TLR4 agonist, application thereof and vaccine composition

Abstract

The invention relates to the technical field of medicinal chemistry, and provides a TLR4 agonist, application thereof and a vaccine composition. The TLR4 agonist is a compound with a structure as shown in a formula 1 or a pharmaceutically acceptable salt thereof, the compound is glucosamine aminoalkyl 4-phosphate series molecules derived from MPL-12 structure modification, and the series molecules have enhanced immunostimulatory activity and selective immune response induction capability.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a TLR4 agonist, its application, and a vaccine composition. Background Technology

[0002] Monophosphorylated lipid A (MPLA) is a component of the inner liposome (lipid) of Gram-negative bacterial cell wall endotoxin (LPS). Lipid A is an amphiphilic structure and a key component in the toxicity and immunogenicity of Gram-negative bacteria, inducing an immune response. Chemically attenuated LPS retains its potent immunostimulatory properties while reducing its original toxicity. As a safe and effective TLR4 agonist, MPLA has been developed as a vaccine adjuvant and used in prophylactic vaccines, such as for the prevention of shingles, malaria, and HIV.

[0003] However, the chemical synthesis of MPLA faces significant challenges, including complex manufacturing processes and high energy consumption, which limit its large-scale production. Although MPLA can be obtained from Gram-negative bacterial cultures, its inherent heterogeneity and potential contamination by pro-inflammatory bacterial components are major obstacles to obtaining pharmaceutical-grade purity and stability of natural lipid A. To overcome these limitations, simplify molecular structures, and reduce synthetic complexity, researchers have developed simplified TLR4 agonists based on non-reducing glucosamine backbones, such as the AGP series (Aminoglucose Phosphates) and the PET-A series (Phosphonooxyethyl Triazole-A). These molecular structures are all derived from the structural modification of MPLA (MPL) from Salmonella Minnesota, and their glycosyl receptors all contain a double-chain acyl fatty acid chain (i.e., a secondary acyl chain branched on a main chain). Meanwhile, another high-potential structure has been overlooked: MPL-12 from Escherichia coli, whose core feature is that its glycosyl receptor contains two independent monoacyl fatty acid chains. Theoretical studies have shown that agonists with this monoacyl chain configuration can also effectively activate the TLR4 immune pathway. However, current development of monosaccharide agonists is almost entirely focused on MPL-type structures, and systematic research on the structure of MPL-12 is still lacking: due to the incomplete understanding of the mechanism of action and the lack of targeted molecular design strategies for this backbone, its unique potential has remained untapped for a long time. Summary of the Invention

[0004] The purpose of this invention is to provide a TLR4 agonist, its application, and a vaccine composition thereof. This invention utilizes a series of glucosamine aminoalkyl 4-phosphate (GAP) molecules derived from the structural modification of MPL-12 as TLR4 agonists, which possess enhanced immunostimulatory activity and selective immune response induction capabilities.

[0005] In a first aspect, the present invention provides a TLR4 agonist, said TLR4 agonist being a compound of the structure shown in Formula 1 or a pharmaceutically acceptable salt thereof:

[0006]

[0007] In Equation 1, the structure of R1 is as follows:

[0008]

[0009] The structure of R2 is shown below:

[0010]

[0011] in, Indicates a connecting bond, where m is an integer from 0 to 25, n is 0 or 1, o is an integer from 0 to 10, p is an integer from 1 to 25, and q is an integer from 0 to 25; X and Y may be the same or different, and each is independently selected from -O-, -NH-, or -CH2-; L is -CH2- or -C(O)-; Z is an oxygen atom or a sulfur atom; E is -O- or -NH-; one of A1 and A2 is -CH2OH, and the other is -CH2NH2, -COOH, -CH2OH, -CH2SH, -CF3, -CHF2, -CH2F, or -CH2OCH3.

[0012] Secondly, the present invention provides the use of the TLR4 agonist described in the first aspect of the present invention in the preparation of immunomodulatory agents. Preferably, the immunomodulatory agent is at least one of the following: vaccines, cell activators for in vitro activation, expansion and differentiation of immune cells, immunotargeting drugs, pharmaceutical preparations in combination with or conjugated with chemotherapeutic drugs, immunomodulatory agents for allergic diseases, pharmaceutical preparations for nervous system diseases, cardiovascular protective agents, antiviral and antibacterial infection agents, and agents for autoimmune diseases.

[0013] Thirdly, the present invention provides a vaccine composition comprising an antigen, an adjuvant, and a pharmaceutical carrier, wherein the adjuvant comprises the TLR4 agonist described in the first aspect of the present invention.

[0014] In the TLR4 agonist of this invention, the compound shown in Formula 1 (GAP series molecule) is an MPL-12 structural derivative, belonging to the structurally simplified monosaccharide MPLA analog. Its core advantage lies in the fact that, on the one hand, this type of compound retains the non-reducing monosaccharide, C-4 phosphate group, and two independent monoacyl chains; on the other hand, the reducing sugar of MPL is replaced with a simpler non-glycocyclic structure. The synthesis difficulty was reduced and the number of reaction steps decreased. Furthermore, the molecular biological activity was improved by systematically modifying the lengths of the two secondary acyl chains (R1). Moreover, Formula 1 contains two secondary acyl chains (-ER2). Due to the conserved key amino acid sequence involved in the binding of MD-2 to the extracellular domain of TLR4, and its hydrophobicity, the agonist's immune activity in mice can be modulated by controlling the length of the secondary acyl chains. Simultaneously, the molecule retains a key phosphate group and the 4+2 acyl chain distribution pattern, ensuring the compound possesses agonist activity. Further, the TLR4 agonist can effectively activate the immune system, exhibiting comparable or even better immune agonistic activity than MPLA in both innate and adaptive immunity. Specifically, vaccines prepared using this TLR4 agonist as an adjuvant showed superior efficacy in activating the mouse immune system, providing a new direction for the design of highly effective TLR4 agonists.

[0015] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0016] Exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0017] Figure 1 These are the results of the DC2.4(A) and RAW264.7(B) immune signaling pathway tests in the test cases;

[0018] Figure 2 The test case shows the secretion of IL-6 (A), IL-12p70 (B), and CD80+CD86+ double positive expression by DC2.4 in the in vitro activation experiment (C).

[0019] Figure 3 The test case shows the secretion of IL-6 (A), IL-12p70 (B), and CD80+CD86+ double positive expression by RAW264.7 in the in vitro activation experiment (C).

[0020] Figure 4 The concentrations of IL-6(A), IL-6(B), and IFN-γ(C) in the serum of mice 2 hours after immunization, 6 hours after immunization, and 2 hours after immunization, are in the test case.

[0021] Figure 5 This test measures the titer of OVA-specific IgG antibodies in the serum of immunized mice on days 14, 28, and 42.

[0022] Figure 6 The titer of OVA-specific IgG subtypes (IgG1, IgG2a, IgG2b, IgG3) antibodies in the serum of immunized mice on day 42 was measured.

[0023] Figure 7 The proportion of CD80 and CD86 double-positive cells in the spleen DC cells of immunized mice (A) and the proportion of CD80 and CD86 double-positive cells in the draining lymph nodes of immunized mice (B) are shown.

[0024] Figure 8 The proportion of IFN-γ and TNF-α double-positive CD4+ T cells secreted by the spleen of immunized mice was tested (A), and the proportion of IFN-γ and TNF-α double-positive CD8+ T cells secreted by the spleen was tested (B).

[0025] Figure 9 The proportion of CD4+ central memory T (Tcm) cells in the spleen of immunized mice (A) and the proportion of CD8+ Tcm cells in the spleen (B) are tested.

[0026] Figure 10 The test cases show the effects of vaccines with different concentrations of adjuvants on the survival rates of HEK293 cells (A), B16F10 cells (B), and tissue sections of vital organs from Balb / c mice (C). Detailed Implementation

[0027] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0028] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] A first aspect of the present invention provides a TLR4 agonist, which is a compound of the structure shown in Formula 1 or a pharmaceutically acceptable salt thereof:

[0030]

[0031] Wherein, Z is an oxygen atom (O) or a sulfur atom (S), preferably an oxygen atom; E is -O- or -NH-, preferably -NH-. One of A1 and A2 is -CH2OH, and the other is -CH2NH2, -COOH, -CH2OH, -CH2SH, -CF3, -CHF2, -CH2F, or -CH2OCH3; preferably, both A1 and A2 are -CH2OH.

[0032] In Equation 1, the structure of R1 is as follows:

[0033]

[0034] Where m is an integer from 0 to 25, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25; X and Y may be the same or different, and each is independently selected from -O-, -NH- or -CH2-; L is -CH2- or -C(O)-.

[0035] As some specific examples, m can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 20, 22, 23, 24, etc.; o can be 0, 1, 2, 3, 4, 5, 7, 8, etc.; p can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 22, 25, etc.

[0036] In some preferred embodiments, m is an integer from 2 to 21, n is 1, o is an integer from 0 to 5, and p is an integer from 1 to 15.

[0037] In some preferred embodiments, X and Y are both -CH2, and L is -C(O)-. More preferably, R1 is selected from groups with the following structures:

[0038]

[0039] In Equation 1, the structure of R2 is as follows:

[0040]

[0041] Where q is an integer from 0 to 25, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 20, 22, 23, 24, 25, etc.

[0042] In some embodiments, q is an integer from 2 to 22. Preferably, q is an integer from 5 to 12. This is beneficial for further improving the activity of the agonist.

[0043] More preferably, R2 is selected from groups with the following structure:

[0044]

[0045] In this invention, All of these represent connecting keys. Additionally, it can be understood that in Equation 1, keys such as... (The asterisk "*" indicates a linking site, and j is a variable representing m, p, q, and o.) In the structures shown, j is always a positive integer, representing the number of directly linked methylene groups (-CH2-). For example, when j is 0, it means that the methylene group within the parentheses does not exist. for This indicates a single methyl group (-CH3); when j is 1, it means there is one methylene group within the parentheses. for This represents an ethyl group (-CH2CH3); when j is 2, it means there are 2 methylene groups inside the parentheses. for This represents a n-propyl group (-CH2CH2CH3).

[0046] In this invention, in the structure shown in Formula 1, the two R1s can be the same or different, and the two R2s can be the same or different. Preferably, the two R1s are the same, and the two R2s are also the same.

[0047] In some embodiments, the TLR4 agonist is selected from at least one of the following compounds or pharmaceutically acceptable salts thereof:

[0048]

[0049]

[0050] As some preferred embodiments, the TLR4 agonist is at least one of compound 8a (also referred to herein as GAP210), compound 8b (also referred to herein as GAP212), and compound 8c (also referred to herein as GAP214). More preferably, the TLR4 agonist is GAP214.

[0051] The present invention does not impose any particular limitation on the synthetic method of the compound shown in Formula 1. Those skilled in the art can select a suitable synthetic route based on the structure of the compound to obtain the compound. As some embodiments, the compound shown in Formula 1 can be prepared according to the synthetic route shown below:

[0052]

[0053] Where R'2 is The italicized "i" in the numbering indicates a variable, intended to distinguish different compounds by assigning different numbers when synthesizing different compounds. For example, when synthesizing compound 4i, depending on the choice of the R1 group, the prepared compounds 4i can be numbered 4a, 4b, 4c, 4d, etc., respectively. When preparing compound 6j from compound 4i, the corresponding compounds 6j can be numbered 6a, 6b, 6c, 6d, etc. Furthermore, exemplary descriptions of the amounts of reactants and operating conditions in this synthetic route are provided in the synthesis section below and will not be repeated here. Additionally, it should be understood that unless otherwise mentioned in the synthesis section below, temperature refers to room temperature.

[0054] A second aspect of the present invention provides the use of the TLR4 agonist described in the first aspect of the present invention in the preparation of an immunomodulatory pharmaceutical formulation. Preferably, the immunomodulatory pharmaceutical formulation is selected from at least one of the following: vaccines, cell activators for in vitro activation, expansion and differentiation of immune cells, immune-targeting drugs, pharmaceutical formulations in combination with or conjugated with chemotherapeutic drugs, immunomodulators for allergic diseases, pharmaceutical formulations for nervous system diseases, cardiovascular protective agents, antibacterial and antiviral agents, and agents for autoimmune diseases.

[0055] According to the present invention, the TLR4 agonist can be used as a vaccine adjuvant when used in vaccine preparation, and the resulting vaccine has a simple molecular structure. As some preferred embodiments, the vaccine is a semi-synthetic vaccine, a fully synthetic sugar vaccine, a peptide vaccine, a glycopeptide vaccine, a subunit vaccine, a nucleic acid vaccine, a virus-like particle vaccine, an inactivated vaccine, or a live-attenuated vaccine.

[0056] According to the present invention, when the TLR4 agonist is used to prepare a cell activator for in vitro activation, expansion and differentiation of immune cells, the immune cells are preferably DC cells, macrophages, NK cells, NKT cells, CAR-T cells, neutrophils, eosinophils, basophils or mast cells.

[0057] According to the present invention, when preparing an immunotargeted drug using the TLR4 agonist, the TLR4 agonist can be conjugated with a targeted drug (such as a monoclonal antibody or an immune agonist) to obtain the immunotargeted drug. The immunotargeted drug can be, for example, a tumor-targeting agent, an anti-infection-targeting agent, etc.; by specifically activating the TLR4 signaling pathway, it targets and regulates the function of immune cells such as dendritic cells and macrophages for use in tumor immunotherapy or infection-targeting therapy, enhancing local immune responses and overcoming the immunosuppressive microenvironment.

[0058] According to the present invention, in addition to the TLR4 agonist, the immunomodulatory drug formulation may further comprise at least one of a drug carrier and a pharmaceutical excipient. The drug carrier may be selected from microcapsules, microspheres, nanoparticles, or liposomes. The pharmaceutical excipient may, for example, comprise an excipient, wherein the excipient includes, but is not limited to, at least one of a solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, absorbent, diluent, flocculant, anti-flocculator, filter aid, and release inhibitor.

[0059] According to the present invention, the dosage form of the immunomodulatory drug preparation may be tablets, powders, granules, capsules, suspensions, emulsions, syrups or extracts.

[0060] A third aspect of the present invention provides a vaccine composition comprising an antigen, an adjuvant, and a pharmaceutical carrier, wherein the adjuvant comprises the TLR4 agonist described in the first aspect of the present invention.

[0061] The present invention does not particularly limit the type of antigen, and it may be at least one of viral antigen, tumor antigen, and bacterial antigen. Preferably, the antigen is a protein antigen or a polypeptide antigen.

[0062] As some specific examples, the antigen may be selected from ovalbumin, bovine serum albumin, tetanus toxoid, diphtheria toxoid, group B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxoid mutant, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filamentous virus structural protein, coronavirus structural protein, or subunits of the proteins listed above or their modified derivatives.

[0063] Preferably, the drug carrier is a liposome. The liposome may be selected from at least one of conventional liposomes, cationic liposomes, stealth liposomes, PEGylated liposomes, and lipid nanoparticles (LNPs).

[0064] As some specific examples, the liposomes are prepared by thin-film dispersion of distearate phosphatidylcholine (DSPC) and cholesterol (Chol). Preferably, the molar ratio of the adjuvant, antigen, DSPC, and Chol is (1-2):1:(4-6):(3-5), for example, 1:1:5:4, 1.5:1:6:5, etc.

[0065] The present invention will be described in detail below through examples, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available.

[0066] 1. Synthesis of compound Sub 2-i

[0067] The synthetic route of compound Sub 2-i is shown below. Depending on the value of p, the synthesized products are denoted as Sub 2-1, Sub 2-2, and Sub 2-3, respectively.

[0068]

[0069] (1) Synthesis of compound 9

[0070]

[0071] Lauroyl chloride (121.6 g, 0.556 mol, 2 eq.) and Michaelis acid (40 g, 0.278 mol, 1 eq.) were dissolved in 200 mL of dry dichloromethane (DCM), and then pyridine (py, 45 mL, 0.556 mol, 2 eq.) was added under an Ar atmosphere and stirred for 3 hours. The organic layer was extracted with 750 mL of 1 M HCl solution and 200 mL of water. The combined organic layers were dried over Na2SO4 and concentrated under vacuum to obtain a mixture. The mixture was dissolved in 200 mL of anhydrous MeOH and refluxed at 60 °C overnight. The solvent was removed under vacuum, and the reaction was detected to be complete by TLC. The crude product was purified by column chromatography (gradient elution, ethyl acetate (EA) / petroleum ether (PE), v / v = 10:90 to 20:80) to give target compound 9 (53.6 g, 0.21 mol, yield 75%).

[0072] The characterization data for compound 9 are shown below.

[0073] 1 H NMR (600MHz, CDCl3) δ3.74(s,3H,OCH3),3.45(s,2H),2.53(t,J=7.4Hz,2H,COCH2CO),2.53(dt,J=10.3 ,5.3Hz,2H,CH2COCH2),1.59(p,J=6.9Hz,2H),1.27(d,J=14.6Hz,16H),0.88(t,J=6.9Hz,3H,CH2CH3).

[0074] 13 C NMR (151MHz, CDCl3) δ202.78(CO),167.61(COOCH3),52.20(OCH3),48.90(COCH2CO),42.99,31.82,29.51,29.35,29.26,23.37,22.60,14.03.

[0075] HRMS (High-Resolution Mass Spectrometry): [M+H] + Theoretical components: C15 H 29 O3 + Theoretical value: 257.2117, measured value: 257.2116.

[0076] (2) Synthesis of compound 10

[0077]

[0078] In a hydrogenation autoclave under an Ar atmosphere, compound 9 (53.6 g, 0.21 mol, 1 eq.) was dissolved in 50 mL of anhydrous methanol (MeOH), and hydrogenation catalyst R-(+)-ruthenium chloride BINAP (Ru[(R)-BINAP]Cl2, 0.21 mmol, 0.1%) and 1 g H were added. + The mixture was purged with Ar three times, then the Ar in the autoclave was replaced with H2. Finally, 10 bar of H2 was introduced and the mixture was heated to 100°C and stirred for 6 hours. The mixture was then cooled to room temperature and the precipitate was filtered off. The crude product was concentrated under vacuum and purified by column chromatography (gradient elution, EA / PE, v / v = 10:90 to 20:80) to give the target compound 10 (44.9 g, 0.174 mol, yield 83%).

[0079] The characterization data for compound 10 are shown below.

[0080] 1 H NMR(400MHz, CDCl3) δ4.01(q,J=8.5,6.5Hz,1H,HOCH),3.72(d,J=3.5Hz,3H,OCH3),2.86(s,1H,CHOH),2.56-2.34( m,2H,CHCH2CO),1.51(d,J=9.7Hz,1H),1.46-1.38(m,2H,CH2CHOH),1.31(s,CH2×9),0.88(q,J=5.6Hz,3H,CH2CH3).

[0081] 13 C NMR(101MHz,CDCl3)δ173.52(COOCH3),68.04(HOCH),51.73(OCH3),41.10(COCH2C O),36.53,31.92,29.65,29.63,29.59,29.57,29.52,29.35,25.48,22.69,14.12.

[0082] HRMS: [M+H] + Theoretical components: C 15 H 31 O3 +Theoretical value: 259.2267, measured value: 259.2268.

[0083] (3) Synthesis of compound 11

[0084]

[0085] Compound 10 (5.7 g, 22.07 mmol, 1 eq.) was dissolved in a mixed solution of 20 mL tetrahydrofuran (THF) and 20 mL H₂O, followed by the addition of lithium hydroxide (LiOH, 4.63 g, 110 mmol, 5 eq.). When TLC analysis indicated the reaction was complete, the pH of the solution was adjusted to neutral with 1 M hydrochloric acid. Extraction was performed with 50 mL of saturated NaHCO₃ aqueous solution and 50 mL of DCM. The combined organic layers were dried with Na2SO4, filtered, and concentrated. The concentrated product was dissolved in 20 mL of dry EA, and then α-bromoacetophenone (PacBr, 4.83 g, 24.28 mmol, 1.1 eq.) and triethylamine (Et3N, 2.7 g, 26.48 mmol, 1.2 eq.) were added and reacted for 3 hours to obtain the crude product. The crude product was concentrated under vacuum and purified by column chromatography (gradient elution, EA / PE, v / v = 5:95 to 15:85) to obtain the target compound 11 (6.9 g, 19 mmol, yield 86%).

[0086] The characterization data for compound 11 are shown below.

[0087] 1 H NMR (600MHz, CDCl3) δ7.91 (t, J=5.3Hz, 2H, Ar-o), 7.62 (q, J=5.8, 4.0Hz, 1H, Ar-

[0088] p),7.53-7.44(m,2H,Ar-m),5.49-5.35(m,2H,OCH2COPh),4.13(dq,J=8.7,4.3,3.9Hz,

[0089] 1H,CH2CHOCH2),3.55-3.20(m,1H),2.73-2.42(m,2H,CH2CH2COO),1.60(dt,J=15.3,8.4Hz,2H,CH2C H2COO),1.54-1.43(m,2H,CH2CHCOO),1.28(d,J=22.4Hz,16H,CH2×8)0.87(q,J=4.8,3.3Hz,3H,CH3).

[0090] 13C NMR(151MHz, CDCl3)δ192.56(PhCO),172.05(CO),134.26,133.78,128.97,127.88(Ph×4),68.52(CHO H),66.03(OCH2CO),42.12(CHOHCH2),36.57,31.94,29.69,29.66,29.62,29.56,25.63,22.72,14.10.

[0091] HRMS: [M+Na] + Theoretical components: C 22 H 34 O4Na + Theoretical value: 385.2354, measured value: 385.2350.

[0092] (4) Synthesis of compound Sub 1-i

[0093] The synthetic route is shown below. Depending on the different secondary acyl chains of R1 (i.e. different p), the corresponding compounds Sub1-i are numbered Sub 1-1, Sub 1-2 and Sub 1-3. Reactant x represents the straight-chain saturated fatty acyl chlorides listed in Table 1.

[0094]

[0095] (4-1) Synthesis of compound Sub 1-1

[0096] Compound 11 (2 g, 5.5 mmol, 1 eq.) and decanoyl chloride (1.72 g, 8.28 mmol, 1.5 eq., p = 7) were dissolved in 20 mL of dry DCM under an Ar atmosphere, and then Py (3.04 g, 38.5 mmol, 7 eq.) was added and reacted for 3 h. Extraction was performed with 50 mL of saturated NaHCO3 solution and 50 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, DCM / PE, v / v = 10:90 to 20:80) to give the target compound Sub 1-1 (2.76 g, 5.34 mmol, 97% yield).

[0097] (4-2) Composition of Sub 1-2 and Sub 1-3

[0098] Sub 1-2 and Sub 1-3 were synthesized using the same method as compound Sub 1-1, except that decanoyl chloride was replaced with each reactant x. The yields of each reactant x and the products are shown in Table 1.

[0099] Table 1

[0100]

[0101] The characterization data of compound Sub 1-1 are shown below.

[0102] 1 H NMR (400MHz, CDCl3) δ7.90 (d, J = 7.7Hz, 2H, Ar-o), 7.61 (t, J = 7.4Hz, 1H, Ar-p), 7.48 (t, J = 7.6Hz, 2H, Ar-m), 5.34 (s, 2H, OCH2COPh), 5.30 (q, J = 6.6Hz, 1H,

[0103] CH2CHOCH2),2.83-2.65(m,2H,CHOCH2CO),2.32(t,J=7.5Hz,2H,CH2CH2COO),1.71-1.61(m,2H,CH2CHCOO), 1.58(dd,J=14.1,7.2Hz,2H,CH2CHO), 1.27(d,J=10.5Hz,30H,CH2×15), 0.87(dt,J=6.7,3.2Hz,6H,CH3×2).

[0104] 13 C NMR(101MHz, CDCl3)δ191.74(PhCO),173.39,173.39(CO×2),134.16,133.94,128.90,127.78(Ph×4),70.21(CHOH),66 .14(OCH2CO),38.97(CHOHCH2),34.54,34.05,31.91,29.68,29.60,29.52,29.40,29.20,25.18,25.05,22.73,14.17.

[0105] HRMS: [M+Na] + Theoretical components: C 32 H 52 O5Na + :539.3712, measured value:539.3815.

[0106] The characterization data of compound Sub 1-2 are shown below.

[0107] 1H NMR (400MHz, CDCl3) δ7.95-7.80(m,2H,Ar-o),7.58(dt,J=13.5,7.4Hz,1H,Ar-p),7.46(dt,J=21.1,7.7H z,2H,Ar-m),5.31(d,J=21.1Hz,2H,OCH2COPh),5.26(dd,J=13.9,6.8Hz,1H,CH2CHOCH2),2.73(dtd,J=20. 6,13.6,11.8,6.4Hz,2H,CHOCH2CO),2.29(dt,J=13.5,7.3Hz,2H,CH2CH2COO),1.71-1.61(m,2H,CH2CHCOO ),1.58(dd,J=14.1,7.2Hz,2H,CH2CHO),1.38-1.12(m,34H,CH2×17),0.85(dt,J=13.9,6.8Hz,6H,CH3×2).

[0108] 13 C NMR(151MHz, CDCl3)δ191.50(PhCO),173.13,169.76(CO×2),134.02,133.72,128.70,127.60(Ph×4),70.02(CHOH) ,65.97(OCH2CO),38.80(CHOHCH2),34.17,33.74,31.69,29.42,29.31,29.13,28.91,24.88,24.75,22.45,13.86.

[0109] HRMS: [M+Na] + Theoretical components: C 34 H 56 O5Na + :567.4020, measured value:567.4018.

[0110] The characterization data of compound Sub 1-3 are shown below.

[0111] 1H NMR (400MHz, CDCl3) δ7.90-7.88(m,2H,Ar-o),7.58(dt,J=13.5,7.4Hz,1H,Ar-p),7.47(dt,J=21.1,7.7H z,2H,Ar-m),5.32(d,J=21.1Hz,2H,OCH2COPh),5.30(dd,J=13.9,6.8Hz,1H,CH2CHOCH2),2.73(dtd,J=20. 6,13.6,11.8,6.4Hz,2H,CHOCH2CO),2.29(dt,J=13.5,7.3Hz,2H,CH2CH2COO),1.71-1.61(m,2H,CH2CHCOO ),1.58(dd,J=14.1,7.2Hz,2H,CH2CHO),1.38-1.12(m,38H,CH2×19),0.85(dt,J=13.9,6.8Hz,6H,CH3×2). 13 CNMR(151MHz, CDCl3)δ191.69(PhCO),173.32,169.91(CO×2),134.2,133.86,128.82,127.71(Ph×4),70.17(CHOH ),66.07(OCH2CO),38.92(CHOHCH2),34.49,33.99,31.86,29.48,29.30,29.14,28.91,24.88,24.75,22.66,14.1.

[0112] HRMS: [M+H] + Theoretical components: C 36 H 61 O5 + :573.4541, measured value:573.4549.

[0113] (5) Synthesis of compound Sub 2-i

[0114] The synthetic route is shown below. Depending on the value of p, the compounds Sub 2-i prepared are specifically numbered Sub 2-1, Sub 2-2, and Sub 2-3.

[0115]

[0116] (5-1) Synthesis of compound Sub 2-1

[0117] Compound Sub 1-1 (2.1 g, 4 mmol, 1 eq.) was dissolved in 10 mL of acetic acid (AcOH), and then zinc powder (2.6 g, 40 mmol, 10 eq.) was added under an Ar atmosphere to obtain a mixture. The mixture was heated to 60 °C for 3 hours, then cooled to room temperature. The precipitate was filtered off and the filtrate was collected. 5 mL of toluene was added to remove AcOH from the filtrate under vacuum, yielding a mixture. The mixture was dissolved in EA and extracted with 50 mL of saturated NaHCO3 aqueous solution and 50 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, MeOH / DCM, v / v = 1:99 to 10:90) to give the target compound Sub 2-1 (1.08 g, 2.72 mmol, yield 68%).

[0118] (5-2) Composition of Sub 2-2 and Sub 2-3

[0119] Sub 2-2 and Sub 2-3 were synthesized using the same method as compound Sub 2-1, except that reactant Sub 1-1 was replaced with another compound Sub 1-i. The yields of Sub 1-i and the products are shown in Table 2.

[0120] Table 2

[0121]

[0122]

[0123] The characterization data of compound Sub 2-1 are shown below.

[0124] 1 H NMR (600MHz, CDCl3) δ5.26-5.16(m,1H,CH2CHOCH2),2.64-2.54(m,2H,CHOCH2CO),2.27(t,J=7.5Hz,2H ,CH2CH2COO),1.64-1.55(m,4H,CH2CH2COO),1.32-1.23(m,30H,CH2×15),0.87(t,J=6.9Hz,6H,CH3×2).

[0125] 13 C NMR (101MHz, CDCl3) δ176.16, 173.31 (CO×2), 70.00 (CHOH), 34.50 (CHOHCH2), 33.98, 31.88, 29.48, 29.30, 29.12, 25.02, 22.68, 14.11.

[0126] HRMS: [M+Na] + Theoretical components: C 24 H 46 O4Na + :421.3288, measured value:421.3278.

[0127] The characterization data of compound Sub 2-2 are shown below.

[0128] 1 H NMR (600MHz, CDCl3) δ5.21 (t, J=6.9Hz, 1H, CH2CHOCH2), 2.61 (qd, J=15.8, 6.3Hz, 2H, CHOCH2CO), 2.28 (t, J=7.5Hz, 2H, CH2CH2COO), 1.61 (h, J=8.2, 7.5Hz, 4H, CH2CH2COO), 1.33-1.21 (m, 34H, CH2×17), 0.88 (t, J=6.9Hz, 6H, CH3×2).

[0129] 13 C NMR(151MHz, CDCl3)δ176.73,173.21(CO×2),69.90(CHOH),38.87(CHOHCH2),34.36, 33.87,31.85,29.57,29.48,29.42,29.29,29.22,29.03,25.03,24.92,22.61,14.01.

[0130] HRMS: [M+Na] + Theoretical components: C 26 H 50 O4Na + :449.3601, measured value:449.3600.

[0131] The characterization data of compound Sub 2-3 are shown below.

[0132] 1 H NMR (600MHz, CDCl3) δ5.20 (q, J=6.4Hz, 1H, CH2CHOCH2), 2.58 (qd, J=15.9, 6.4Hz, 2H, CHOCH2CO), 2.26 (t, J=7.5H z,2H,CH2CH2COO),1.59(dt,J=14.8,8.3Hz,4H,CH2CH2COO),1.23(m,38H,CH2×19),0.86(t,J=7.0Hz,6H,CH3×2).

[0133] 13C NMR(151MHz, CDCl3)δ176.75,173.28(CO×2),69.92(CHOH),38.89(CHOHCH2),34.42,33.91, 31.89,29.67,29.64,29.60,29.51,29.45,29.32,29.25,29.07,25.06,24.96,22.65,14.06.

[0134] HRMS: [M+Na] + Theoretical components: C 28 H 54 O4Na + :477.3914, measured value:477.3899.

[0135] 2. Synthesis of Compound 5

[0136] The synthetic route for compound 5 is as follows:

[0137]

[0138] Compound 10 (24 g, 93 mmol, 1 eq.), 2-naphthaldehyde (43.5 g, 279 mmol, 3 eq.), and hexamethyldisiloxane ((TMS)₂O, 118 mL, 558 mmol, 6 eq.) were added to 400 mL of THF and stirred for 15 minutes to dissolve. Then, trimethylsilyl trifluoromethanesulfonate (TMSOTf, 14.4 mL, 74 mmol, 0.8 eq.) was added and stirring continued for 15 minutes. Finally, triethylsilane (Et₃SiH, 51.8 mL, 325 mmol, 3.5 eq.) was added and the mixture was reacted for 3 hours. The mixture was then dissolved in DCM and extracted with 500 mL of saturated NaHCO₃ aqueous solution and 500 mL of DCM. The combined organic layers were dried with Na2SO4, filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography (gradient elution, DCM / PE, v / v = 17:83 to 33:67) to give target compound 11 (23.5 g, 55.8 mmol, yield 60%).

[0139] The characterization data for compound 11 are shown below.

[0140] 1H NMR(600MHz,CDCl3)δ7.90-7.46(q,J=8.7,7.9Hz,7H,aromatic(Nap)),4.71(s,2H,CH2(Na p)),3.94(p,J=6.2Hz,1H,CHCH2COOMe),3.67(s,3H,OCH3),2.65(dd,J=14.9,7.3Hz,1H,CH 2a COOMe),2.52(dd,J=15.0,5.4Hz,1H,CH 2b COOMe), 1.66 (d, J = 8.4Hz, 2H, CH2CHCH2COO), 1.45-1.14 (m, 20H, CH2 x10), 0.89 (t, J = 7.0Hz, 3H, CH3). 13 C NMR(151MHz, CDCl3)δ172.16(CO),135.83,133.09,132.77,127.88,127.69,126.21,125.83,125.74,125.60(aromatic (Nap)),75.93(CHCH2COOMe),71.45(CH2(Nap)),51.48,39.64,34.23,31.77,29.48,29.42,29.20,25.02,22.54,13.99.

[0141] HRMS: [M+Na] + Theoretical components: C 26 H 38 O3Na + :421.2719, measured value:421.2716.

[0142] (2) Synthesis of compound 12

[0143]

[0144] Compound 11 (23.5 g, 55.8 mmol, 1 eq.) was dissolved in a mixed solution of 100 mL THF and 100 mL H2O under an Ar atmosphere. Lithium hydroxide (LiOH, 11.7 g, 279 mmol, 5 eq.) was added, and the mixture was reacted at room temperature for 6 hours. The pH of the system was adjusted to neutral with 1.5 M HCl solution, and the mixture was extracted with 200 mL saturated NaHCO3 aqueous solution and 200 mL DCM solution. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (DCM / PE gradient elution, DCM / PE, v / v = 20:80 to 40:60) to give the target compound 12 (16.3 g, 42.4 mmol, yield 76%).

[0145] The characterization data for compound 12 are shown below.

[0146] 1 H NMR(600MHz,CDCl3)δ7.79-7.37(dd,7H,aromatic(Nap)),4.72(d,J=5.7Hz,2HCH2(Nap)),3.91(q,J=7.3,6.7Hz,1H,CHCH2COOMe),2.65(dt,J=14.2,7.0Hz,1H,CH 2a COOMe),2.58(dd,J=15.6,5.5Hz,1H,CH 2b COOMe),1.76-1.51(m,2H,CH2CHCH2COO),1.42-1.08(m,20H,CH2×10),0.86(p,J=8.0,7.3Hz,3H,CH3).

[0147] 13 C NMR(151MHz, CDCl3)δ177.31(CO),135.47,127.97,127.72,127.48,126.34,125.86,125.70,125.66(aromatic(Na p)),75.62(CHCH2COOMe),71.48(CH2(Nap)),39.45,34.04,31.77,29.49,29.42,29.20,24.99,22.54,13.99(CH3).

[0148] HRMS:[MH] - Theoretical components: C 25 H 35 O3 - 383.2592, measured value: 383.2591.

[0149] (3) Synthesis of compound Sub 3

[0150]

[0151] Compound 12 (16.3 g, 42.4 mmol, 1 eq.) and pentafluorophenol (PFP, 9.36 g, 50.9 mmol, 1.2 eq.) were dissolved in dry DCM under an Ar atmosphere. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 12.2 g, 63.6 mmol, 1.5 eq.) and 4-dimethylaminopyridine (DMAP, 1.04 g, 8.48 mmol, 0.2 eq.) were added, and the mixture was reacted at room temperature for 2 hours. Extraction was performed with 50 mL of saturated NaHCO3 aqueous solution and 50 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, DCM / PE, v / v = 5:95 to 10:90) to give the target compound Sub 3 (21.7 g, 39.4 mmol, 93% yield).

[0152] The characterization data of compound Sub 3 are shown below.

[0153] 1 H NMR (400MHz, CDCl3) δ7.92-7.52 (dd, J=6.9, 2.9Hz, 7H, aromatic (Nap)), 4.88-4.75 (m,2 H,CH2(Nap)),4.10(dt,J=11.3,5.7Hz,1H,CHCH2COOMe),3.04(dd,J=15.4,7.5Hz,1H,CH 2a COOMe),2.91(dd,J=15.4,5.1Hz,1H,CH 2b COOMe), 1.88-1.50 (m, 2H, CH2CHCH2COO), 1.43–1.13 (m, 16H, CH2×8), 0.94 (t, J=7.0Hz, 3H, CH3).

[0154] 13 C NMR(101MHz, CDCl3)δ167.78(CO),133.35,128.21,127.94,127.73,126.56,126.12,125.92,75.77(CHCH 2COOMe),71.97(CH2(Nap)),39.07,34.26,32.02,29.74,29.66,29.63,29.45,25.14,22.79,14.20(CH3).

[0155] HRMS: [M+Na] + Theoretical components: C 31 H 35 O3F5Na + :573.2420, measured value:573.2475.

[0156] (4) Synthesis of compound 14

[0157]

[0158] Compound 13 (pentaerythritol, 146.8 mmol, 20 g, 1 eq.), benzaldehyde dimethyl acetal (BDMA, 146.8 mmol, 22 g, 1 eq.), and p-toluenesulfonic acid monohydrate (TsOH·H2O, 44 mmol, 10.2 g, 0.3 eq.) were dissolved in dry acetonitrile (CH3CN) under an Ar atmosphere and reacted at 50 °C for 1 h. The reaction was then quenched with 6 mL of Et3N, followed by extraction with 50 mL of saturated NaHCO3 aqueous solution and 50 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, EA / PE, v / v = 20:80 to 55:45) to obtain target compound 14 (6.6 g, 30 mmol, yield 20%).

[0159] The characterization data for compound 14 are shown below.

[0160] 1 H NMR (400MHz, CDCl3) δ7.63-7.29(m,5H,Ph×5),5.44(s,1H,PhCH),4.27-4.05(m,4H,OCH2CH×2),3.83-3.68(m,2H,CHCH 2a OH), 3.56(s, 2H, CHCH) 2b OH), 2.22(s, 2H, CH2OH×2).

[0161] 13 C NMR (151MHz, CDCl3) δ129.07,128.33,125.99(Ph×5),102.03(PhCH),69.98(OCH2CH×2),65.68(CHCH 2a OH), 64.25(CHCH 2b OH), 41.71(CH2CHCH2).

[0162] HRMS: [M+H] + Theoretical components: C12 H 17 O4 + :225.1121, measured value:225.1119.

[0163] (5) Synthesis of compound 15

[0164]

[0165] Compound 14 (6.6 g, 30 mmol, 1 eq.) was dissolved in Et3N (208 mL, 1.5 mol, 50 eq.) under ice bath conditions. p-Toluenesulfonyl chloride (TsCl, 57.2 g, 300 mmol, 10 eq.) was added, and the reaction was carried out under Ar atmosphere for 8 hours. The reaction product was extracted with 500 mL of saturated NaHCO3 aqueous solution and 500 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, EA / PE, v / v = 20:80 to 33:67) to give target compound 15 (15.0 g, 28.2 mmol, 94% yield).

[0166] The characterization data for compound 15 are shown below.

[0167] 1 H NMR (600MHz, CDCl3) δ7.76-7.14(m,13H,Ph×13),5.29(s,1H,PhCH),4.27(s,2H,OCH 2a CH), 3.95 (d, J = 11.7 Hz, 2H, OCH) 2b CH),3.86-3.69(m,4H,CHCH2OH×2),2.48(s,3H,PhCH 3a ),2.41(s,3H,PhCH 3b ).

[0168] 13 C NMR (151MHz, CDCl3) δ145.55,137.06,130.18,129.94,129.20,128.18,128.10,127.95,1 25.92,101.93(aromatic(PhCH)),68.30,67.88,67.26,31.67(CH2CHCH2),21.68(CH3×2).

[0169] HRMS:[MH] - Theoretical components: C 26 H 27 O8S2 -:531.1153, measured value:531.1174.

[0170] (6) Synthesis of compound 16

[0171]

[0172] Compound 15 (15 g, 28.2 mmol, 1 eq.), trimethyl azidosilane (TMSN3, 23.5 mL, 169.2 g, 6 eq.), and cesium fluoride (CsF, 25 g, 169.2 mmol, 16 eq.) were dissolved in 20 mL of DMF (N,N-dimethylformamide). The mixture was heated to 90 °C and refluxed under an Ar atmosphere for 4 days. After the reaction was completed by TLC, a white solid was filtered off and the filtrate was collected. The filtrate was extracted with 500 mL of saturated NaHCO3 aqueous solution and 500 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography (gradient elution, EA / PE, v / v = 20:80 to 25:75) to obtain the target compound 16 (6.4 g, 23.4 mmol, yield 83%).

[0173] The characterization data for compound 16 are shown below.

[0174] 1 H NMR (600MHz, CDCl3) δ7.60-7.31(m,5H,Ph×5),5.43(d,J=4.1Hz,1H,PhCH),4.16-4.00(m,2H,OCH 2a C), 3.85(t, J = 3.8 Hz, 2H, OCH 2b C), 3.78-3.70 (m, 2H, CCH) 2a N3), 3.21(t, J = 3.7 Hz, 2H, CCH 2b N3).

[0175] 13 C NMR (151MHz, CDCl3) δ137.64,129.25,128.41,126.09,102.05(aromatic(PhCH)),70.42(OCH2C),52.20,52.10,51.55(CCH2N3),38.03(CH2CCH2).

[0176] HRMS: [M+Na] + Theoretical components: C 12 H 14 N6O2Na + :297.1079, measured value:297.1066.

[0177] (7) Synthesis of compound 17

[0178]

[0179] Compound 16 (6.4 g, 23.4 mmol, 1 eq.) was dissolved in a mixed solvent of 16 mL trifluoroacetic acid (TFA), 6.4 mL DCM, and 0.4 mL H2O. The reaction was carried out in an ice bath under an Ar atmosphere for 20 min. The resulting reaction system was extracted with 50 mL of saturated NaHCO3 aqueous solution and 50 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, EA / PE, v / v = 30:70 to 60:40) to obtain target compound 17 (4.0 g, 21.5 mmol, yield 92%).

[0180] The characterization data for compound 17 are shown below.

[0181] 1 H NMR (400MHz, CDCl3) δ3.60(s,4H,CCH2OH×2), 3.41(s,4H,CCH2N3×2).

[0182] 13 C NMR (101MHz, CDCl3) δ63.18(CCH2OH), 51.52(CCH2N3), 44.90(CH2CCH2).

[0183] HRMS:[MH] - Theoretical composition: C5H9N6O2 - :185.0792, measured value:185.0808.

[0184] (8) Synthesis of Compound 5

[0185]

[0186] R'2 in compound 5 represents

[0187] a) Compound 17 (1 g, 5.4 mmol, 1 eq.) and trimethylphosphine (PMe3, 4.3 mL, 4.3 mmol, 8 eq.) were dissolved in a mixed solvent of 15 mL DCM and 1 mL H2O and reacted at room temperature under nitrogen protection. When TLC analysis showed that the reaction was complete, the resulting system was dried with Na2SO4, filtered and evaporated to dryness to give intermediate IM-1 with the structure shown below.

[0188]

[0189] b) Under an Ar atmosphere, the obtained intermediate IM-1 and compound Sub 3 (7.4 g, 13.5 mmol, 2.5 eq.) and N,N-diisopropylethylamine (DIPEA, 2.3 mL, 13.5 mmol, 2.5 eq.) were dissolved in 20 mL of dry DCM and reacted at room temperature. After the reaction was detected by TLC, the crude product was concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, MeOH / DCM, v / v = 1:99 to 3:97) to obtain the target compound 5 (3.8 g, 4.4 mmol, two-step yield 82%).

[0190] The characterization data for compound 5 are shown below.

[0191] 1 H NMR (400MHz, CDCl3) δ7.91-7.38 (m, 14H, aromatic (Nap)), 7.08 (t, J = 6.8Hz, 2H, NH × 2), 4.74 (d, J = 11.5Hz, 2H, CH 2a (Nap)),4.65(d,J=11.6Hz,2H,CH 2b (Nap)),3.93(p,J=6.0Hz,2H,CH2CHCH2×2),3.59(p,J=6.7Hz,1H,CCH2OH),3.15(s,4H,CCH2NH×2),2.99(dh,J=20.9,6.9,6.5Hz,5H,CCH 2OH×2,CCH2OH),2.58-2.41(m,4H,CCH2CH×2),1.72(dq,J=14.3,6.8Hz,2H,CCH2CH2),1.57(td,J=13.6,12.9,6.6Hz,3H),1.38(d,J=6.8

[0192] Hz,53H),0.92(t,J=6.7Hz,6H CH3×2).

[0193] 13C NMR(101MHz, CDCl3)δ173.72(CO),133.32,128.16,127.91,127.73,126.53,126.16,125.96,125.91(aromatic(Nap)),76.53(CH2CHCH2),71.53 (CH2(Nap)×2),60.52(CHCH2OH),46.42(CCH2NH),41.73(CCH2CH),38.19,33.99,32.03,29.81,29.77,29.72,29.47,25.26,22.81,14.26(CH3).

[0194] HRMS: [M+Na] + Theoretical components: C 55 H 82 N2O6Na + :889.6071, measured value:889.5814.

[0195] 3. Synthesis of Compound 2

[0196]

[0197] a) Compound 1 (D-glucosamine hydrochloride, 40 g, 184 mmol, 1 eq.) and NaHCO3 (46.3 g, 552 mmol, 3 eq.) were added to 500 mL of H2O. The resulting mixture was stirred vigorously under an Ar atmosphere until the solution became clear. Then, 2,2,2-trichloroethyl chloroformate (Troc-Cl, 30.4 mL, 221 mmol, 1.2 eq.) was added and the mixture was stirred for another 3 hours, producing a white, fluffy solid. The liquid was filtered from the mixture, and the resulting white solid was washed with 300 mL of H2O and dried to give intermediate IM-2, with the structure shown below.

[0198]

[0199] b) The intermediate IM-2 was dissolved in a mixed solvent of Py (448 mL, 5.52 mol, 30 eq.) and acetic anhydride (Ac2O, 262 mL, 2.78 mol, 15 eq.), and stirred vigorously for 8 hours under an Ar atmosphere to allow the reaction to complete. The reaction product was extracted with 500 mL of saturated NaHCO3 aqueous solution and 500 mL of DCM. The combined organic layers were dried with Na2SO4, filtered, and concentrated under vacuum to obtain the intermediate IM-3 with the structure shown below.

[0200]

[0201] c) Intermediate IM-3, p-toluenethiophenol (TolSH, 45.7 g, 368 mmol, 2 eq.), and boron trifluoride diethyl ether (BF3·Et2O, 72.7 mL, 1.1 mol, 6 eq.) were dissolved in 500 mL of dry DCM and refluxed at 40 °C for 8 hours under an Ar atmosphere. The resulting reaction mixture was extracted with 500 mL of saturated NaHCO3 aqueous solution and 500 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography (gradient elution, EA / PE, v / v = 10:90 to 25:75) to obtain the target product, compound 2 (50.7 g, 101.2 mmol, overall yield of 55% in three steps).

[0202] The characterization data for compound 2 are shown below.

[0203] 1 H NMR (400MHz, CDCl3) δ7.44 (dt, J=8.2, 2.6Hz, 2H, (pht)), 7.14 (d, J=7.6Hz, 2H,

[0204] (pht)),5.41(d,J=8.9Hz,1H,H-1),5.30(t,J=9.6Hz,1H,H-3),5.11-4.98(m,1H,H-4),

[0205] 4.90-4.79(m,2H,CH2(Troc)),4.74(m,1H,H-2),4.34-4.10(m,2H,H-6),3.81-3.56(m,

[0206] 2H),2.43-2.30(m,3H,CH3(Tol)),2.11(t,J=2.6Hz,3H,CH3),2.04(s,6H,CH3×2).

[0207] 13 C NMR(101MHz, CDCl3)δ170.70,169.53,153.95(COCH3×4),138.77,133.68,

[0208] 129.79,127.94,95.46(CCCl3(Troc)),86.74(C-1),75.77(CCCl3(Troc)),74.55(C-5),73.27

[0209] (C-3),68.55(C-4),62.35(C-6),55.01(C-2),21.24(PhCH3,Tol),20.82,20.71,20.65

[0210] (COCH3×3).

[0211] HRMS: [M+Na] + Theoretical Components C 22 H 26 Cl3NO9SNa + :608.0292, measured value:608.0183.

[0212] 4. Synthesis of compound 3i

[0213] The synthetic route is shown below. Depending on R1, the compounds 3i prepared are specifically numbered 3a, 3b, and 3c.

[0214]

[0215] (1) Synthesis of compound 3a

[0216] a) Compound 2 (50.7 g, 86.4 mmol, 1 eq.) was dissolved in 500 mL of methanol (MeOH), and then sodium methoxide (NaOMe, 22 g, 345.6 mmol, 4 eq.) was added and reacted for 20 minutes. After the reaction was completed by TLC, the reaction was quenched with AcOH and concentrated under vacuum to obtain intermediate IM-4 with the structure shown below.

[0217]

[0218] b) Under an Ar atmosphere, intermediate IM-4 (32.4 g, 70.6 mmol, 1 eq.), benzaldehyde dimethyl acetal (BDMA, 31.7 mL, 211.8 mmol, 3 eq.), and TsOH·H₂O (4 g, 21.2 mmol, 0.3 eq.) were dissolved in 200 mL of dry CH₃CN and reacted. After the reaction was completed by TLC, the reaction was quenched with 5 mL of Et₃N and concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, MeOH / DCM, v / v = 0:100 to 5:95) to give intermediate IM-5 (28.2 g, 51.5 mmol, two-step yield 60%), as shown below.

[0219]

[0220] c) Under an Ar atmosphere, intermediate IM-5 (3 g, 5.5 mmol, 1 eq.) and compound Sub 2-1 (8.25 mmol, 1.5 eq.) were dissolved in dry DCM. EDC·HCl (2.64 g, 13.75 mmol, 2.5 eq.), 4-dimethylaminopyridine (DMAP, 0.336 g, 2.75 mmol, 0.5 eq.), and 1-hydroxybenzotriazole (HOBT, 1.49 g, 11 mmol, 2 eq.) were refluxed at 50 °C for 8 hours. The organic phases were extracted with 50 mL of saturated NaHCO3 solution and 50 mL of DCM to combine the organic phases. The organic phases were then dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (gradient elution, EA / PE, v / v = 4:96 to 8:92) to give compound 3a (3.5 g, yield 69%).

[0221] (2) Synthesis of compounds 3b and 3c

[0222] Compounds 3b and 3c were synthesized using the same method as compound 3a, except that the compound Sub 2-1 used was replaced with each Sub 2-i. The replaced reactants Sub 2-i and the corresponding compounds and their yields are shown in Table 3.

[0223] Table 3

[0224]

[0225] The characterization data of compound 3a are shown below.

[0226] 1 H NMR(400MHz, CDCl3)δ7.43(d,J=8.1Hz,4H,(pht)),7.37(d,J=4.5Hz,3H,(pht)),

[0227] 7.18(d,J=7.8Hz,2H(pht)),5.54(d,J=5.0Hz,1H,PhCHO2),5.51-5.39(m,1H,H-3),5.21

[0228] (s,1H,CH2CHOCH2),4.98(d,J=10.2Hz,1H,H-1),4.81(t,J=9.7Hz,2H,CH2(Troc)),

[0229] 4.41(dd,J=10.8,5.1Hz,1H,H-4),3.83(t,J=10.2Hz,1H,H-6a),3.69(dd,J=17.4,9.3Hz,

[0230] 2H,H-6b,H-2),3.59(s,1H,H-5),2.68-2.49(m,2H,CHOCH2CO),2.39(s,3H,CH3(To l)), 2.22 (t, J=7.6Hz, 2H, CH2CH2COO), 1.29 (s, 28H), 0.92 (t, J=6.6Hz, 6H, CH3×2).

[0231] 13 C NMR(101MHz, CDCl3)δ173.53,169.99,154.1(CO×3),138.67,136.81,133.45,129.88,12 9.18,128.26,126.19,101.50(PhCHO),95.46(CCl3(Troc)),87.92(C-1),78.56(C-4),74 .59(C-5),72.23(C-6),70.58,70.01(C-3),68.54,55.86(C-2),39.36,34.39,33.91,31. 97,29.75,29.70,29.59,29.41,29.37,29.34,29.18,25.10,25.03,22.74,21.24,14.18.

[0232] HRMS: [M+Na] + Theoretical Components C 47 H 68 Cl3NO9SNa + :950.3578, measured value:950.3562.

[0233] The characterization data for compound 3b are shown below.

[0234] 1H NMR (400MHz, CDCl3) δ7.47-7.40(m,4H,(pht)),7.34(dd,J=6.4,3.1Hz,3H,(pht)),7.16(d,J=7.9Hz,2H,(pht)),5.75(dd,J=9.6,3 .7Hz,1H,PhCHO2),5.51(s,1H,H-3),5.22(tt,J=12.7,6.6Hz,1H,CH2CHOCH2),4.98(d,J=10.2Hz,1H,H-1),4.80(s,1H,CH2(Troc)), 4.36(dd,J=10.5,4.9Hz,1H,H-4),3.80(t,J=10.1Hz,1H,H-6a),3.69(q,J=9.5Hz,1H,H-2),3.55(tt,J=9.1,4.5Hz,1H,H-5),2.73- 2.50(m,2H,CHOCH2CO),2.38(s,3H,CH3(Tol)),2.20(t,J=7.5Hz,2H,CH2CH2COO),1.40-1.14(m,38H),0.92(t,J=6.7Hz,6H,CH3×2).

[0235] 13 C NMR(101MHz, CDCl3)δ173.51,170.12,154.22(CO×3),138.57,136.87,133.48,129.84 ,129.16,128.25,126.19,101.44(PhCHO),95.55(CCl3(Troc)),87.83(C-1),78.56(C -4),74.61(C-5),72.47(C-6),70.48,70.01(C-3),68.52,55.73(C-2),39.32,34.39, 33.89,31.97,29.69,29.58,29.40,29.36,29.18,25.10,25.03,22.74,21.23,14.17.

[0236] HRMS: [M+Na] + Theoretical Components C 49 H 72 Cl3NO9SNa + :978.3891, Measured value:978.3890.

[0237] The characterization data of compound 3c are shown below.

[0238] 1H NMR(400MHz, CDCl3)δ7.57-7.41(m,4H,(pht)),7.38(dq,J=6.5,3.3Hz,3H,(pht)),7.17(d,J=7.7Hz,2H,(pht)),5.53(s,1H,PhCHO2),5.43(t, J=9.7Hz,1H,H-3),5.21(p,J=6.4Hz,1H,CH2CHOCH2),4.97(d,J=10.5Hz,1H,H-1),4.87-4.71(m,2H,CH2(Troc)),4.36(ddd,J=24.0,10.2,5.0H z,1H,H-4),3.83(t,J=10.2Hz,1H,H-6a),3.76-3.64(m,2H,H-6b,H-2),3.58(td,J=9.5,4.9Hz,1H,H-5),2.59(qd,J=15.3,6.4Hz,2H,CHOCH2CO ),2.38(d,J=8.9Hz,3H,CH3(Tol)),2.22(t,J=7.5Hz,2H,CH2CH2COO),1.71-1.49(m,5H),1.23(d,J=8.0Hz,45H),0.92(t,J=6.6Hz,6H,CH3×2).

[0239] 13 C NMR(101MHz, CDCl3)δ173.51,170.12,154.22(CO×3),138.68,136.82,133.48,129.84 ,129.16,128.25,126.19,101.44(PhCHO),95.55(CCl3(Troc)),87.83(C-1),78.56(C -4),74.61(C-5),72.47(C-6),70.48,70.01(C-3),68.52,55.73(C-2),39.32,34.39, 33.89,31.97,29.69,29.58,29.40,29.36,29.18,25.10,25.03,22.74,21.23,14.17.

[0240] HRMS: [M+Na] + Theoretical Components C 51 H 76 Cl3NO9SNa + :1006.4204, Measured value:1006.4202.

[0241] 5. Synthesis of compounds 4a, 4b and 4c

[0242] The specific synthesis route is shown below.

[0243]

[0244] (1) Synthesis of compound 4a

[0245] a) Compound 3a (3.71 g, 4 mmol, 1 eq.) was dissolved in a mixed solvent of 1.6 mL TFA, 6.4 mL DCM, and 0.4 mL H2O. The reaction was carried out under Ar atmosphere at 0 °C for 20 min. After the reaction was completed by TLC, the product was extracted with 50 mL of saturated NaHCO3 aqueous solution and 50 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (gradient elution, MeOH / DCM, v / v = 3:97 to 5:95) to obtain the intermediate IM-6a with the structure shown below:

[0246]

[0247] b) At 0 °C, tert-butyldiphenylchlorosilane (TBDPS-Cl, 1.1 g, 4 mmol, 2 eq.) and imidazole (0.27 g, 4 mmol, 2 eq.) were added to 20 mL of anhydrous DCM solution of intermediate IM-6a (2 mmol, 1 eq.) and reacted for 8 hours. After the reaction was completed by TLC, the crude product was subjected to column chromatography (gradient elution, EA / PE, v / v = 1:99 to 8:92) to obtain intermediate IM-7a with the structure shown below:

[0248]

[0249] c) Dissolve intermediate IM-7a (1.3 mmol, 1 eq.) in dry DCM, then add 500 mg of activated [product name] under an Ar atmosphere. Molecular sieves were added and stirred for 30 minutes. Diallyl diisopropylphosphonamide (i-Pr2NP(OAllyl)2, 0.96 g, 3.9 mmol, 3 eq.) was added, followed by 1H-tetrazole (0.55 g, 7.8 mmol, 6 eq.). The reaction was carried out at room temperature for 2 hours. After the reaction was detected by TLC, tert-butyl hydroperoxide (TBHP, 0.88 mL, 11.7 mmol, 9 eq.) was added and stirred for 30 minutes. After the reaction was detected by TLC, the crude product was concentrated under vacuum and purified by column chromatography (gradient elution, EA / PE, v / v = 5:95 to 12:88) to give the target compound 4a (1.4 g, yield 89%).

[0250] (2) Synthesis of compounds 4b and 4c

[0251] Compounds 4b and 4c were synthesized using the same method as compound 4a, except that compound 3a was replaced with compounds 3b and 3c, respectively. The resulting compounds, their masses, and yields are shown in Table 4.

[0252] Table 4

[0253]

[0254]

[0255] The characterization data for compound 4a are shown below.

[0256] 1 H NMR(400MHz, CDCl3)δ7.76(q,J=6.1,4.5Hz,4H,(pht)),7.52-7.33(m,8H,(pht)),7.10-6.98(m,2H,(pht)),5.70(dd,J=12.9,5. 4Hz,1H,NH),5.62(d,J=8.2Hz,1H,H-1),5.36-5.10(m,6H,CH2=CHCH2O×2,H-3,CHCH2COO(Lipid)),4.83(dd,J=12.0,2.5Hz,1H,CH 2a (Troc)),4.72(dd,J=12.1,2.5Hz,1H,CH 2b (Troc)),4.47(q,J=8.9,7.9Hz,1H,),4.37(q,J=8.7,7.7Hz,3H,CH2=CHCH2O×2,H-4),4.25(dt,J=13.2,6 .7Hz,1H),4.03(d,J=11.6Hz,1H,H-6a),3.90(dd,J=12.5,5.3Hz,1H,H-2),3.72-3.59(m,1H,H-6b),3.47 (q,J=8.3Hz,1H),2.73-2.55(m,2H,CHOCH2CO),2.31(dd,J=9.3,4.6Hz,5H,CH3(Tol)),1.49(dq,J=12.0, 6.5, 6.0Hz, 4H), 1.30 (d, J = 9.4Hz, 32H), 1.11 (d, J = 2.7Hz, 9H, CH3 (TBDPS) × 3), 0.95-0.86 (m, 6H, CH3 × 2).

[0257] 13C NMR (101MHz, CDCl3) δ173.74,170.30,153.97(CO×3),138.05,135.87,135.72,132.85,132.18,132.11(CH2=CH-CH2× 2),129.81,129.71,127.75,127.72,118.49(CH2=CH×2),95.52(CCl3(Troc)),86.50(C-1),79.33(C-4),74.53(C-5) ,73.55,70.13(C-6),68.57(C-3),68.51,68.29,68.23(CH=CHCH2×2),62.83,55.75(C-2),39.79,34.54,34.42,31.9 7,31.93,29.70,29.62,29.59,29.54,29.41,29.39,29.35,29.24,26.85,25.20,25.09,22.74,21.19,19.31,14.19.

[0258] 31 P NMR (162MHz, CDCl3): δ-2.04.

[0259] HRMS: [M+Na] + Theoretical Components C 62 H 91 Cl3NO 12 SPSiNa + :1260.4732, measured value:1260.4730.

[0260] The characterization data for compound 4b are shown below.

[0261] 1 H NMR(400MHz, CDCl3)δ7.65(ddt,J=8.7,4.6,2.3Hz,4H,(pht)),7.37-7.25(m,8H,( pht)), 6.93 (d, J=7.9Hz, 2H, (pht)), 5.75 (ddt, J=16.2, 10.8, 5.6Hz, 1H, NH), 5.62 (d,J=8.2Hz,1H,H-1),5.73(s,1H,H-3),5.66-5.50(m,2H),5.35(t,J=9.5Hz,1H), 5.24-4.98(m,6H,CH2=CHCH2O×2,H-3,CHCH2COO(Lipid)),4.72(d,J=12.1Hz,1H,CH 2a(Troc)),4.62(d,J=12.1Hz,1H,CH 2b (Troc)),4.43-4.12(m,5H,CH2=CHCH2O×2,H-4),3.92(dd,J=11.6,1.8Hz,1H,H-6a),3.80( dd,J=11.6,5.4Hz,1H,H-2),3.54(ddd,J=10.0,5.5,1.8Hz,1H,H-6b),3.44-3.32(m,1H),2 .64-2.46(m,2H,CHOCH2CO),2.33-2.11(m,5H,CH3(Tol)),1.49(dq,J=12.0,6.5,6.0Hz,5H ),1.19(dd,J=8.3,3.5Hz,38H),1.00(s,9H,CH3(TBDPS)×3),0.80(t,J=6.6Hz,6H,CH3×2).

[0262] 13 C NMR (101MHz, CDCl3) δ173.79,170.34,153.97(CO×3),138.11,135.88,135.74,133.13,132.86,132.18(CH2 =CHCH2×2),129.83,129.73,127.77,127.74,118.52(CH2=CH×2),95.51(CCl3(Troc)),86.57(C-1),79.30(C -4),74.55(C-5),73.54,70.17(C-6),68.60,68.54(C-3),68.26(CH=CH-CH2×2),62.86,55.78(C-2),39.82 ,34.56,34.45,31.99,29.71,29.64,29.61,29.42,29.27,26.87,25.22,25.11,22.76,21.21,19.33,14.20. 31 P NMR (162MHz, CDCl3): δ-2.22.

[0263] HRMS: [M+Na] + Theoretical Components C 64 H 95 Cl3NO 12 SPSiNa + :1288.5045, measured value:1288.5033.

[0264] The characterization data for compound 4c are shown below.

[0265] 1 H NMR(400MHz,CDCl3)δ7.81-7.71(m,4H,(pht)),7.51-7.34(m,8H,(pht),7.04(d,J=7.8Hz,2H,(pht)),5.85(ddt,J=16.3,10.8,5.7Hz,1H,NH),5.71(ddt,J=16.3,10.8,5.6Hz,1H,H-1),5.61(t,J=8.1Hz,1H,H-3),5.45(q,J=9.2Hz,1H),5.35–5.11(m,6H,CH2=CHCH2O×2,H-3,CHCH2COO(Lipid)),4.83(d,J=12.1Hz,1H,CH 2a (Troc)),4.72(d,J=12.1Hz,1H,CH 2b (Troc)),4.47-4.25(m,5H,CH2=CHCH2O×2,H-4),4.08-3.97(m,1H),3.90(dd,J=11.6,5.5Hz,1H,H-6a),3.65(dd,J=10.4,5.2Hz,1H,H-2),2.74-2.58(m,2H,CHOCH2CO),2.32(d,J=6.9Hz,5H,CH3(Tol)),1.68-1.53(m,4H),1.25(s,42H),1.19(dd,J=8.3,3.5Hz,42H),1.10(d,J=10.2Hz,9H,CH3(TBDPS)×3),0.91(t,J=6.7Hz,6H,CH3×2).

[0266] 13C NMR (101MHz, CDCl3) δ173.80,170.32,153.95(CO×3),135.88,135.73,132.84,132.23,132.16(CH2=CH-CH2 ×2),129.83,129.72,127.76,127.73,118.52(CH2=CH×2),95.49(CCl3(Troc)),86.54(C-1),79.35(C-4),7 4.52(C-5),73.50,70.15(C-6),68.59,68.28(C-3),62.83(CH=CHCH2×2),62.83,55.77(C-2),39.84,34.56 ,34.45,31.99,29.77,29.73,29.64,29.61,29.43,29.27,26.85,25.21,25.11,22.77,21.22,19.32,14.21.

[0267] 31 P NMR (162MHz, CDCl3): δ-2.28.

[0268] HRMS: [M+Na] + Theoretical Components C 66 H 99 Cl3NO 12 SPSiNa + :1316.5358, measured value:1316.5352.

[0269] 6. Synthesis of compounds 6a, 6b and 6c

[0270] The synthesis route is shown below:

[0271]

[0272] (1) Synthesis of compound 6a

[0273] Compound 4a (1.45 g, 1.17 mmol, 1.5 eq.) was dissolved in 10 mL of anhydrous DCM, and 1 g of activated [agent / concentrate] was added. Molecular sieves were added and stirred for 30 minutes. Then, diphenyl sulfoxide (Ph₂SO₄, 173.6 mg, 0.86 mmol, 1.1 eq.) was added and stirred for 10 minutes. Trifluoromethanesulfonic anhydride (Tf₂O, 144.7 μL, 0.86 mmol, 1.1 eq.) was added and stirring was continued for 20 minutes. Finally, compound 5 (673 mg, 0.78 mmol, 1 eq.) was added and the reaction was carried out for 1 hour. After the reaction was completed by TLC, the mixture was filtered through molecular sieves and extracted with 150 mL of saturated NaHCO₃ aqueous solution and 150 mL of DCM. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (gradient elution, MeOH / DCM, v / v = 5:95 to 11:89) to obtain the target compound 6a (1.3 g, yield 56%).

[0274] (2) Synthesis of compounds 6b and 6c

[0275] Compounds 6b and 6c were synthesized using the same method as compound 6a, except that compound 4a was replaced with compounds 4b and 4c, respectively. The synthesized compounds, their masses, and yields are shown in Table 5.

[0276] Table 5

[0277]

[0278]

[0279] The characterization data for compound 6a are shown below.

[0280] 1 H NMR(400MHz, CDCl3)δ7.83(qd,J=13.5,8.1Hz,8H,(pht),aromatic(Nap)),7.70(t,J=6.0Hz,4H,(pht),aromatic(Nap)),7.45(ddt,J=17.3,14.0,5.9Hz,11H,(pht) ,aromatic(Nap)),7.16(s,1H,NH),6.91(s,1H,NH),5.88(ddd,J=16.5,10.8,5.6H z,1H,NH),5.82-5.68(m,1H,H-1),5.39-5.31(m,1H,H-3),5.25(t,J=11.9Hz,3H,CH 2a =CHCH2O),5.17(d,J=10.4Hz,1H,CH 2b(CHCH2O), 5.12(s,1H,CHCH2COO(Lipid)), 4.71(ddt,J 19.0, 14.8, 9.9Hz, 6H,CH2(Troc),CH2(Nap)×2), 4.42(tt,J 13.1, 6.7Hz, 4H,CH2 CHCH2O×2), 4.30(dd,J 12.5, 6.9Hz, 2H, H-4), 4.00(dd,J 12.8, 6.2Hz, 1H, H-6a), 3.96-3.89(m,2H,CH2CHCH2×2), 3.85(dd,J 11.6, 5.2Hz, 1H, H-6b), 3.48-3.2 6(m,4H,H-2),3.15(dt,J=26.2,12.1Hz,2H,CHOCH2CO),2.70-2.57(m,3H) ,2.51(dp,J=14.2,5.7,5.0Hz,3H),2.41(dd,J=14.3,4.4Hz,1H),2.30(t,J =7.6Hz,3H),1.63(tdd,J=23.9,18.3,15.4,7.7Hz,8H),1.49-1.17(m,69H) ,1.10(d,J=4.5Hz,9H,CH3(TBDPS)×3),0.91(q,J=4.5,2.7Hz,12H,CH3×4).

[0281] 13C NMR(101MHz, CDCl3)δ173.43,173.18,170.67,154.84(CO×4),136.07,135.7 0,135.59,133.35,133.11(CH2=CH-CH2×2),129.86,129.76,128.13,128.07, 127.88,127.73,126.41,126.12,125.94,125.85(aromatic(Nap),Ph),118.5 4(CH2=CH),101.27(C-1),95.46(CCl3(Troc)),76.49(C-4),75.13,74.48(C- 5),72.59(C-3),71.51,71.41(CH2(Nap)×2),70.02(C-6),68.57,68.51(CH= CHCH2×2),62.38,56.30(C-2),45.42,41.87,39.38,38.46,34.53,34.27,31. 95, 29.77, 29.70, 29.67, 29.65, 29.64, 29.61, 29.59, 29.56, 29.43, 29.39, 29.36, 29.22, 26.77, 25.28, 25.24, 25.18, 25.09, 22.73, 19.35, 14.18 (CH3×7). 31 P NMR (162MHz, CDCl3): δ-2.08.

[0282] HRMS: [M+Na] + Theoretical Components C 110 H 165 Cl3N3O 18 PSiNa + :2004.0592, Measured value:2004.0586.

[0283] The characterization data for compound 6b are shown below.

[0284] 1H NMR(400MHz,CDCl3)δ7.78-7.63(m,8H,(pht),aromatic(Nap)),7.63-7.53(m,4H,(pht),aromatic(Nap)),7.42-7.23(m,11H,(pht),aromatic(Nap)),7.05(s,1H,NH),6.81(s,1H,NH),5.78(ddt,J=16.3,10.8,5.6Hz,1H,H-1),5.63(ddt,J=16.2,10.8,5.6Hz,1H,H-3),5.23(d,J=17.1Hz,1H,CH 2a =CHCH2O),5.17-5.09(m,3H,CH 2b =CHCH2O),5.05(d,J=10.4Hz,1H,CHCH2COO(Lipid)),4.71(ddt,J=19.0,14.8,9.9Hz,6H,CH2(Troc),CH2(Nap)×2),4.29(dt,J=13.3,3.4Hz,4H,CH2=CHCH2O×2),4.19(dd,J=12.8,7.2Hz,2H,H-4),3.93-3.85(m,1H,H-6a),3.85-3.78(m,2H,CH2CHCH2×2),3.73(dd,J=11.6,5.2Hz,1H,H-6b),3.35-3.16(m,3H,CHOCH2CO,H-2),2.63(dd,J=14.1,4.9Hz,1H),2.58-2.45(m,3H),2.45-2.34(m,3H),2.30(dd,J=14.2,4.4Hz,1H),2.19(t,J=7.6Hz,2H),1.49(dd,J=13.3,6.8Hz,8H),1.35-1.08(m,68H),0.98(s,9H,CH3(TBDPS)×3),0.80(t,J=6.7Hz,12H.CH3×4).

[0285] 13C NMR (101MHz, CDCl3) δ173.46,173.21,170.65,154.87(CO×4),135.72,135.61,133.28(CH2=CH-CH2×2),132.93,132.17,129.88,1 28.14,127.89,127.75,126.43,126.13,125.96(aromatic(Nap),Ph),118.57(CH2=CH),101.33(C-1),95.45(CCl3(Troc)),76.86 (C-4),76.51,74.52,74.52(C-5),72.66(C-3),71.54,71.52,71.45(CH2(Nap)×2),70.05(C-6),68.60,61.05(CH=CHCH2×2),56.3 9(C-2),45.46,42.14,42.05,38.79,38.42,34.55,34.55,34.29,31.97,29.69,29.41,26.80,25.22,22.74,19.38,14.18(CH3×7).

[0286] 31 P NMR (162MHz, CDCl3): δ-2.14.

[0287] HRMS: [M+Na] + Theoretical Components C 112 H 169 Cl3N3O 18 PSiNa + :2032.0905, measured value:2032.0736.

[0288] The characterization data for compound 6c are shown below.

[0289] 1 H NMR(400MHz, CDCl3)δ7.87-7.75(m,8H(pht),aromatic(Nap)),7.69(t,J=6.6Hz,4H,(pht),aromatic(Nap)),7.53-7.34(m,11H,(pht),ar omatic(Nap)),7.16(s,1H,NH),6.91(s,1H,NH),5.89(ddd,J=16.9,10.7,5.4Hz,1H,H-1),5.81-5.67(m,1H,H-3),5.33(d,J=17.0Hz,1H,CH 2a=CHCH2O),5.28-5.14(m,3H,CH 2b =CHCH2O),5.08(t,J=9.9Hz,1H,CHCH2COO(Lipid)),4.80-4.58(m,6H,CH2(Troc),CH2(Nap)×2),4.50-4.34(m,4H,CH2=CHCH2O×2),4.28(q,J=11.2,9.9Hz,1H,H-4),3.99(dd,J=13.4,6.9Hz,1H,H-6a),3.95-3.84(m,J=11.7,5.2Hz,3H,CH2CHCH2×2,H-2),3.70(d,J=9.6Hz,1H,H-6b),3.55(d,J=9.6Hz,1H),3.46-3.26(m,3H),3.23-3.02(m,2H),2.73(dd,J=14.2,4.9Hz,1H),2.68-2.54(m,3H),2.49(ddd,J=17.9,9.3,5.8Hz,3H),2.42(d,J=4.2Hz,1H),2.29(t,J=7.5Hz,2H),1.66-1.50(m,6H),1.48-1.17(m,73H),1.08(s,9H,CH3(TBDPS)×3),0.91(t,J=6.6Hz,12H.CH3×4).

[0290] 13C NMR (101MHz, CDCl3) δ173.60,173.21,170.69,154.91(CO×4),135.74,135.63,133.30,133.14(CH2=CH-CH2×2),132.94,129.90,129.81,128.18,1 28.09,127.91,127.77,126.45,126.15,125.98(aromatic(Nap),Ph),118.60(CH2=CH),101.31(C-1),95.48(CCl3(Troc)),76.53(C-4),75.18,74 .53(C-5),73.47,72.68(C-3),71.54,71.44(CH2(Nap)×2),70.10(C-6), 68.60,68.37,62.42,61.15,61.14(CH=CHCH2×2),56.48(C-2),45.45,39. 50,38.81,38.51,34.57,34.30,33.97,31.99,29.76,29.72,29.68,29.61 ,29.43,29.26,26.81,25.31,25.22,25.13,22.76,19.40,14.21(CH3×7).

[0291] 31 P NMR (162MHz, CDCl3): δ-2.28.

[0292] HRMS: [M+Na] + Theoretical Components C 114 H 173 Cl3N3O 18 PSiNa + :2060.1218, measured value:2060.1223.

[0293] 7. Synthesis of compounds 7a, 7b and 7c

[0294]

[0295] (1) Synthesis of compound 7a

[0296] a) Compound 6a (1.26 g, 0.63 mmol, 1 eq.) was dissolved in a mixed solvent of 4 mL MeOH, 2 mL AcOH, and 2 mL DCM. Zinc powder (0.742 g, 11.3 mmol, 18 eq.) was added, and the mixture was reacted at room temperature for 2 hours to complete N-Troc deprotection. The reaction mixture was then filtered using diatomaceous earth as the filter medium. The filtrate was extracted with 40 mL of saturated NaHCO3 aqueous solution and 40 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (gradient elution: MeOH / DCM, v / v = 3:97 to 5:95) to obtain the glucosamine intermediate IM-8a, with the structure shown below:

[0297]

[0298] b) Dissolve IM-8a in 10 mL of dry DCM, and add 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 1.05 mmol, 2.5 eq.) and compound Sub 2-1 (0.76 mmol, 1.2 eq.) and 100 mg of activated [reagent / concentrate]. The molecular sieve was stirred for 5 hours. The resulting reaction system was filtered and the filtrate was collected. The filtrate was concentrated under vacuum. The crude product was purified by column chromatography (gradient elution, MeOH / DCM, v / v = 0:100 to 8:92) to obtain the target compound 7a (1.13 g, two-step yield 82%).

[0299] (2) Synthesis of compounds 7b and 7c

[0300] Compounds 7b and 7c were synthesized using the same method as compound 7a, except that compound 6a was replaced with compounds 6b and 6c respectively, and the compound Sub 2-i was adjusted. The synthesized compounds and their masses and yields are shown in Table 6.

[0301] Table 6

[0302]

[0303]

[0304] The characterization data for compound 7a are shown below.

[0305] 1H NMR(400MHz,CDCl3)δ7.80(t,J=8.2Hz,8H,(pht),aromatic(Nap)),7.69(d,J=6.7Hz,4H,(pht),aromatic(Nap)),7.50-7.37(m,13H,(pht),aromatic(Nap)),7.21(s,1H,NH),6.37(s,1H,NH),5.86(s,1H,H-1),5.73(s,1H,H-3),5.32(d,J=17.2Hz,1H),5.28-5.03(m,7H),4.72(s,4H,CH2(Nap)×2),4.42(dt,J=18.9,8.9Hz,6H,CH2=CHCH2O×2,H-4),4.01(t,J=6.3Hz,1H,H-6a),3.94(d,J=10.4Hz,2H),3.84(dd,J=12.9,6.1Hz,2H),3.75(t,J=7.7Hz,1H,H-6b),3.39(ddt,J=24.0,13.9,7.7Hz,3H),3.25(dd,J=24.1,8.0Hz,2H),2.70-2.53(m,5H),2.45(dd,J=19.9,14.2Hz,3H),2.28(dt,J=15.2,7.7Hz,5H),1.61(s,14H),1.28(s,110H),1.08(s,9H,CH3(TBDPS)×3),0.90(d,J=7.2Hz,18H,CH3×6). 13C NMR (101MHz, CDCl3) δ173.60,173.55,173.40,173.22,171.02,171.00 (CO×6) ,136.22,135.76,135.64,133.29,132.90,132.21(CH2=CHCH2×2),129.86,129 .75,128.07,128.00,127.89,127.75,127.71,126.36,126.03,125.76(aroma tic(Nap),Ph),118.56,118.51(CH2=CH×2),118.51,101.50(C-1),77.28(C-4) ,75.23,75.25(C-5),73.54,73.06(C-3),71.39,70.77(CH2(Nap)×2),70.76( C-6),68.35,62.38,61.41(CH=CHCH2×2),54.71(C-2),42.03,41.75,38.91,34 .54,34.10,31.99,31.95,29.83,29.76,29.73,29.70,29.59,29.44,29.39,29.29,26.78,25.42,25.38,25.33,25.24,25.11,22.76,19.36,14.20(CH3×7).

[0306] 31 P NMR (162MHz, CDCl3): δ-2.17.

[0307] HRMS: [M+Na] + Theoretical Components C 131 H 208 N3O 19 PSiNa + :2210.4840, Measured value:2210.4726.

[0308] The characterization data for compound 7b are shown below.

[0309] 1H NMR(400MHz,CDCl3)δ7.81(q,J=8.3,5.9Hz,8H,(pht),aromatic(Nap)),7.70(t,J=7.2Hz,4H,(pht),aromatic(Nap)),7.55-7.33(m,13H,(pht),aromatic(Nap)),7.26-7.17(m,1H,NH),6.30(d,J=7.6Hz,1H,NH),5.88(ddt,J=16.2,10.6,5.5Hz,1H,H-1),5.74(ddt,J=16.2,10.8,5.5Hz,1H,H-3),5.33(d,J=17.2Hz,1H),5.29-5.04(m,6H),4.81-4.66(m,4H,CH2(Nap)×2),4.52-4.36(m,6H,CH2=CHCH2O×2,H-4),4.34-4.22(m,1H),4.06-3.91(m,3H,H-6a),3.88-3.73(m,3H,H-6b),3.41(ddt,J=22.2,14.9,8.3Hz,3H),3.27(dd,J=14.3,10.6Hz,2H),3.09(d,J=12.2Hz,1H),2.82(dd,J=13.2,4.1Hz,1H),2.71-2.54(m,5H),2.53-2.39(m,3H),2.29(dt,J=14.9,7.6Hz,5H),1.59(tt,J=27.6,13.3Hz,13H),1.28(s,106H),1.08(s,9H,CH3(TBDPS)×3),0.92(t,J=6.7Hz,18H,CH3×6).

[0310] 13C NMR(101MHz, CDCl3)δ173.59,173.51,173.36,173.18,171.05,171.00(CO×6),136.29 ,136.26,135.77,135.64,133.30,132.93,132.21(CH2=CHCH2×2),129.86,129.74,128 .05,127.99,127.92,127.89,127.75,127.70,126.35,126.02,125.98,125.74(aroma tic(Nap),Ph),118.54,118.48(CH2=CH×2),101.50(C-1),76.58(C-4),76.52,75.25(C -5),73.56,73.07(C-3),71.38,70.88(CH2(Nap)×2),70.76(C-6),70.13,68.54,68.4 0,62.37,61.54(CH=CHCH2×2),54.79(C-2),45.33,42.05,41.76,41.57,39.53,38.96, 38.59, 34.54, 34.12, 31.99, 29.81, 29.76, 29.73, 29.70, 29.66, 29.63, 29.53, 29.47, 29.44, 29.32, 29.29, 26.79, 25.44, 25.36, 25.25, 25.12, 22.76, 19.36, 14.20 (CH3×7).

[0311] 31 P NMR (162MHz, CDCl3): δ-2.20.

[0312] HRMS: [M+Na] + Theoretical Components C 135 H 216 N3O 19 PSiNa + :2266.5511, Measured value:2266.5548.

[0313] The characterization data for compound 7c are shown below.

[0314] 1H NMR(400MHz,CDCl3)δ7.80(t,J=7.1Hz,8H,(pht),aromatic(Nap)),7.70(t,J=6.6Hz,5H,(pht),aromatic(Nap)),7.42(dt,J=23.2,8.8Hz,14H,(pht),aromatic(Nap)),7.27-7.15(m,1H,NH),5.88(ddt,J=16.3,10.8,5.6Hz,1H,H-1),5.74(ddt,J=16.3,10.9,5.5Hz,1H,H-3),5.33(d,J=17.1Hz,1H),5.29-5.03(m,6H),4.82-4.67(m,4H,CH2(Nap)×2),4.42(dt,J=17.7,9.2Hz,6H,CH2=CHCH2O×2,H-4),4.30(p,J=6.7Hz,1H),4.02(dd,J=8.1,4.7Hz,1H,H-6a),3.99-3.91(m,2H,H-6b),3.90-3.80(m,2H),3.75(t,J=9.2Hz,1H),3.39(ddt,J=21.4,14.4,6.6Hz,3H),3.25(dt,J=11.9,8.8Hz,2H),3.09(d,J=11.8Hz,1H),2.86-2.75(m,1H),2.71-2.54(m,5H),2.48(dd,J=14.6,4.4Hz,3H),2.37-2.23(m,5H),1.76-1.48(m,14H),1.28(s,132H),1.08(s,9H,CH3(TBDPS)×3)),0.91(t,J=6.7Hz,18H,CH3×6).

[0315] 13C NMR(101MHz, CDCl3)δ173.69,173.65,173.47,173.30,171.13,171.08(CO×6),136.31,135.85,135.72,133 .38,133.01,132.30(CH2=CHCH2×2),129.94,129.83,128.15,128.09,127.97,127.84,127.79,126.44,126. 12,126.06,125.85(aromatic(Nap),Ph),118.65,118.59(CH2=CH×2),101.58(C-1),77.38(C-4),76.66,75 .28(C-5),73.60(C-3),71.47,70.80(CH2(Nap)×2),70.75(C-6),68.64,68.49,62.42,61.27(CH=CHCH2×2),

[0316] 54.65(C-2),45.42,42.11,41.84,39.68,39.54,39.00,38.57,36.72,34.63,34.19,32.08,

[0317] 29.91, 29.83, 29.57, 29.53, 29.38, 26.87, 25.20, 22.85, 19.45, 14.29 (CH3×7).

[0318] 31 P NMR (162MHz, CDCl3): δ-2.18.

[0319] HRMS: [M+Na] + Theoretical Components C 139 H 224 N3O 19 PSiNa + :2322.6092, measured value:2322.6048.

[0320] Synthesis Examples 1 to 3

[0321] The synthetic examples illustrate the preparation of compounds 8a, 8b, and 8c, and their synthetic routes are shown below.

[0322]

[0323] (1) Synthesis of compound 8a

[0324] a) Compound 7a (190 mg, 0.087 mmol, 1 eq.) was dissolved in 1 mL of 3 M HF CH3CN solution and stirred for 3 hours. After the reaction was completed by TLC, the reaction product was extracted with 10 mL of saturated NaHCO3 solution and 10 mL of DCM. The combined organic layers were dried with Na2SO4, filtered, concentrated under vacuum, and the crude product was purified by silica gel column chromatography (gradient elution: MeOH / DCM, v / v = 3:97 to 20:80) to give intermediate IM-9a (76 mg, yield 45%) with the structure shown below.

[0325]

[0326] b) Under a H2 atmosphere, the iridium complex [Ir(cod)(PMe(C6H5)2)2]PF6 (0.0435 mmol, 1 eq.) was dissolved in 2 mL of dry THF and stirred until the solution became clear. The H2 in the system was replaced three times with Ar, and then IM-9a (84.8 mg, 43.5 μmol, 1 eq.) was added. The mixture was stirred at room temperature for 2 hours. Then, 4 mL of water and iodine (271.5 mg, 1.07 mmol) were added at 0 °C and stirred for 1 hour. Excess iodine was quenched with a 5 wt% Na2S2O3 aqueous solution, and the reaction mixture was extracted with 10 mL of saturated NaHCO3 aqueous solution and 10 mL of DCM. The resulting organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by Sephadex LH-20 column chromatography (CH2Cl2 / MeOH, v / v = 1:1) to obtain the free phosphate IM-10a with the structure shown below:

[0327]

[0328] c) Phosphate IM-10a was dissolved in a mixed solvent of 4 mL DCM and 1 mL MeOH, and 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ, 130.5 μmol, 1 eq.) was added. The mixture was sonicated at room temperature for 2 hours. Unreacted DDQ was washed away with CH3CN. The crude product was diluted with 2 mL MeOH and purified by transferring it to a Sephadex LH-20 column (CH2Cl2 / MeOH, v / v = 1:1) to obtain target compound 8a (17.3 mg, b) and c) in a total yield of 25%.

[0329] (2) Synthesis of compounds 8b and 8c

[0330] Compounds 8b and 8c were synthesized using the same method as compound 8a, except that compound 7a was replaced with compounds 7b and 7c, respectively. The synthesized compounds, their masses, and yields are shown in Table 7.

[0331] Table 7

[0332]

[0333]

[0334] The characterization data for compound 8a are shown below.

[0335] 1 H NMR (600MHz, CDCl3) δ5.19(s,4H,H-1,H-3),4.50(q,J=7.5Hz,2H),4.36(dt,J=28.1,8.8Hz,1H,H-6a),4.00-3.96(dt,J=53.9Hz,7 H),3.56-3.52(dt,J=23.3Hz,6H),2.56(s,5H),2.29(t,J=15.1Hz,7H),1.59(s,14H),1.39-1.11(m,109H),0.87(d,J=7.7Hz,20H).

[0336] 13 C NMR (101MHz, CDCl3 / CD3OD) δ173.41,173.13,171.69,170.73,170.04,100.07,72.62,70.21,66.50,60.91,60.76,38. 18,33.75,33.71,33.67,33.58,31.21,31.17,28.94,28.84,28.66,28.60,24.64,24.47,24.35,24.30,21.89,13.01.

[0337] 31 P NMR (162MHz, CDCl3) δ -0.53.

[0338] HRMS:[MH] - Theoretical Components C 87 H 165 N3O 19 P - :1587.1775, measured value:1587.3224.

[0339] The characterization data for compound 8b are shown below.

[0340] 1H NMR (600MHz, CDCl3) δ5.16 (t, J=13.4Hz, 4H, H-1, H-3), 4.53 (q, J=7.5Hz, 2H), 4.34 (dt, J=28.1, 8.8Hz, 1H, H-6a), 4.02-3.91 (dt, J= 65.3Hz,7H),3.57-3.46(dt,7H),2.56(s,5H),2.28(t,J=7.6Hz,7H),1.58(s,10H),1.39-1.09(m,94H),0.88(q,J=4.9,3.1Hz,16H).

[0341] 13 C NMR (101MHz, CDCl3 / CD3OD) δ173.50,173.48,173.18,171.74,170.78,170.10,100.78,72.67,70.26,66.55,60.96,60.81 ,38.23,33.80,33.76,33.72,33.63,31.26,31.22,28.99,28.89,28.71,28.65,24.69,24.52,24.40,24.35,21.94,13.06.

[0342] 31 P NMR (162MHz, CDCl3) δ 0.07.

[0343] HRMS:[MH] - Theoretical Components C 91 H 173 N3O 19 P - :1643.2406, Measured value:1643.2496.

[0344] The characterization data for compound 8c are shown below.

[0345] 1 H NMR (600MHz, CDCl3) δ5.20 (s, 4H, H-1, H-3), 4.51 (q, J=7.5Hz, 2H), 4.36 (dt, J=28.1, 8.8Hz, 1H, H-6a), 4.04-3.93 (dt, 7H ),3.58-3.45(dt,8H),2.57(s,6H),2.29(s,9H),1.60(s,17H),1.28(q,J=7.6,7.0Hz,147H),0.89(q,J=8.4,7.9Hz,20H).

[0346] 13C NMR (101MHz, CDCl3 / CD3OD) δ173.42,173.40,173.11,170.71,170.02,100.70,74.32,72.60,70.66,70.19,66.48,60.88,60. 73,38.15,33.72,33.68,33.65,33.56,31.18,31.15,28.91,28.81,28.64,28.58,24.62,24.44,24.33,24.28,21.86,12.99.

[0347] 31 P NMR (162MHz, CDCl3) δ -0.43.

[0348] HRMS:[MH] - Theoretical Components C 95 H 181 N3O 19 P - :1699.3021, measured value:1699.3738.

[0349] The following examples illustrate vaccines prepared using compounds 8a, 8b, and 8c as adjuvants in the above-described synthesis examples and their preparation methods. In the following text, compound 8a is referred to as GAP210, compound 8b as GAP212, and compound 8c as GAP214.

[0350] Examples 1-3

[0351] Vaccines were prepared by encapsulating ovalbumin antigen (OVA) with GAP210, GAP212, and GAP214 as adjuvants via liposomes. The liposomes were prepared using a thin-film dispersion method, with a molar ratio of DSPC, Chol, OVA, and adjuvants of 5:4:1:1.

[0352] The vaccine preparation method is as follows: First, DSPC, Chol and adjuvant are dissolved in a mixed solvent of DCM and MeOH (v:v = 1:1), and then the solvent is removed by vacuum evaporation to leave a thin lipid membrane; then, phosphate buffered saline (PBS) containing OVA is added to it, and the mixture is sonicated for 3 minutes to obtain the vaccine.

[0353] Comparative Example 1

[0354] The vaccine was prepared according to the method of Example 1, except that the adjuvant GAP210 was replaced with MPLA (CAS:1246298-63-4).

[0355] Comparative Example 2

[0356] The vaccine was prepared according to the method of Example 1, except that the adjuvant GAP210 was not used.

[0357] The main components and dosages of the vaccines in the above embodiments and comparative examples are shown in Table 8.

[0358] Table 8

[0359]

[0360] Test case

[0361] The test examples are used to illustrate the immunological performance and safety of the vaccines prepared in the examples and comparative examples.

[0362] 1. Drug administration to mice

[0363] Before injection, the prepared vaccine was sonicated for 20 minutes. Immunization method: Mice were intraperitoneally injected on days 1, 15, and 29. Blood was collected by tail amputation at the following times: 2 hours and 6 hours after injection on day 1, and on days 14, 28, and 42. The blood was then centrifuged to separate the serum.

[0364] 2. Validation of the signal path

[0365] Use RAW264.7 or DC2.4 at 5×10 per hole 5 Cells were seeded in 96-well plates and pre-incubated for 1 hour with or without TAK-242 (DMSO, 5 μM). Then, they were incubated with different vaccines at 37°C for 12 hours. The supernatant was collected by centrifugation and the concentration of IL-6 cytokine was measured.

[0366] The results are as follows Figure 1 As shown in the figure, "+" indicates pre-incubation, and "-" indicates no pre-incubation. Figure 1 It can be seen that the vaccines in Examples 1-3, when used as adjuvants with GAP210, GAP212, and GAP214, can stimulate DC2.4 cells (Figure A) and RAW264.7 cells (Figure B) to secrete IL-6. Furthermore, after pre-incubation with TAK-242 to inhibit the TLR4 signaling pathway, the secretion of IL-6 by GAP210, GAP212, and GAP214 was significantly reduced, reflecting that GAP210, GAP212, and GAP214 activate RAW264.7 or DC2.4 cells through the TLR4 pathway.

[0367] 3. Activation with DC2.4 or RAW264.7

[0368] 1) Use DC2.4 or RAW264.7 at 5×10 mm per hole. 5Cells were seeded in 96-well plates, with each cell type seeded in triplicate. 500 μL of the corresponding cell culture medium and vaccine were added to each well, and the cells were incubated at 37°C for 20 hours. The supernatant was aspirated using a pipette to measure cytokines. For DC2.4 cells, 0.5 mL of trypsin was added and allowed to stand for 5 minutes, followed by 0.5 mL of DC2.4 cell culture medium. The DC2.4 cells were then pipetted to obtain a DC2.4 cell suspension. For RAW264.7 cells, 0.5 mL of RAW264.7 culture medium was added directly, and the cells were pipetted to obtain a RAW264.7 cell suspension.

[0369] 2) Transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 2500 rpm for 5 minutes, discard the supernatant, add staining solution (phosphate buffer containing 1 vol% fetal bovine serum (FBS) and 1 wt% bovine serum albumin (BSA)) to the remaining cell pellet, wash once, centrifuge and discard the supernatant.

[0370] Dilute 1 μL of anti-CD11c (0.5 mg / mL), 1 μL of anti-CD80 (0.2 mg / mL), and 1 μL of anti-CD86 (0.2 mg / mL) antibody into 100 μL of staining solution. Add the antibody-containing staining solution to the cell pellet, 100 μL per well. Mix well with a pipette and incubate on ice for 30 minutes. Centrifuge to remove the supernatant. Add 1 mL of staining solution to the cell pellet to wash it twice.

[0371] 3) After washing, the cell pellet was resuspended in PBS (phosphate buffer) containing 0.1 wt% BSA and read on a flow cytometer.

[0372] The results are as follows Figure 2 and Figure 3 As shown, GAP214 significantly increased the secretion of IL-6 and IL-12p70 cytokines by DC2.4 or RAW264.7, and the levels were higher than those of MPLA. Simultaneously, GAP210, GAP212, and GAP214 induced slightly higher expression of CD80 and CD86 in DC2.4 than in MPLA, and induced similar levels of CD80 and CD86 in RAW264.7 as in MPLA, indicating that these three GAP molecules successfully promoted the activation of DC2.4 and RAW264.7 in vitro.

[0373] 4. Cytokine testing

[0374] The concentrations of cytokines (IL-6, IL-12p70, IFN-γ) in serum were detected using an ELISA kit. The specific steps are as follows:

[0375] 1) Coating: Add 100 μL of diluted capture antibody (40 μL capture antibody diluted in 10 mL coating buffer) to each well of a 96-well plate and incubate overnight at 4°C. Wash three times with 250 μL of PBST (PBS containing 0.05 vol% Tween-20). The coating buffer was prepared as follows: Dissolve 8.4 g NaHCO3 and 3.56 g Na2CO3 in deionized water, bring the volume to 1 L, and adjust the pH to 9.5.

[0376] 2) Blocking: Add 200 μL of dilution buffer (PBS containing 10 vol% FBS) to each well of the 96-well plate and incubate at room temperature for 1 hour. Wash each well three times with 250 μL of PBST.

[0377] 3) Add serum: Add 100 μL of diluted standard (diluted with PBS buffer containing 10 vol% FBS) or serum to each well of a 96-well plate, incubate at room temperature for 1 hour, and then add 250 μL of PBST to each well for washing 5 times.

[0378] 4) Add detection antibody and HRP enzyme: Add 100 μL of diluted detection antibody and HRP enzyme (20 μL detection antibody and 40 μL HRP enzyme diluted in 10 mL diluent) to each well of the 96-well plate, incubate at room temperature for 1 hour, and then add 250 μL PBST to each well for washing 7 times.

[0379] 5) Color development: Add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color development solution to each well of the 96-well plate and incubate at room temperature in the dark for 30 minutes.

[0380] 6) Termination: Add 50 μL of sulfuric acid (2M) to each well of the 96-well plate and measure the absorbance at 450 nm using an ELISA reader.

[0381] The results are as follows Figure 4 As shown in the figures, Figure A represents the serum IL-6 concentration in mice 2 hours after immunization, while Figures B and C represent the serum IL-12p70 and IFN-γ concentrations 6 hours after immunization, respectively. The figures indicate that GAP210, GAP212, and GAP214 induce IL-6 levels lower than MPLA, while IFN-γ and IL-12p70 levels are significantly higher than MPLA. Furthermore, IL-6 is a Th2-biased cytokine, while IFN-γ and IL-12p70 are Th1-biased cytokines. Therefore, the three GAP molecules are more inclined to promote Th1-type immune responses, and all three molecules are Th1-type immune agonists.

[0382] 5. Antibody titer testing

[0383] 1) Coating: Add 100 μL of diluted OVA (20 μL of 1 μg / mL OVA solution diluted in 10 mL of coating buffer) to each well of a 96-well plate, incubate overnight at 4°C, and then wash each well four times with 200 μL of PBST. The coating buffer was prepared as follows: Dissolve 1.59 g Na₂CO₃ and 2.93 g NaHCO₃ in deionized water, bring the volume to 1 L, and adjust the pH to 9.5.

[0384] 2) Blocking: Add 100 μL of 1 wt% casein PBS solution to each well of a 96-well plate and incubate at 37°C for 1 hour. Wash each well three times with 200 μL of PBST.

[0385] 3) Add serum: Add 100 μL of serum diluted with PBS containing 1 wt% casein to each well of a 96-well plate, incubate at 37°C for 1 h, and then add 200 μL of PBST to each well for washing 3 times.

[0386] 4) Add enzyme-labeled secondary antibody: Add 100 μL of enzyme-labeled secondary antibody IgG, IgM, IgG1, IgG2a, IgG2b, and IgG3 diluted in PBST to each well of a 96-well plate (3 μL of the corresponding enzyme-labeled secondary antibody diluted in 10 mL of PBST), incubate at 37°C for 1 hour, and then add 200 μL of PBST to each well for washing 3 times.

[0387] 5) Color development: Add 100 μL of color development solution to each well of the 96-well plate and incubate at room temperature in the dark for 5 minutes.

[0388] Colorimetric solution: 9.5 mL substrate buffer solution + 0.5 mL solution A + 32 μL solution B.

[0389] The substrate buffer solution was prepared by dissolving 18.66 g of citric acid monohydrate (C6H8O7·H2O) and 36.82 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·H2O) in deionized water, bringing the volume to 2 L, and adjusting the pH to 5.

[0390] Solution A is prepared by dissolving 160 mg TMB and 200 mg β-cyclodextrin in 80 mL of dimethyl sulfoxide (DMSO) and 20 mL of ethanol.

[0391] Solution B is prepared by dissolving 300 mg of hydrogen peroxide urea in deionized water and bringing the volume to 10 mL.

[0392] 6) Termination: Add 50 μL of 2M sulfuric acid to each well of the 96-well plate and measure the absorbance at 450 nm using an ELISA reader.

[0393] Figure 5The results showed antibody titers after three immunizations (days 14, 28, and 42). The results indicated that the antibody-inducing effect of the candidate vaccines (with GAP210, GAP212, and GAP214 as adjuvants, respectively) in mice increased with the growth of the secondary acyl chain, and the overall effect was comparable to or better than that of MPLA, indicating that all three GAP molecules could effectively stimulate humoral immunity in mice.

[0394] Figure 6 The antibody subtype results were obtained after the third immunization (day 42). The dominant IgG subtype characteristic in all groups was IgG1. The levels of all IgG subtypes induced in the three candidate vaccine groups were comparable to those in the MPLA control group. These results suggest that the candidate vaccines induced a broader and more diverse IgG subtype response, potentially enhancing protection against viral infection.

[0395] 6. In vivo DC activation

[0396] 1) Two days after the completion of the second immunization (day 28), the mice were euthanized by cervical dislocation. The spleen and draining lymph nodes of the mice were removed and placed in 2 mL PBS. They were then ground with a cell sieve, centrifuged at 2500 rpm for 2 minutes, and the supernatant was discarded.

[0397] 2) Add 2 mL of erythrocyte lysis buffer to the splenic cells and draining lymph node cells pellet, repeatedly aspirate and mix with a pipette, let stand for 5 minutes, centrifuge at 2500 rpm for 5 minutes, discard the supernatant, add 1 mL of PBS to wash once, centrifuge and discard the supernatant.

[0398] 3) Dilute 1 μL of anti-CD11c (0.5 mg / mL), 2 μL of anti-CD80 (0.2 mg / mL), and 0.3 μL of anti-CD86 (0.2 mg / mL) into 50 μL of staining solution (PBS containing 1 vol% FBS and 1 wt% BSA), then add the solution to a centrifuge tube containing the cell pellet. Mix well with a pipette, incubate on ice for 30 minutes, and then wash twice with 1 mL of staining solution.

[0399] 4) Add 400 μL of staining solution to the centrifuge tube and resuspend it, then read the values ​​on a flow cytometer.

[0400] The results are as follows Figure 7 As shown in the figure, all three GAP molecules can effectively induce the activation of DC cells in the spleen and draining lymph nodes of mice.

[0401] 7. Intracellular cytokine staining test

[0402] 1) Isolation of mouse spleen: After immunization, mice were euthanized by cervical dislocation, and the spleen was removed and placed in a 40μm cell sieve. 2mL of PBS was added, and the spleen was ground using a 2.5mL syringe plunger. The filtrate was collected, centrifuged at 2500rpm for 2 minutes, and the supernatant was discarded.

[0403] 2) Add 2 mL of red blood cell lysis buffer, pipette repeatedly to mix, let stand for 5 minutes, centrifuge at 2500 rpm for 5 minutes, discard the supernatant, add 1 mL of 1640 medium to wash once, and then add 1 mL of 1640 complete medium (containing 10 vol% FBS + 1 vol% antibiotics) to resuspend the cells to obtain spleen cell suspension.

[0404] 3) Add 200 μL of spleen cell suspension to a 24-well plate, then add 400 μL of 1640 complete culture medium, and then add 10 μL of LOVA solution (1 μg / μL). Incubate at 37°C for 3 h. Add 2 μL of brevidin A (BFA) and incubate at 37°C for 21 h.

[0405] 4) Transfer the cell suspension from the 24-well plate to a 1.5 mL centrifuge tube, centrifuge at 2500 rpm for 1 minute, discard the supernatant, and wash once with 1 mL of staining solution (PBS containing 1 wt% BSA and 1 vol% FBS).

[0406] 5) Dilute 0.8 μL of anti-CD3 antibody, 0.8 μL of anti-CD4 antibody, and 0.8 μL of anti-CD8 antibody in 500 μL of staining solution, then add the solution to a centrifuge tube, mix well with a pipette, and incubate the spleen cells on ice for 30 minutes. Add 1 mL of staining solution to the spleen cells and wash them twice.

[0407] 6) Add 100 μL of fixation and dialysis buffer, incubate at 4°C for 20 minutes, centrifuge at 2500 rpm for 1 minute, discard the supernatant, and wash once with 1 mL of staining solution.

[0408] 7) Dilute 1 μL of anti-IFN-γ antibody (0.2 mg / mL) + 1 μL of anti-TNF-α antibody (0.5 mg / mL) into 250 μL of membrane permeabilization buffer, add it to a centrifuge tube, mix well with a pipette, and incubate at 4°C for 30 minutes. Add 400 μL of membrane permeabilization buffer to the spleen cells to wash them once.

[0409] 8) Add 400 μL of staining solution to the centrifuge tube to resuspend the cells, and read the values ​​using a flow cytometer.

[0410] The results are as follows Figure 9As shown, compared to MPLA, these three GAP molecules induce more CD4 and CD8 cells to produce IFN-γ and TNF-α, indicating that these three GAP molecules have a stronger T cell-dependent immune response.

[0411] 8. Central Memory T Cell Test

[0412] 1) After euthanizing the mice by cervical dislocation, remove the spleen and place it in a 40μm cell strainer. Add 2mL of PBS and grind the spleen using a 2.5mL syringe plunger. Collect the filtrate, centrifuge at 2500rpm for 2 minutes, and discard the supernatant.

[0413] 2) Add 2 mL of erythrocyte lysis buffer to the centrifuge tube containing spleen cell pellet, pipette repeatedly to mix, let stand for 5 minutes, centrifuge at 2500 rpm for 5 minutes, discard the supernatant, add 1 mL of PBS to wash once, centrifuge and discard the supernatant, then add 1 mL of 1640 complete medium (1640 medium containing 10 vol% FBS + 1 vol% penicillin antibody) to resuspend the cells.

[0414] 3) Dilute 1 μL of anti-CD3, 1 μL of anti-CD4, 1 μL of anti-CD8, 1 μL of anti-CD44 (0.2 mg / mL), and 1 μL of anti-CD62L (0.5 mg / mL) antibody into 100 μL of staining solution (PBS containing 1 vol% FBS and 1 wt% BSA), then add the solution to a centrifuge tube, mix well with a pipette, and incubate on ice for 30 minutes. Add 1 mL of staining solution to the spleen cells and wash them twice.

[0415] 4) Add 400 μL of staining solution to the centrifuge tube and resuspend it, then read the values ​​on a flow cytometer.

[0416] like Figure 9 As shown, in the spleen, compared with MPLA, these three GAP molecules induced a higher proportion of CD4+ Tcm cells and CD8+ Tcm cells, indicating that these three GAP molecules can generate high levels of central memory T cells, reflecting their good long-term T cell immunity.

[0417] 9. Security Testing

[0418] (1) Safety assessment of adjuvants at different concentrations: 9 μg, 45 μg, and 90 μg of each of the four adjuvants (MPLA, three GAP molecules, etc.) were dissolved in 0.9 μL of DMSO to prepare DMSO solutions. Each DMSO solution was then diluted to 60 μL with 59.1 μL of PBS buffer to obtain the test solution. B16F10 and HEK293 cells were cultured at 2.5 × 10⁻⁶ cells per well. 6Cells were seeded in 96-well plates (containing 280 μL of culture medium), and 20 μL of the test solution was added. Three wells were prepared for each cell type and concentration. Cells were incubated at 37°C for 24 hours. After incubation, the culture medium was aspirated and 50 μL of MTT solution (5 mg / L) was added. The plates were then incubated at 37°C for another 8 hours. The supernatant was removed by centrifugation, and 100 μL of DMSO was added to each well to dissolve the methylenezin crystals. The absorbance of each well was measured at 490 nm using a microplate reader. Results are shown below. Figure 10 A and Figure 10 B.

[0419] (2) Vaccine safety testing: Mice immunized on day 42 were euthanized by cervical dislocation, and vital organs (heart, liver, spleen, lungs, kidneys, and brain) were removed and preserved in a universal tissue fixative for tissue section testing. Results are shown below. Figure 10 C.

[0420] like Figure 10 As shown, the adjuvants in all groups, at different concentrations, had no significant effect on the cell viability of HEK293 and B16F10 cells. Furthermore, all groups exhibited normal tissue characteristics, with no abnormal cell death, tissue reactions, or inflammatory infiltration. This reflects the good safety profile of the three GAP molecules.

[0421] In summary, GAP210, GAP212, and GAP214, as TLR4 agonists, possess simpler structures compared to MPLA and exhibit potent immunostimulatory properties both in vitro and in vivo. Experimental results demonstrate that these analogues exhibit comparable or even superior immunogenicity to MPLA in both innate and adaptive immunity. Furthermore, the three candidate vaccines in Examples 1-3 induced strong cellular immune responses (demonstrated by more effective antigen-specific T cell activation). In addition, they significantly enhanced the activation of splenic CD4+ Tcm and CD8+ Tcm. Moreover, in vitro and in vivo safety assessments indicated that these candidate vaccines did not cause any identifiable toxicity. In conclusion, the TLR4 agonists provided by this invention are characterized by simple structure, strong immunostimulatory activity, and high safety, showing promising application potential in vaccine adjuvant and immunomodulator development, and possessing broad clinical application prospects.

Claims

1. A TLR4 agonist, characterized in that, The TLR4 agonist is a compound of the structure shown in Formula 1 or a pharmaceutically acceptable salt thereof: In Formula 1, the structure of R1 is shown as follows: The structure of R2 is shown as follows: wherein, represents a bond, m is an integer of 0 to 25, n is 0 or 1, o is an integer of 0 to 10, p is an integer of 1 to 25, and q is an integer of 0 to 25. X and Y are the same or different, and each is independently selected from -O-, -NH- or -CH2-; L is -CH2- or -C(O)-; Z is an oxygen atom or a sulfur atom; E is -O- or -NH-; One of A1 and A2 is -CH2OH, and the other is -CH2NH2, -COOH, -CH2OH, -CH2SH, -CF3, -CHF2, -CH2F or -CH2OCH3.

2. The TLR4 agonist of claim 1, characterized in that, m is an integer from 2 to 21, n is 1, o is an integer from 0 to 5, and p is an integer from 1 to 15; Preferably, X and Y are both -CH2-, and L is -C(O)-; Preferably, R1 is selected from the group consisting of the structures shown as follows:

3. The TLR4 agonist of claim 1, wherein, A1 and A2 are both -CH2OH; Preferably, R2 is a group of the structure shown as follows:

4. The TLR4 agonist of claim 1, wherein, q is an integer from 2 to 22.

5. The TLR4 agonist of claim 1, wherein, The TLR4 agonist is selected from at least one of the following compounds or a pharmaceutically acceptable salt thereof:

6. Use of the TLR4 agonist of any one of claims 1-5 in the preparation of an immunomodulatory pharmaceutical preparation, preferably the immunomodulatory pharmaceutical preparation is at least one of a vaccine, a cell activator for in vitro activation, expansion and differentiation of immune cells, an immunotargeting drug, a pharmaceutical preparation combined or conjugated with a chemotherapeutic drug, an immunomodulator for allergic diseases, a drug for nervous system diseases, a cardiovascular protection preparation, an anti-bacterial and anti-viral infection preparation, and an autoimmune disease preparation.

7. Use according to claim 6, characterized in that, The vaccine is a semi-synthetic vaccine, a fully synthetic saccharide vaccine, a peptide vaccine, a saccharide-peptide vaccine, a subunit vaccine, a nucleic acid vaccine, a virus-like particle vaccine, an inactivated vaccine or a attenuated vaccine; Preferably, the immune cells are DC cells, NK cells, NKT cells, macrophages, CAR-T cells, neutrophils, eosinophils, basophils or mast cells.

8. A vaccine composition, characterized in that, The vaccine composition comprises an antigen, an adjuvant and a pharmaceutical carrier, wherein the adjuvant comprises the TLR4 agonist of any one of claims 1-5.

9. The vaccine composition of claim 8, wherein, The antigen is a protein antigen or a polypeptide antigen; Preferably, the pharmaceutical carrier is a microcapsule, a microsphere, a nanoparticle or a liposome.

10. The vaccine composition of claim 9, wherein, The pharmaceutical carrier is a liposome, and the protein antigen is ovalbumin; Preferably, the liposome is prepared by a film dispersion method using distearoylphosphatidylcholine and cholesterol; More preferably, the molar ratio of the adjuvant, the antigen, distearoylphosphatidylcholine and cholesterol is (1-2) : 1 : (4-6) : (3-5).

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