Globoh hexasaccharide, methods of making, linker compounds and protein conjugates

Abstract

The present application relates to the field of medicine biotechnology, and in particular to GloboH hexasaccharide, its preparation method, linker compound and protein conjugate. The present application provides a compound shown in formula I or its stereoisomer, solvate, hydrate, prodrug, stable isotopic derivative and pharmaceutically acceptable salt: the GloboH hexasaccharide provided by the embodiment of the present application is introduced with diethyleneglycol amine at the end group anomeric position, which can improve the reaction efficiency of subsequent amide condensation, the monoester formed with activated ester linker has higher stability, which is beneficial to subsequent glycoprotein coupling reaction and improves the reaction efficiency.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and more specifically, to GloboH hexasaccharide, its preparation method, linker compounds, and protein conjugates. Background Technology

[0002] Abnormal tumor glycosylation is a key marker in cancer biology. The overexpression of tumor-associated carbohydrate antigens (TACAs) on the surface of tumor cells contrasts sharply with their absence or low expression in normal cells, making TACA research a current focus for therapeutic antibodies and vaccines against tumors. TACA globular serine polysaccharide (Globo H) is a structurally complex hexasaccharide compound. Globo H antigen is overexpressed in various epithelial cell tumors (such as breast, ovarian, colon, stomach, pancreas, and lung cancer), while its expression is extremely low in normal cells. It primarily exerts its pro-cancer effects through tumor immunosuppression, microangiogenesis, and tumor protein kinases. Developing therapeutic vaccines targeting Globo H, by activating the human immune system, particularly activating specific antibodies and cellular immune responses against Globo H, holds promise for blocking these pro-cancer mechanisms. However, tumor-associated glycoantigens have small molecular weights, lack size and cluster effects, and possess strong immunogenicity. They can only stimulate B cells to produce low-affinity IgM antibodies and have no immune memory. Therefore, they need to be coupled with carrier proteins to form glycoconjugates in order to induce the body to produce durable and enhanced IgG antibodies.

[0003] The existing glycoprotein conjugation strategies used in GloboH have several drawbacks: 1) Reductive amination: These require stringent reaction conditions, alter the basic antigen structure, and are accompanied by overreduction and other side effects; 2) Sulfide addition: Sulfide bonds are unstable in vivo and easily reduced or oxidized, affecting the long-term stability of the conjugated product; 3) Disulfide bond method: Disulfide bonds are highly reducing and risk breaking in vivo; 4) Cycloaddition method: Cyclic linker units may present immunogenicity issues. These problems with the conjugation methods affect glycoprotein binding efficiency, easily leading to other side effects or immune side effects, thus limiting the immunogenicity of the existing GloboH vaccine.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide GloboH hexasaccharide, its preparation method, linker compound, and protein conjugate. The GloboH hexasaccharide provided in this invention introduces diethylene glycolamine at the anomeric position of the terminal group, which can significantly improve the efficiency of subsequent amide condensation reactions and result in higher stability of the monoester formed with the activated ester linker, thus facilitating subsequent glycoprotein coupling reactions and improving reaction efficiency.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a compound of Formula I or a stereoisomer thereof, a solvate, a hydrate, a prodrug, a stable isotopic derivative, and a pharmaceutically acceptable salt thereof:

[0008]

[0009] Secondly, the present invention provides a method for preparing the compound of Formula I as described in the foregoing embodiments, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives, and pharmaceutically acceptable salts, comprising: synthesizing according to the following synthetic route:

[0010]

[0011] In an optional embodiment, the step of forming compound D includes: mixing compound A, compound C, an imine and a Lewis acid and reacting them;

[0012] Wherein, the molar ratio of compound A to compound C is (1.2-1.5):1, and the molar ratio of compound C, the imine substance and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

[0013] In an optional embodiment, the step of forming compound F includes: reacting a mixture of compound D, compound E, an imine, and a Lewis acid;

[0014] Wherein, the molar ratio of compound E to compound D is (1.2-1.5):1, and the molar ratio of compound D, the imine substance and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

[0015] In an optional embodiment, the step of forming compound G includes: mixing and stirring compound F, tetrahydrofuran and acid, followed by a primary post-treatment to form a first residue;

[0016] Next, the first residue, tetrahydrofuran, and alkaline compound are mixed and stirred, and then subjected to a second post-treatment to form the second residue;

[0017] Then, the second residue is mixed with an alcohol solvent, an organic base and an acid anhydride and stirred, and then subjected to three post-treatments to form a third residue;

[0018] Next, the third residue, alcohol solvent, and palladium on carbon are mixed and reacted.

[0019] In an optional embodiment, the step of forming compound I includes: mixing compound G, GDP-Fuc, alkaline phosphatase and transferase and reacting them;

[0020] The molar ratio of compound G to GDP-Fuc is 1:(1.2-1.5).

[0021] Thirdly, the present invention provides a linker compound comprising a bis-activated ester linker and a compound of Formula I as described in the foregoing embodiments, or a stereoisomer thereof, solvate, hydrate, prodrug, stable isotope derivative, or pharmaceutically acceptable salt thereof, wherein the bis-activated ester linker is condensed with an aminoamide of the compound of Formula I.

[0022] In an optional embodiment, the dual-activated ester linker is selected from compounds shown in the following structural formulas:

[0023]

[0024] The linker compound is selected from compounds shown in the following structural formulas:

[0025]

[0026] Fourthly, the present invention provides a protein conjugate comprising a protein and the linker compound described in the foregoing embodiments, wherein the protein and the linker compound are coupled via amide condensation.

[0027] In an optional embodiment, the protein conjugate has the following structural formula:

[0028]

[0029] The present invention has the following beneficial effects: The embodiments of the present invention provide a new Globo H hexasaccharide, which introduces diethylene glycolamine at the anomeric position (1 position) of the Globo H hexasaccharide. The monoester formed by this compound and the activated ester linker has higher stability, which is beneficial to the subsequent glycoprotein coupling reaction and improves the reaction efficiency. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The 1H NMR spectrum of the compound of Formula 1 provided in Example 1 of this invention;

[0032] Figure 2 The carbon NMR spectrum of the compound of Formula 1 provided in Example 1 of this invention;

[0033] Figure 3 The spectrum of MALDI-TOF provided in Embodiment 1 of the present invention;

[0034] Figure 4 The spectrum of MALDI-TOF provided in Embodiment 2 of the present invention;

[0035] Figure 5 The image shows the detection results provided for an experimental example of this invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0037] Existing glycoprotein coupling methods have problems, affecting glycoprotein binding efficiency and easily causing other side effects or immune side effects, thus limiting the immunogenicity of existing GloboH vaccines. Therefore, this invention employs amide condensation to achieve glycoprotein coupling. Amide condensation for glycoprotein coupling has advantages such as fewer side effects, strong biocompatibility, and no additional immunogenicity. However, amide condensation suffers from problems such as low stability of the linker in the coupling reaction and difficulty in binding. Therefore, this invention modifies GloboH to obtain a new GloboH hexasaccharide to solve the aforementioned technical problems.

[0038] In a first aspect, the present invention provides a compound of Formula I or a stereoisomer thereof, a solvate, a hydrate, a prodrug, a stable isotopic derivative, and a pharmaceutically acceptable salt thereof:

[0039]

[0040] Secondly, the present invention provides a method for preparing the compound of Formula I as described in the foregoing embodiments, or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives, and pharmaceutically acceptable salts, comprising: synthesizing according to the following synthetic route:

[0041]

[0042] The specific process is as follows:

[0043] (1) Synthesis of compound A:

[0044] Perform the synthesis according to the following synthesis path:

[0045] Dissolve p-methylphenylthio-β-D-galactopyranoside in a chlorinated C1-C3 alkane and a pyridine solvent (e.g., dichloromethane / pyridine (1:1, v / v)). Add 1-1.1 equivalents of tert-butylsilylsulfonic acid (e.g., di-tert-butylsilylbis(trifluoromethanesulfonic acid)) and stir at room temperature (e.g., 20-30°C) for 2-4 hours. Cool (e.g., to -20°C), add 1-1.1 equivalents of an ester compound (e.g., 9-fluorenyl chloroformate), and stir. Then add 1-1.2 equivalents of an acyl chloride-substituted benzene (e.g., benzoyl chloride), and stir the reaction at room temperature. After the reaction is complete, perform post-treatment.

[0046] (2) Synthesize compound B as shown in the following structural formula:

[0047] Compound A is dissolved in a solvent (e.g., DMF), and 1.2–1.5 equivalents of an acetal (e.g., benzaldehyde dimethyl acetal) and 0.1–0.4 equivalents of a Lewis acid (camphor sulfonic acid) are added. The mixture is stirred overnight at 35–45 °C, then the reaction is quenched and concentrated. The residue is then dissolved in a solvent, and 5–6 equivalents of a halobenzyl (e.g., benzyl bromide) and 5–6 equivalents of a hydride (sodium hydrogen hydride) are added at low temperature (e.g., below 0 °C). The mixture is stirred at room temperature, and post-treatment is performed after the reaction is complete.

[0048] (3) Synthesis of compound C:

[0049]

[0050] Compound B and a solvent (e.g., tetrahydrofuran) were added to 3.5-4.5 equivalents of a borane compound (e.g., borane-trimethylamine complex), and 5.5-6.5 equivalents of aluminum trichloride and 1.5-2.5 equivalents of water were added under ice bath conditions. The mixture was stirred at room temperature, and post-treatment was performed after the reaction was completed.

[0051] (4) Synthesize compound D;

[0052] Perform the synthesis according to the following synthesis path:

[0053]

[0054] Compound A and Compound C are dissolved in a solvent (e.g., toluene) and stirred. Then, the mixture is cooled and an imine (e.g., N-iodosuccinimide) and a Lewis acid (e.g., tert-butyldimethylsilyltrifluoromethanesulfonate) are added and reacted at low temperature. The reaction is then quenched using an alcohol solvent and an organic base (e.g., pyridine). Finally, an amine is added and the mixture is stirred at room temperature. After the reaction is complete, post-treatment is performed.

[0055] Wherein, the molar ratio of compound A to compound C is (1.2-1.5):1, and the molar ratio of compound C, the imine substance and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

[0056] (5) Synthesis of compound E:

[0057] Perform the synthesis according to the following synthesis path:

[0058] In this process, 1.2-1.5 equivalents of an ester donor (e.g., trichloroimine ester) and 1 equivalent of a glycoside acceptor (e.g., thioglycoside) are dissolved in a solvent (e.g., dichloromethane / toluene (1:1, v / v)), a molecular sieve is added and stirred, and after cooling (e.g., cooling to 0°C), 0.2-0.4 equivalents of a Lewis acid (e.g., trifluoromethanesulfonic acid) are added. Then, the reaction is stirred at low temperature, and post-processing is performed after the reaction is completed.

[0059] (6) Synthesis of compound F:

[0060] Perform the synthesis according to the following synthesis path:

[0061]

[0062] Dissolve 1.2-1.5 equivalents of compound E and 1 equivalent of compound D in a solvent (e.g., dichloromethane / toluene (1:1, v / v)), add molecular sieves and stir for 30 minutes, cool to -50°C, add (2.5-3.5) equivalents of N-iodosuccinimide and (0.3-0.5) equivalents of trifluoromethanesulfonic acid, and stir at -50°C for 3 hours.

[0063] (7) Synthesis of compound G:

[0064] Perform the synthesis according to the following synthesis path:

[0065]

[0066] Compound F, tetrahydrofuran, and an acid (e.g., pyridine hydrofluoric acid) were mixed and stirred overnight at room temperature. The mixture was then diluted with an ether solvent (e.g., diethyl ether), washed, and dried to form the first residue. Next, the first residue was dissolved in a mixture of tetrahydrofuran and a basic compound and stirred overnight at room temperature. The solution was then adjusted to neutral, followed by two post-treatment processes: filtration, extraction, and concentration, to form the second residue. Then, the second residue was mixed with an alcohol solvent, an organic base (e.g., triethylamine), and an acid anhydride (e.g., acetic anhydride) and stirred at room temperature. This mixture was then subjected to three post-treatment processes, including filtration and concentration, to form the third residue. Finally, the third residue was dissolved in an alcohol solvent / water, and palladium on carbon was added to initiate a debenzylation reaction. After the reaction was complete, further post-treatment was performed.

[0067] (8) Synthesize the compound shown in Formula I:

[0068] One equivalent of compound G, 1.2-1.5 equivalents of guanosine diphosphate-L-fucose (GDP-Fuc), and 2 equivalents of magnesium chloride were dissolved in a buffer solution. Alkaline phosphatase and transferase were added and the mixture was reacted. After the reaction was completed, post-treatment was performed.

[0069] It should be noted that the post-processing described in steps (1) to (8) above includes, but is not limited to, centrifugation, drying, washing, concentration and chromatographic purification.

[0070] Thirdly, the present invention provides a linker compound comprising a bis-activated ester linker and a compound of Formula I as described in the foregoing embodiments, or a stereoisomer thereof, solvate, hydrate, prodrug, stable isotope derivative, or pharmaceutically acceptable salt thereof, wherein the bis-activated ester linker is condensed with an aminoamide of the compound of Formula I.

[0071] For example, the dual-activated ester linker is selected from compounds with the following structural formulas: The structural formula of the linker compound formed using this dual-activated ester linker is shown below:

[0072]

[0073] Fourthly, the present invention provides a protein conjugate comprising a protein and the linker compound described in the foregoing embodiments, wherein the protein and the linker compound are condensed via an amide condensation. This protein conjugate can be coupled with a vaccine adjuvant to form a novel tumor vaccine, and the resulting vaccine exhibits excellent antibody performance.

[0074] Specifically, the structural formula of the protein conjugate in this embodiment of the invention is shown below:

[0075]

[0076] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0077] Example 1

[0078] This embodiment provides a compound of Formula I, the structural formula of which is shown below:

[0079]

[0080] This embodiment also provides a method for preparing the compound shown in Formula I above, including:

[0081] (1) Synthesis of compound A:

[0082] Perform the synthesis according to the following synthesis path:

[0083] p-Methylphenylthio-β-D-galactopyranoside was dissolved in dichloromethane / pyridine (1:1, v / v), and 1.05 equivalents of di-tert-butylsilylbis(trifluoromethanesulfonic acid) were added. The mixture was stirred at room temperature for 3 hours. After cooling to -20°C, 1.05 equivalents of fluorenyl chloroformate were added, and the mixture was stirred for 1 hour. Then, 1.2 equivalents of benzoyl chloride were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, methanol was added to quench the reaction, and the mixture was concentrated. The residue was dissolved in ethyl acetate, washed with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate). Compound A was finally obtained in 89% yield.

[0084] The characterization data of compound A are as follows: 1 H NMR (600MHz, CDCl3) δ8.10 (dd, J=8.4, 1.4Hz, 1H), 7.76-7.69 (m, 1H), 7.60-7.56 (m, 1H), 7.54 (dd, J= 7.4,1.1Hz,1H),7.50-7.44(m,2H),7.41-7.32(m,4H),7.25-7.21(m,1H),7.11(td,J=7.5,1.1Hz,1H ),7.08(d,J=8.0Hz,1H),5.85(t,J=10.0Hz,1H),4.91(dd,J=9.8,3.1Hz,1H),4.87-4.80(m,2H),4.3 6-4.26(m,4H),4.19(t,J=7.7Hz,1H),3.56(d,J=2.2Hz,1H),2.33(s,3H),1.19(s,9H),1.05(s,9H). 13C NMR (150MHz, CDCl3) δ165.42,154.55,143.40,143.17,141.25,138.25,1 33.40,133.26,130.08,130.07,129.78,129.73,128.56,127.90,127.26 ,127.23,125.34,125.29,120.03,88.00,78.49,74.96,70.53,70.46,68 .26,67.16,46.56,27.66,27.65,23.42,21.24,20.84.HRMS(ESI):[M+Na] + calcd for C 43 H 48 O8SSiNa,775.2731; found,775.2732.

[0085] (2) Synthesize compound B as shown in the following structural formula:

[0086] Compound A was dissolved in DMF, and 1.5 equivalents of benzaldehyde dimethyl acetal and 0.3 equivalents of camphor sulfonic acid were added. The mixture was stirred overnight at 40°C, then quenched with triethylamine and concentrated. The residue was dissolved in DMF, and 5.5 equivalents of benzyl bromide and 5.5 equivalents of sodium hydride were added at 0°C, followed by stirring at room temperature for 3 hours. After the reaction was complete, methanol was added to quench the residue and the mixture was concentrated. The solution was purified by silica gel column chromatography (eluent: toluene and acetone) to give compound B in 78% yield.

[0087] The characterization data for compound B are as follows: 1H NMR(600MHz,CDCl3)δ7.57-7.54(m,2H),7.51-7.47(m,2H),7.42-7.38(m,4H),7.38-7.29(m,16H),7.27-7.24(m,3H),7.22-7.19(m,3H),5.49(s,1H),5.21(d,J=10.6Hz,1H),4.96(d,J=11.0Hz,1H),4.88(d,J=11.2Hz,1H),4.82(d,J=10.7Hz,1H),4.79(dd,J=11.2,2.6Hz,2H),4.76-4.74(m,2H),4.57(d,J=12.0Hz,1H),4.51-4.47(m,2H),4.36(d,J=12.1Hz,1H),4.24(dd,J=12.3,1.5Hz,1H),4.08-4.04(m,2H),4.01(t,J=9.3Hz,1H),3.91(dd,J=10.9,4.1Hz,1H),3.87(dd,J=12.4,1.9Hz,1H),3.83-3.78(m,2H),3.76-3.70(m,3H),3.68(d,J=9.0Hz,1H),3.66-3.63(m,2H),3.48(dd,J=9.2,7.8Hz,1H),3.46-3.38(m,2H),3.34-3.25(m,2H),2.96(d,J=1.3Hz,1H)。 13 C NMR(150MHz,CDCl3)δ139.07,138.95,138.93,138.61,138.52,138.21,128.94,128.69,128.46,128.35,128.32,128.29,128.20,128.18,128.03,127.86,127.79,127.70,127.58,127.54,127.48,127.35,126.65,103.92,102.94,101.44,83.07,81.83,79.76,78.91,77.66,75.86,75.39,75.18,74.90,73.75,73.07,71.71,70.58,70.05,69.15,69.05,68.39,66.44,50.77。HRMS(ESI):[M+NH4] + calcd for C 58 H 67 N4O 12 ,1011.4750;found,1011.4748。

[0088] (3) Synthesize compound C as shown in the following structural formula:

[0089] Compound B was dissolved in tetrahydrofuran, and 4 equivalents of a borane-trimethylamine complex were added. 6 equivalents of aluminum trichloride and 2 equivalents of water were added under ice bath conditions, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was diluted with ethyl acetate, washed with 1M hydrochloric acid solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: toluene and acetone) to give compound C in 79% yield.

[0090] The characterization data for compound C are as follows: 1 H NMR (600MHz, CDCl3) δ7.44-7.40(m,2H),7.39-7.22(m,28H),5.01(d,J=10.8Hz,1H),4.94(d,J=11.1Hz,1H),4.83-4. 75(m,4H),4.74(d,J=11.8Hz,1H),4.69(d,J=11.8Hz,1H),4.58(d,J=12.1Hz,1H),4.50-4.45(m,3H),4.44-4.39(m,2H ),4.07-4.03(m,2H),4.00(t,J=9.4Hz,1H),3.85-3.78(m,2H),3.76-3.71(m,3H),3.71-3.67(m,2H),3.66-3.59(m,4 H), 3.51 (dd, J=9.7, 5.3Hz, 1H), 3.45 (dd, J=9.2, 7.8Hz, 1H), 3.43-3.38 (m, 2H), 3.37-3.33 (m, 1H), 3.33-3.26 (m, 2H). 13 CNMR (150MHz, CDCl3) δ139.19,138.87,138.71,138.37,138.28,138.02,128.54,128.44,128.35, 128.34,128.18,128.12,128.01,127.93,127.88,127.84,127.73,127.71,127.68,127.61,127.58 ,127.56,127.31,103.88,102.60,82.89,81.76,81.19,79.46,76.59,75.41,75.32,75.16,74.83 ,73.58,73.21,72.87,72.09,70.56,70.03,69.11,68.54,68.35,66.23,50.76; HRMS(ESI):[M+Na]+ calcd for C 58 H 65 N3O 12 Na,1018.4460; found,1018.4463.

[0091] (4) Synthesize compound D;

[0092] Perform the synthesis according to the following synthesis path:

[0093]

[0094] 1.5 equivalents of compound A (donor) and 1 equivalent of compound C (acceptor) were dissolved in toluene. A molecular sieve was added and the mixture was stirred for 30 minutes. After cooling to 0°C, 3 equivalents of N-iodosuccinimide and 0.5 equivalents of tert-butyldimethylsilyltrifluoromethanesulfonate were added. The mixture was stirred at low temperature for 5 hours, then quenched with a small amount of pyridine and methanol. 10V of triethylamine was then added, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was filtered, concentrated, and purified by silica gel column chromatography (eluent: acetone and petroleum ether) to obtain compound D in 83% yield.

[0095] The characterization data of compound D are as follows: 1H NMR(600MHz,CDCl3)δ8.02-7.99(m,2H),7.43-7.39(m,1H),7.31-7.22(m,18H),7.22-7.13(m,12H),7.04-6.99(m,2H),5.43(dd,J=10.3,3.5Hz,1H),5.17(d,J=3.5Hz,1H),4.90(d,J=11.0Hz,1H),4.86(d,J=12.3Hz,1H),4.82-4.75(m,2H),4.72-4.66(m,3H),4.59(d,J=12.2Hz,1H),4.48(d,J=12.1Hz,1H),4.42(d,J=7.7Hz,1H),4.40(d,J=7.7Hz,1H),4.38(dd,J=3.4,1.2Hz,1H),4.33(d,J=12.1Hz,1H),4.21-4.20(m,1H),4.14(dd,J=10.3,3.4Hz,1H),4.06(d,J=3.0Hz,1H),4.04-3.97(m,3H),3.92(t,J=9.2Hz,1H),3.78-3.69(m,5H),3.68-3.64(m,3H),3.60-3.56(m,4H),3.56-3.52(m,2H),3.41(dd,J=9.0,7.7Hz,1H),3.37-3.32(m,1H),3.26-3.22(m,3H),3.22-3.17(m,2H),1.09(s,9H),1.00(s,9H); 13C NMR (125MHz, CDCl3) δ167.00,139.12,138.80,138.52,138.40,138.14,138 .09,137.57,133.21,130.19,129.94,129.73,128.62,128.52,128.50,128. 47,128.36,128.31,128.27,128.21,128.17,128.14,128.09,128.06,128.03,127.94,127.87,127.83,127.78,127.72,127.68,127.62,127.59,127.43 ,127.25,127.12,103.84,102.98,98.31,81.92,81.86,80.88,79.06,77.36,76.99,75.07,74.96,74.79,74.72,74.35,74.04,73.42,73.24,73.20,73 .06,72.97,72.53,71.91,70.59,70.05,69.17,69.11,68.65,68.51,68.45, 67.41,66.90,66.76,50.78,27.62,27.47,23.41,20.90; HRMS(ESI):[M+Na] + calcd for C 79 H 95 N3O 18 SiNa,1424.6272; found,1424.6267.

[0096] (5) Synthesis of compound E:

[0097] Perform the synthesis according to the following synthesis path:

[0098] 1.5 equivalents of trichloroimine ester donor and 1 equivalent of thioglycoside acceptor were dissolved in dichloromethane / toluene (1:1, v / v), and a molecular sieve was added and stirred for 30 minutes. The mixture was then cooled to 0°C and 0.3 equivalents of trifluoromethanesulfonic acid were added. The mixture was stirred at low temperature for 3 hours. After the reaction was complete, triethylamine was added to quench the reaction, and the mixture was filtered, concentrated, and purified by silica gel column chromatography (elution: acetone and petroleum ether) to give compound E in 76% yield.

[0099] The characterization data for compound E are as follows: 1H NMR (600MHz, CDCl3) δ7.57-7.53(m,2H),7.44-7.34(m,3H),7.16-7.12(m,1H),7.09(d,J=7.9Hz,2H),5.52(s,1H),5.48(d,J=7.2Hz,1 H),5.36(dd,J=3.6,1.2Hz,1H),5.20(dd,J=10.4,7.9Hz,1H),5.07(d,J=10.1Hz,1H),4.96(dd,J=10.4,3.5Hz,1H),4.79(d,J=8.0Hz,1 H),4.75(d,J=12.0Hz,1H),4.70(d,J=12.0Hz,1H),4.45(dd,J=10.8,3.3Hz,1H),4.30(d,J=3.2Hz,1H),4.19-4.08(m,3H),3.93(dd,J= 12.2,1.6Hz,1H),3.89-3.83(m,1H),3.74-3.64(m,1H),3.32(s,1H),2.56(s,6H),2.15(s,3H),2.07(s,3H),2.05(s,3H),1.97(s,3H); 13 C NMR (150MHz, CDCl3) δ169.88,169.81,169.60,168.85,153.29,144.08,1 37.50,129.97,128.57,128.53,127.77,127.68,125.85,100.99,100.26, 94.70,85.59,75.90,75.61,74.09,70.44,70.37,69.07,68.87,68.43,66 .56,61.11,52.82,22.13,20.30,20.26,20.24,20.08.HRMS(ESI):[M+Na] + calcdfor C 38 H 44 Cl3NO 15 SNa,914.1389; found,914.1385.

[0100] (6) Synthesis of compound F:

[0101] Perform the synthesis according to the following synthesis path:

[0102]

[0103] 1.5 equivalents of the donor compound E and 1 equivalent of the acceptor compound D were dissolved in dichloromethane / toluene (1:1, v / v), and a molecular sieve was added. The mixture was stirred for 30 minutes, cooled to -50°C, and then 3 equivalents of N-iodosuccinimide and 0.3 equivalents of trifluoromethanesulfonic acid were added. The mixture was stirred at -50°C for 3 hours. After the reaction was complete, triethylamine was added to quench the reaction, and the mixture was filtered, concentrated, and purified by silica gel column chromatography (elution: acetone and petroleum ether) to give compound F in 76% yield.

[0104] The characterization data of compound F are as follows: 1 H NMR (500MHz, CDCl3) δ8.25-8.19(m,2H),7.59-7.54(m,1H),7.51-7.46(m,5H),7.40-7.23(m,32H),7.22-7.17(m,6H ),7.06-7.01(m,2H),5.76(dd,J=10.7,3.7Hz,1H),5.48(s,1H),5.33(d,J=3.5Hz,1H),5.29-5.22(m,2H),5.13(dd,J =10.4,7.9Hz,1H),5.00(d,J=7.9Hz,1H),4.96(d,J=11.1Hz,1H),4.88(d,J=12.1Hz,1H),4.86-4.79(m,3H),4.78-4. 70(m,5H),4.65-4.57(m,4H),4.49(d,J=12.1Hz,1H),4.44(d,J=7.6Hz,1H),4.41(d,J=7.6Hz,1H),4.38-4.28(m,5H) ,4.26(s,1H),4.19-4.11(m,4H),4.11-4.07(m,2H),4.05-4.00(m,2H),3.99-3.91(m,3H),3.91-3.85(m,4H),3.85- 3.80(m,2H),3.80-3.74(m,2H),3.74-3.67(m,5H),3.67-3.64(m,1H),3.64-3.61(m,2H),3.54(t,J=8.8Hz,1H),3.48 (d,J=8.1Hz,1H),3.46-3.40(m,2H),3.37-3.31(m,2H),3.31-3.25(m,4H),3.16(dd,J=8.7,5.5Hz,1H),3.06(dd,J=9 .1,5.4Hz,1H),2.84(t,J=6.2Hz,2H),2.15(s,3H),2.06(s,3H),2.00(s,3H),1.95(s,3H),1.14(s,9H),0.97(s,9H); 13C NMR (125MHz, CDCl3) δ170.38,170.35,170.02,169.42,165.92,153.40,139.25 ,138.71,138.65,138.26,138.08,138.02,137.54,133.53,130.61,129.80,128 .93,128.84,128.42,128.33,128.29,128.26,128.21,128.15,128.07,128.03,127.83,127.80,127.68,127.60,127.53,127.51,127.46,127.20,126.50,103. 75, 102.69, 101.75, 100.80, 99.55, 98.31, 95.45, 82.00, 80.89, 80.66, 79.45, 77.36, 76.02, 75.85, 75.02, 74.84, 74.56, 74.32, 73.63, 73.12, 72.97, 72.84, 72 .31,70.88,70.68,70.54,69.99,69.86,69.02,68.64,67.76,67.00,65.86,61. 38,54.36,50.72,27.54,23.33,20.89,20.80,20.76,20.56; HRMS(ESI):[M+Na] + calcd for C 109 H 129 Cl3N4O 33 SiNa,2177.7266; ​​found,2177.7250.

[0105] (7) Synthesis of compound G:

[0106] Perform the synthesis according to the following synthesis path:

[0107]

[0108] Compound F was dissolved in tetrahydrofuran / pyridine hydrofluoric acid (2:1, v / v), stirred overnight at room temperature, diluted with diethyl ether, washed with copper sulfate solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in tetrahydrofuran / 1M sodium hydroxide solution (3:1, v / v), stirred overnight at room temperature, adjusted to neutral with hydrogen-form resin, filtered, extracted, and concentrated. The residue was dissolved in methanol / triethylamine / acetic anhydride (12:2:1, v / v / v), stirred at room temperature for 3 hours, filtered, and concentrated. The residue was dissolved in n-butanol / water (1:1, v / v), palladium on carbon was added, and hydrogenation debenzylation was performed. After the reaction was complete, the residue was filtered, concentrated, and purified by size exclusion chromatography (BioGel P-2, 45-90 μm, eluent: 0.1M ammonium bicarbonate solution). The eluents containing the product were combined and lyophilized to give compound G (79%).

[0109] The characterization data of compound G are as follows: 1 H NMR(600MHz,D2O)δ4.90(d,J=4.0Hz,1H),4.68(d,J=8.4Hz,1H),4.53(d,J=8.1Hz,1H),4.50(d,J=7.8Hz,1H),4.4 4(d,J=7.8Hz,1H),4.37(t,J=6.6Hz,1H),4.24-4.23(m,1H),4.17(d,J=3.2Hz,1H),4.10-4.06(m,2H),4.05-4.01 (m,2H),4.01-3.94(m,2H),3.93-3.87(m,4H),3.87-3.80(m,4H),3.80-3.72(m,11H),3.71-3.66(m,4H),3.66-3. 62(m,3H),3.62-3.55(m,4H),3.51(dd,J=9.9,7.7Hz,1H),3.35-3.30(m,1H),3.21(t,J=5.1Hz,2H),2.01(s,3H); 13 C NMR(150MHz,D2O)δ174.65,104.34,102.82,102.43,101.55,99.90,79.08,78.18,76.68,74.99,74.51,74.34,74.12,73.99,7 2.40,71.95,70.37,70.09,69.77,69.11,68.45,68.07,67.49,67.10,65.91,60.51,60.46,59.85,59.47,51.00,38.59,21.77.

[0110] (8) Synthesize the compound shown in Formula I:

[0111] One equivalent of compound G, 1.5 equivalents of guanosine diphosphate-L-fucose (GDP-Fuc), and 2 equivalents of magnesium chloride were dissolved in 0.1 M Tris-HCl buffer. Alkaline phosphatase (CIAP, 1 U / μL) and fucosyltransferase (α1,2FucT, 1 mg / mL) were added. The mixture was incubated at 37 °C for 16 hours, and the reaction progress was monitored by mass spectrometry. After the reaction was complete, the mixture was centrifuged and purified by size exclusion chromatography (BioGel P-2, 45-90 μm, eluent: 0.1 M ammonium bicarbonate solution). The eluents containing the product were combined and lyophilized to give compound H (87%).

[0112] The characterization spectrum of the compound shown in Formula I can be found in [reference needed]. Figure 1 and Figure 2 Characterization data: 1 H NMR(600MHz,D2O)δ5.21(d,J=4.1Hz,1H),4.87(d,J=4.0Hz,1H),4.60(d,J=7.7Hz,1H),4.54-4.51(m,2H),4.50(d,J =7.8Hz,1H),4.37(t,J=6.6Hz,1H),4.24-4.19(m,2H),4.09-4.05(m,2H),4.01(d,J=3.2Hz,1H),4.00-3.95(m,3H),3 .95-3.91(m,2H),3.90-3.84(m,4H),3.84-3.81(m,3H),3.81-3.80(m,1H),3.79-3.71(m,12H),3.70-3.66(m,4H),3. 66-3.60(m,6H),3.60-3.55(m,2H),3.32(t,J=8.5Hz,1H),3.16(t,J=5.1Hz,2H),2.03(s,3H),1.20(d,J=6.6Hz,3H); 13 CNMR(150MHz,D2O)δ174.28,103.96,103.30,102.02,100.41,99.26,78.65,7 8.22,77.11,76.32,76.08,75.48,75.04,74.79,74.59,74.44,73.54,72.87,7 2.06,71.82,70.81,70.10,69.55,69.48,69.16,69.08,68.90,68.45,67.99,67.80,66.91,66.76,60.92,60.32,59.94,51.61,39.11,23.21,22.20,15.28.

[0113] This embodiment provides a method for preparing a protein conjugate, comprising: synthesizing according to the following synthetic pathway:

[0114]

[0115] 3 mg (one equivalent) of Formula I and three equivalents of PNP activated diester were stirred in ten times the amount of triethylamine at room temperature for 2 hours. The reaction was completed by TLC. The reaction solution was concentrated and chromatographically analyzed by C18 reverse column chromatography to obtain Globo H-linker (Formula II), which is the linker compound, with a yield of ~95%.

[0116] Dissolve Globo H-linker (Formula II) (3 mg) and CRM197 (2 mg) in PBS buffer (320 μL, 100 mM, pH 7.4), incubate at room temperature for 16 hours, and purify the reaction solution using a 0.5 mL centrifugal filter membrane. Ultra-0.5 (10Kd), eluent: 300μL×3 ultrapure water, centrifugation conditions: 12000r×5min.

[0117] According to MALDI-TOF analysis ( Figure 3 CRM197 peaked at m / z 58710.416, while Globo H-CRM197 peaked at m / z 63669.512. Calculations show that each carrier protein CRM197 is loaded with an average of 4.1 oligosaccharide units.

[0118] Example 2

[0119] This embodiment provides a method for preparing GloboH-BSA glycoprotein, including:

[0120] Dissolve Globo H-linker (Formula II) (3 mg) and BSA (2 mg) in PBS buffer (320 μL, 100 mM, pH 7.4), incubate at room temperature for 16 hours, and purify the reaction solution using a 0.5 mL centrifugal filter membrane. Ultra-0.510Kd), eluent: 300 μL × 3 of ultrapure water, centrifugation conditions: 12000 r × 5 min, to form GloboH-BSA glycoprotein. According to MALDI-TOF analysis (… Figure 4 BSA peaks at m / z 66889.351, while Globo H-BSA peaks at m / z 81603.957. Calculations show that the average number of Globo H hexasaccharides on each BSA is: n = (81603.957 - 66889.351) / 1212.47 = 12.14 ≈ 12.

[0121] In terms of biocompatibility, diethylene glycolamine-type linkers also have better biocompatibility and are less likely to cause additional immune side effects compared to 2-aminoethanol and 5-amino-1-pentanol. Therefore, they were chosen as glycoprotein linkers for tumor vaccines.

[0122] Experimental Example – Immunological Evaluation

[0123] Six- to eight-week-old SPF female Balb / C mice were used and grouped into eight groups of ten mice each, according to the table below. The antigen dose (Globo H) was 5 μg / dose, and the S34 dose was 2 μg / dose. Blank liposomes and Al adjuvant (aluminum hydroxide) were used as negative and positive controls, respectively. Before injection, the adjuvant and vaccine were vortexed for 30 seconds. Immunization was performed subcutaneously on the back according to the schedule of days 1, 14, and 28. Blood samples were collected from the orbital sinus on days 0, 28, and 42, and the serum antibody titer was measured (ELISA method) to evaluate the level of Globo H antibody production by the adjuvant.

[0124]

[0125] (1) ELISA detection method

[0126] In this experiment, the GloboH-BSA glycoprotein prepared in Example 2 was used to coat an ELISA plate for detecting anti-GloboH antibodies in serum. The GloboH-BSA antigen was diluted to 1 μg / mL with PBS, and coated at a rate of 100 μL / well, incubated overnight at 4°C. The plate was washed three times with PBST (250 μL / well), and then 2% BSA solution prepared with PBS was added, incubated at room temperature for 2 hours. The serum to be tested was diluted proportionally with diluent (1% BSA / PBST). The blocking buffer was discarded, and the diluted serum was added, incubated at room temperature for 2 hours. The plate was washed three times with PBST, and 100 μL / well of diluted HRP-labeled secondary antibody was added, incubated at room temperature for 1 hour. The plate was washed five times with PBST, and 100 μL / well of TMB chromogenic solution was added, incubated at room temperature in the dark for 10-15 minutes. 100 μL / well of 1M HCl was added. The OD450 value was read within 15 minutes.

[0127] Serum was obtained by collecting blood from the orbital cavity 13 days after the first, second, and third immunizations of mice to detect antibody titers post-immunization. Results are as follows: Figure 5 ,according to Figure 5 It can be seen that the antibody titer was higher after 3 doses of immunization than after 2 doses of immunization, indicating that the antibody production effect was best after 3 doses of immunization.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound of Formula I: or a pharmaceutically acceptable salt thereof. ； The preparation of the compound of Formula I comprises synthesizing according to the following synthetic route: The specific procedure is as follows: The step of forming compound D comprises mixing compound A, compound C, an imine and a Lewis acid and reacting; The step of forming compound F comprises mixing compound D, compound E, an imine and a Lewis acid and reacting; The step of forming compound G comprises mixing compound F, tetrahydrofuran and an acid, stirring and primary work-up to form a first residue; Then, mixing the first residue, tetrahydrofuran and a basic compound, stirring and secondary work-up to form a second residue; Then, mixing the second residue, an alcoholic solvent, an organic base and an acid anhydride, stirring and tertiary work-up to form a third residue; Then, mixing the third residue, an alcoholic solvent and palladium on carbon and reacting; The step of forming compound I comprises mixing compound G, GDP-Fuc, alkaline phosphatase and a transferase and reacting.

2. The compound of claim 1, represented by Formula I, wherein The molar ratio of compound A to compound C is (1.2-1.5):1, and the molar ratio of compound C, the imine and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

3. The compound of Formula I according to claim 1, wherein: The molar ratio of compound E to compound D is (1.2-1.5):1, and the molar ratio of compound D, the imine and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

4. The compound of Formula I according to claim 1, wherein: The molar ratio of compound G to GDP-Fuc is 1:(1.2-1.5).

5. A linker compound, characterized in that, It comprises a bis-activated ester linker and the compound of Formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the bis-activated ester linker is condensed with the amino amide of the compound of Formula I.

6. The linker compound of claim 5, wherein, The bis-activated ester linker is selected from the following compounds: ; The linker compound is selected from the following compounds: 。 7. A protein conjugate, characterized in that, It comprises a protein and the linker compound according to claim 5, wherein the protein is condensed with the linker compound by amide.

8. The protein conjugate of claim 7, wherein, The structure of the protein conjugate is as follows: 。

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