GloboH hexasaccharide, preparation method thereof, linker compound and protein conjugate

Abstract

The invention relates to the technical field of medical biology, in particular to GloboH hexasaccharide, a preparation method thereof, a linker compound and a protein conjugate. The invention provides a compound as shown in a formula I or a stereoisomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and pharmaceutically acceptable salt thereof: # imgabs0 #. According to the GloboH hexasaccharide provided by the embodiment of the invention, diethylene glycol amine is introduced at an anomeric position of a terminal group, so that the reaction efficiency of subsequent amide condensation can be improved; monoester formed by the compound and an activated ester linker is higher in stability, subsequent glycoprotein coupling reaction is facilitated, and the reaction efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to GloboH hexasaccharide, a preparation method thereof, a linker compound and a protein conjugate. Background Art

[0002] Abnormal tumor glycosylation is an important hallmark in cancer biology. The overexpression of tumor-associated carbohydrate antigens (TACAs) on the surface of tumor cells is in sharp contrast to their absence or low expression in normal cells. The study of related carbohydrate antigens is also the focus of current tumor therapeutic antibodies and therapeutic vaccines. TACA globular serine glycan (Globo H) is a complex hexasaccharide compound. Globo H antigen is overexpressed in various epithelial cell tumors (such as breast, ovarian, colon, gastric, pancreatic and lung cancer) and is extremely low in normal cells. It mainly promotes cancer through tumor immunosuppression, microangiogenesis and tumor protein kinase. The development of therapeutic vaccines against Globo H is expected to block these cancer-promoting mechanisms by activating the human immune system, especially activating specific antibodies and cellular immune responses against Globo H. However, tumor-associated carbohydrate antigens have a small molecular weight, lack size effect and clustering effect, have strong immunogenicity, 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 carbohydrate conjugates to induce the body to produce persistent and enhanceable IgG antibodies.

[0003] The existing sugar-protein coupling strategy used by GloboH has many disadvantages: 1) Reductive amination method: The reaction conditions are harsh, which changes the basic structure of the antigen, accompanied by over-reduction and other side reactions; 2) Sulfur addition method: Sulfur-containing bonds are unstable in the body environment and are easily reduced or oxidized, affecting the long-term stability of the coupling product; 3) Disulfide bond method: Disulfide bonds are highly reducing and have the risk of breaking in the body; 4) Cycloaddition method: The cyclic linker unit may have immunogenicity problems. Due to problems in the coupling method, the sugar-protein binding efficiency is affected, and other side reactions or immune side effects are easily generated, which limits the immune effect of the existing GloboH vaccine.

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

[0005] The object of the present invention is to provide GloboH hexasaccharide, a preparation method thereof, a linker compound and a protein conjugate. The GloboH hexasaccharide provided in the embodiment of the present invention introduces diethylene glycolamine at the terminal anomeric position, which can improve the reaction efficiency of the subsequent amide condensation, and the monoester formed with the activated ester linker has higher stability, which is beneficial to the subsequent glycoprotein coupling reaction and improves the reaction efficiency.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a compound of formula I or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts:

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the compound represented by formula I described in the above embodiment or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, comprising: synthesizing according to the following synthesis route:

[0010]

[0011] In an optional embodiment, the step of forming compound D comprises: mixing compound A, compound C, an imine substance and a Lewis acid to react;

[0012] The molar ratio of the compound A to the compound C is (1.2-1.5):1, and the molar ratio of the 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 comprises: mixing compound D, compound E, an imine substance and a Lewis acid to react;

[0014] The molar ratio of the compound E to the compound D is (1.2-1.5):1, and the molar ratio of the 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 comprises: mixing compound F, tetrahydrofuran and acid and performing a primary post-treatment to form a first residue;

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

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

[0018] Next, the third residue, an alcohol solvent and palladium carbon are mixed to react.

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

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

[0021] In a third aspect, the present invention provides a linker compound, which includes a biactivated ester linker and a compound represented by Formula I described in the aforementioned embodiment or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, wherein the biactivated ester linker is condensed with the aminoamide of the compound represented by Formula I.

[0022] In an optional embodiment, the di-activated ester linker is selected from the compounds represented by the following structural formula:

[0023]

[0024] The linker compound is selected from the compounds represented by the following structural formula:

[0025]

[0026] In a fourth aspect, the present invention provides a protein conjugate, comprising a protein and the linker compound described in the aforementioned embodiment, wherein the protein and the linker compound are condensed by amide.

[0027] In an optional embodiment, the structural formula of the protein conjugate is as follows:

[0028]

[0029] The present invention has the following beneficial effects: The embodiment of the present invention provides a new Globo H hexasaccharide, in which diethylene glycolamine is introduced at the anomeric position (position 1) of the GloboH hexasaccharide. The monoester formed by the compound and the activated ester linker has higher stability, which is beneficial to the subsequent glycoprotein coupling reaction and improves the reaction efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

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

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

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

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

[0035] Figure 5 This is a test result diagram provided for an experimental example of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0037] There are problems with the sugar-protein coupling method in the prior art, which affects the sugar-protein binding efficiency and is prone to other side effects or immune side effects, limiting the immune effect of the existing GloboH vaccine. Therefore, the embodiment of the present invention uses amide condensation to achieve sugar-protein coupling. The amide condensation method for sugar-protein coupling has the advantages of few side reactions, strong biocompatibility, and no additional immunogenicity. However, amide condensation has the problem that the linker is less stable and difficult to bind in the coupling reaction. Therefore, the embodiment of the present invention modifies GloboH to obtain a new GloboH hexasaccharide to solve the above technical problems.

[0038] In a first aspect, the present invention provides a compound of formula I or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts:

[0039]

[0040] In a second aspect, the present invention provides a method for preparing the compound represented by formula I described in the above embodiment or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, comprising: synthesizing according to the following synthesis route:

[0041]

[0042] The specific process is as follows:

[0043] (1) Synthesis of Compound A:

[0044] The synthesis was carried out according to the following synthesis route:

[0045] Dissolve p-methylphenylthio-β-D-pyranogalactoside 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-fluorenylmethyl chloroformate), and stir. Then add 1-1.2 equivalents of an acyl chloride-substituted benzene (e.g., benzoyl chloride), stir the reaction at room temperature, and perform post-treatment after the reaction is completed.

[0046] (2) Synthesize compound B represented by the following structural formula:

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

[0048] (3) Synthesis of Compound C:

[0049]

[0050] Add 3.5-4.5 equivalents of borane compound (such as borane-trimethylamine complex) to compound B and solvent (such as tetrahydrofuran), add 5.5-6.5 equivalents of aluminum chloride and 1.5-2.5 equivalents of water under ice bath condition, stir and react at room temperature, and perform post-treatment after the reaction is completed.

[0051] (4) synthesizing compound D;

[0052] The synthesis was carried out according to the following synthesis route:

[0053]

[0054] Compound A and compound C are dissolved in a solvent (such as toluene) and stirred, then cooled and an imine substance (such as N-iodosuccinimide) and a Lewis acid (such as tert-butyldimethylsilyl trifluoromethanesulfonate) are added to mix and react at low temperature, then the reaction is quenched with an alcohol solvent and an organic base (such as pyridine), and then an amine substance is added to react by stirring at room temperature, and post-treatment is performed after the reaction is completed.

[0055] The molar ratio of the compound A to the compound C is (1.2-1.5):1, and the molar ratio of the 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] The synthesis was carried out according to the following synthesis route:

[0058] Among them, 1.2-1.5 equivalents of ester donor (such as trichloroimidate) and 1 equivalent of glycoside acceptor (such as thioglycoside) are dissolved in a solvent (such as dichloromethane / toluene (1:1, v / v)), molecular sieves are added and stirred, and after cooling (for example, cooling to 0°C), 0.2-0.4 equivalents of Lewis acid (such as trifluoromethanesulfonic acid) are added, and then the reaction is stirred at low temperature, and post-treatment is performed after the reaction is completed.

[0059] (6) Synthesis of Compound F:

[0060] The synthesis was carried out according to the following synthesis route:

[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] The synthesis was carried out according to the following synthesis route:

[0065]

[0066] , after Compound F, tetrahydrofuran and acid (e.g. pyridine hydrofluoride) are mixed at room temperature and stirred overnight, an ether solvent (e.g. ether) is added to dilute, and then washed and dried to form a first residue. Then, the first residue is dissolved in a mixed solution of tetrahydrofuran and an alkaline compound and stirred overnight at room temperature, and then adjusted to neutrality, filtered, extracted and concentrated to form a second residue. Then, the second residue is mixed with an alcoholic solvent, an organic base (e.g. triethylamine) and an acid anhydride (e.g. acetic anhydride) and stirred at room temperature, and then filtered and concentrated to form a third residue. Then, the third residue is dissolved in an alcoholic solvent / water, palladium carbon is added to carry out a debenzylation reaction, and after the reaction is finished, a post-treatment is carried out.

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

[0068] 1 equivalent of compound G, 1.2-1.5 equivalents of guanosine diphosphate-L-fucose (GDP-Fuc), and 2 equivalents of magnesium chloride are dissolved in a buffer solution, and alkaline phosphatase and transferase are added to mix and react, and post-treatment is performed after the reaction is completed.

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

[0070] In a third aspect, the present invention provides a linker compound, which includes a biactivated ester linker and a compound represented by Formula I described in the aforementioned embodiment or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, wherein the biactivated ester linker is condensed with the aminoamide of the compound represented by Formula I.

[0071] For example, the diactivated ester linker is selected from the compounds represented by the following structural formula: The structural formula of the linker compound formed by the double activated ester linker is shown below:

[0072]

[0073] In a fourth aspect, the present invention provides a protein conjugate, which comprises a protein and the linker compound described in the above embodiment, wherein the protein and the linker compound are condensed by amide. The protein conjugate can be combined with a vaccine adjuvant to form a new tumor vaccine, and the vaccine antibody has excellent performance.

[0074] Specifically, the structural formula of the protein conjugate in the embodiment of the present invention is as follows:

[0075]

[0076] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0077] Example 1

[0078] This embodiment provides a compound represented by formula I, and its structural formula is as follows:

[0079]

[0080] This embodiment also provides a method for preparing the compound represented by the above formula I, comprising:

[0081] (1) Synthesis of Compound A:

[0082] The synthesis was carried out according to the following synthesis route:

[0083] Dissolve p-methylphenylthio-β-D-pyranogalactoside in dichloromethane / pyridine (1:1, v / v), add 1.05 equivalents of di-tert-butylsilylbis(trifluoromethanesulfonic acid), and stir at room temperature for 3 hours. Cool to -20°C, add 1.05 equivalents of 9-fluorenylmethyl chloroformate, and stir for 1 hour. Add 1.2 equivalents of benzoyl chloride and stir at room temperature for 1 hour. After the reaction is completed, methanol is added to quench and concentrate. The residue is dissolved in ethyl acetate, washed with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate), and finally compound A is obtained with a yield of 89%.

[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). 13CNMR(150MHz,CDCl3)δ165.42,154.55,143.40,143.17,141.25,138.25, 133.40,133.26,130.08,130.07,129.78,129.73,128.56,127.90,127.26 ,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] + Calculate for C 43 H 48 O8SSiNa,775.2731; found,775.2732.

[0085] (2) Synthesize compound B represented by the following structural formula:

[0086] Compound A was dissolved in DMF, 1.5 equivalents of benzaldehyde dimethyl acetal and 0.3 equivalents of camphorsulfonic acid were added, and the mixture was stirred at 40°C overnight, and then triethylamine was added to quench the mixture and the mixture was 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, respectively, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, methanol was added to quench the mixture and the mixture was concentrated, and the mixture was purified by silica gel column chromatography (eluent: toluene and acetone) to obtain compound B with a yield of 78%.

[0087] The characterization data of 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 represented by the following structural formula:

[0089] Compound B was dissolved in tetrahydrofuran, 4 equivalents of borane-trimethylamine complex was added, 6 equivalents of aluminum chloride and 2 equivalents of water were added under ice bath, and stirred at room temperature for 3 hours. After the reaction was completed, ethyl acetate was added for dilution, 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 obtain compound C with a yield of 79%.

[0090] The characterization data of 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]+ Calculate for C 58 H 65 N3O 12 Na,1018.4460; found,1018.4463.

[0091] (4) synthesizing compound D;

[0092] The synthesis was carried out according to the following synthesis route:

[0093]

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

[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] + Calculate for C 79 H 95 N3O 18 SiNa,1424.6272; found,1424.6267.

[0096] (5) Synthesis of Compound E:

[0097] The synthesis was carried out according to the following synthesis route:

[0098] 1.5 equivalents of trichloroimidate donor and 1 equivalent of thioglycoside acceptor were dissolved in dichloromethane / toluene (1:1, v / v), molecular sieves were added and stirred for 30 minutes, and 0.3 equivalents of trifluoromethanesulfonic acid were added under cooling to 0°C. Stirring was carried out at low temperature for 3 hours. After the reaction was completed, triethylamine was added to quench, filtered, concentrated, and purified by silica gel column chromatography (eluent: acetone and petroleum ether) to obtain compound E with a yield of 76%.

[0099] The characterization data of 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] The synthesis was carried out according to the following synthesis route:

[0102]

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

[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] + Calculate for C 109 H 129 Cl3N4O 33 SiNa,2177.7266; ​​found,2177.7250.

[0105] (7) Synthesis of Compound G:

[0106] The synthesis was carried out according to the following synthesis route:

[0107]

[0108] , Compound F was dissolved in tetrahydrofuran / pyridine hydrofluoride (2:1, v / v), stirred at room temperature overnight, then diluted with 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 at room temperature overnight, adjusted to neutrality with hydrogen 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 carbon was added, and hydrogenation was performed to remove the benzyl group. After the reaction was completed, it was filtered, concentrated, and purified by exclusion chromatography (BioGel P-2, 45-90μm, eluent: 0.1M ammonium bicarbonate solution), and the eluent containing the product was combined and freeze-dried to obtain 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] 1 equivalent of compound G, 1.5 equivalents of guanosine diphosphate-L-fucose (GDP-Fuc) and 2 equivalents of magnesium chloride were dissolved in 0.1M Tris-HCl buffer, alkaline phosphatase (CIAP, 1U / μL) and fucosyltransferase (α1,2FucT, 1mg / mL) were added, and the mixture was incubated at 37°C for 16 hours and the reaction progress was monitored by mass spectrometry. After the reaction was complete, centrifugation was performed and purified by exclusion chromatography (BioGel P-2, 45-90μm, eluent: 0.1M ammonium bicarbonate solution), and the eluent containing the product was combined and lyophilized to obtain compound H (87%).

[0112] The characterization spectrum of the compound shown in Formula I is shown in 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 synthesis path:

[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. TLC showed that the reaction was complete. The reaction solution was concentrated and chromatographed on a C18 reverse chromatographic column to obtain Globo H-linker (Formula II), i.e., linker compound, with a yield of 95%.

[0116] The Globo H-linker (Formula II) (3 mg) and CRM197 (2 mg) were dissolved in PBS buffer (320 μL, 100 mM, pH 7.4) and incubated at room temperature for 16 hours. The reaction solution was purified by 0.5 mL centrifugal filter membrane ( Ultra-0.5 10Kd), eluent: ultrapure water 300μL×3, centrifugation condition: 12000r×5min.

[0117] According to MALDI-TOF analysis ( Figure 3 ), CRM197 peaks at m / z 58710.416, and the highest peak of Globo H-CRM197 is at m / z 63669.512. Through calculation, it can be seen 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, comprising:

[0120] The Globo H-linker (Formula II) (3 mg) and BSA (2 mg) were dissolved in PBS buffer (320 μL, 100 mM, pH 7.4), incubated at room temperature for 16 hours, and the reaction solution was purified by 0.5 mL centrifugal filter membrane ( Ultra-0.510Kd), eluent: ultrapure water 300μL×3, centrifugation conditions: 12000r×5min, to form GloboH-BSA glycoprotein. According to MALDI-TOF analysis ( Figure 4 ), BSA peaks at m / z 66889.351, and the highest peak of Globo H-BSA is m / z 81603.957. Through calculation, it can be known 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 glycol amine type linkers also have better biocompatibility and are less likely to cause additional immune side effects than 2-aminoethanol and 5-amino-1-pentanol. Therefore, they are selected as the sugar-protein linker for tumor vaccines.

[0122] Experimental example - immune evaluation

[0123] 6-8 week old SPF female Balb / C mice were used and grouped according to the following table. There were 8 groups with 10 mice in each group. The antigen dose (Globo H) was 5μg / dose and S34 was 2μg / dose. Blank liposomes and Al adjuvant (aluminum hydroxide) were used as negative and positive controls, respectively. The adjuvant and vaccine were vortexed for 30 seconds before injection. Immunization was performed by subcutaneous administration on the back according to the schedule of 1, 14, and 28 days. Blood was collected from the eye sockets on days 0, 28, and 42, and then the mouse serum antibody titer (ELISA method) was detected to evaluate the level of Globo H antibody produced by the adjuvant.

[0124]

[0125] (1) ELISA method

[0126] In this experiment, the GloboH-BSA glycoprotein prepared in Example 2 was used to coat the ELISA plate to detect anti-GloboH antibodies in serum. The GloboH-BSA antigen was diluted to 1ug / mL with PBS, coated at 100μL / well, and incubated overnight at 4°C. Wash three times with PBST, 250μL / well, then add a 2% BSA solution prepared with PBS, and incubate at room temperature for 2 hours. Dilute the serum to be tested with the diluent (1% BSA / PBST) in proportion. Pour out the blocking solution, add the diluted serum to be tested, and incubate at room temperature for two hours. Wash three times with PBST, add the diluted HRP-labeled secondary antibody, 100μL / well, and incubate at room temperature for 1 hour. Wash five times with PBST, add TMB colorimetric solution 100μL / well, and incubate at room temperature in the dark for 10-15 minutes. Add 1M HCl, 100μL / well. Read the OD450 value within 15 minutes.

[0127] On the 13th day after the first, second and third injections of the mice, blood was collected from the eye sockets to obtain serum to detect the antibody titer after immunization. Figure 5 ,according to Figure 5 It can be seen that after three injections of immunization, the antibody titer is higher than that after two injections of immunization, indicating that the antibody production effect is best after three injections of immunization.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A compound of formula I or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts:

2. A method for preparing the compound of formula I according to claim 1 or its stereoisomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, characterized in that: include: The synthesis was carried out according to the following synthesis route:

3. The preparation method according to claim 2, characterized in that: The step of forming compound D comprises: mixing compound A, compound C, an imine substance and a Lewis acid to react, The molar ratio of the compound A to the compound C is (1.2-1.5):1, and the molar ratio of the compound C, the imine substance and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

4. The preparation method according to claim 2, characterized in that: The step of forming compound F comprises: mixing compound D, compound E, imine substance and Lewis acid for reaction, The molar ratio of the compound E to the compound D is (1.2-1.5):1, and the molar ratio of the compound D, the imine substance and the Lewis acid is 1:(2.5-3.5):(0.3-0.5).

5. The preparation method according to claim 2, characterized in that: The step of forming compound G comprises: mixing compound F, tetrahydrofuran and acid and performing a primary post-treatment to form a first residue; Next, the first residue, tetrahydrofuran and the basic compound are mixed and stirred and then subjected to secondary post-treatment to form a second residue; Then, the second residue is mixed with an alcohol solvent, an organic base and an acid anhydride, stirred and post-treated three times to form a third residue; Next, the third residue, an alcohol solvent and palladium carbon are mixed to react.

6. The preparation method according to claim 2, characterized in that: The step of forming compound I comprises: mixing compound G, GDP-Fuc, alkaline phosphatase and transferase for reaction; Wherein, the molar ratio of the compound G to the GDP-Fuc is 1:(1.2-1.5).

7. A linker compound, characterized in that It comprises a double activated ester linker and the compound represented by formula I according to claim 1 or its stereoisomer, solvate, hydrate, prodrug, stable isotope derivative and pharmaceutically acceptable salt, wherein the double activated ester linker is condensed with the aminoamide of the compound represented by formula I.

8. The linker compound according to claim 7, characterized in that The diactivated ester linker is selected from the compounds represented by the following structural formula: The linker compound is selected from the compounds represented by the following structural formula:

9. A protein conjugate, characterized in that: The method comprises a protein and the linker compound according to claim 7, wherein the protein and the linker compound are condensed by amide.

10. The protein conjugate according to claim 9, characterized in that The structural formula of the protein conjugate is shown below:

Images