C in the dark. After incubation, add 1 mL of FACS buffer to each tube, centrifuge at 2000 g for 5 min, discard the supernatant, resuspend in 400 μL of FACS buffer, load onto a flow cytometer, and analyze the flow cytometry results using FlowJo software. The results are as follows: Figure 5 , Figure 6 As shown.

Claims

1. A novel glycolipid compound, and its pharmaceutically acceptable salts, isotopes, and isomers, having the structure shown in general formula I: in, R 1 For CO(CH2)nCH3, R 2 For hydrogen, CO(CH2)mCH3, R 3 The formula is CO(CH2)mCH3, where X is O or S, Y is O or NH, and n and m are aliphatic chains with 8-18 methylene groups.

2. The compound according to claim 1, characterized in that, X is O or S, Y is O or NH, and when X is S, Y is not NH.

3. The compound according to claim 2, characterized in that, The compounds are selected from the following structures: 。 4. A pharmaceutical composition, characterized in that, The composition comprises any novel glycolipid compound of any one of claims 1-3 and its pharmaceutically acceptable carrier or diluent.

5. The use of the novel glycolipid compound and its pharmaceutically acceptable composition as described in any one of claims 1-3 in the preparation of a medicament, characterized in that, The drug can activate the TLR2 receptor and its mediated signaling pathway.

6. The application according to claim 5, characterized in that, The drug can be used alone or in combination with others, and can be used as a vaccine adjuvant, in tumor immunotherapy, and for immune modulation.

7. The compound or combination thereof according to any one of claims 1-3 may be used in combination with other drugs, preferably PD-L1 and PD1 antibodies.

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