WO2022016755A1 - 一种海藻糖衍生物与糖抗原的缀合物及其制备方法与应用 - Google Patents

一种海藻糖衍生物与糖抗原的缀合物及其制备方法与应用 Download PDF

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WO2022016755A1
WO2022016755A1 PCT/CN2020/130230 CN2020130230W WO2022016755A1 WO 2022016755 A1 WO2022016755 A1 WO 2022016755A1 CN 2020130230 W CN2020130230 W CN 2020130230W WO 2022016755 A1 WO2022016755 A1 WO 2022016755A1
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compound
conjugate
trehalose
preparation
nhc
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PCT/CN2020/130230
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French (fr)
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廖国超
刘中秋
李文伟
练庆海
杨德盈
吴鹏
卢琳琳
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广州中医药大学(广州中医药研究院)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • the present invention relates to the technical field of chemistry and medicine, in particular to a conjugate of a trehalose derivative and a saccharide antigen and a preparation method and application thereof.
  • TACAs Tumor-associated carbohydrate antigens
  • TACAs tumor-associated carbohydrate antigens
  • TACAs tumor-associated carbohydrate antigens
  • the classic strategy is to conjugate carbohydrate antigens with carrier proteins to enhance their immunogenicity, but glycoprotein vaccines have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components.
  • the total synthesis strategy of tumor-related carbohydrate antigen vaccines has become one of the new research hotspots; the fully synthetic carbohydrate antigen vaccines have the advantages of clear structure, stable and controllable quality, etc., which are convenient for various immunological and clinical studies.
  • sugar molecules on the surface of various pathogenic bacteria can be used as embedded adjuvants for fully synthesized sugar antigen tumor vaccines, which can not only overcome the shortcomings of the weak immunogenicity of sugar antigens, but also avoid the "epitope" caused by proteins. suppress” effect.
  • Trehalose-6,6-dimycolate (TDM, cord factor) is an important glycolipid component in the cell wall of Mycobacterium tuberculosis, consisting of trehalose and two mycolate chains; this glycolipid can cause inflammatory responses , and has certain cytotoxicity to tumor cells. Studies have shown that the derivatives of trehalose-6,6-dimycolate, Vizantin and TDE, have high immune activity and low toxicity, and can be used as effective adjuvants for vaccine research.
  • the object of the present invention is to provide a conjugate of a trehalose derivative and a carbohydrate antigen.
  • the present invention uses potent immunostimulators trehalose derivatives Vizantin and TDE as built-in adjuvants to conjugate sugar antigens (STn, Tn) respectively to obtain the conjugates, wherein trehalose derivatives can improve the immunogenicity of sugar antigens , the conjugate can induce T cell-mediated humoral immunity, produce high-concentration high-affinity IgG antibody, and achieve the purpose of specifically killing tumor cells.
  • Another object of the present invention is to provide a method for preparing the conjugate of the trehalose derivative and carbohydrate antigen.
  • Another object of the present invention is to provide the application of the conjugate of the trehalose derivative and the saccharide antigen in the preparation of tumor vaccine.
  • Another object of the present invention is to provide the application of the conjugate of the trehalose derivative and the carbohydrate antigen in the preparation of antitumor drugs.
  • X represents a carbohydrate antigen, selected from any one of the carbohydrate antigens in the following formula, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • L represents a linker, selected from the following (CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -(OCH 2 CH 2 ) a -CH 2 CH 2 -C(O)NH-, (CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a -C(O)NH-, -NHC(O)-(CH 2 ) a Any of , a is any integer from 0 to 20;
  • Y represents a trehalose derivative, selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • R 1 and R 2 are each independently selected from hydrogen, -CH 2 -CH(OR 3 )-(CH 2 ) m -CH 3 , -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , R 3 is -(CH 2 ) m -CH 3 or -C(O)-(CH 2 ) m -CH 3 , m is an integer selected from 8-26.
  • the L is selected from (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 (CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a Any of , where a is any integer from 0 to 20.
  • the L is selected from (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 ) a Any of , where a is any integer from 0 to 20.
  • the Y is selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • R 1 and R 2 are each independently selected from hydrogen, -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , and R 3 is -(CH 2 ) m -CH 3 or -C(O )-(CH 2 ) m -CH 3 , m is an integer selected from 8-26.
  • the Y is selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • R 1 and R 2 are each independently selected from hydrogen or -(CH 2 ) m CH 3 , and m is an integer selected from 8-26.
  • the conjugate is selected from any of the following structures or a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • the preparation method of the conjugate of trehalose derivative and saccharide antigen when Y is , the preparation process includes:
  • compound 10 reacts with compound Tn or STn, compound 11 and compound STn respectively under the action of a catalyst to obtain compound 19 or 20, compound 21;
  • the preparation process includes;
  • Compound 22 or 23 are respectively subjected to debenzylation protection reaction to obtain the target product;
  • the catalyst of the debenzylation protection reaction is hydrogen/palladium carbon/palladium hydroxide, hydrogen/palladium carbon or hydrogen/palladium hydroxide, etc., preferably hydrogen/palladium carbon;
  • the solvent used in the reaction is Dichloromethane/methanol/water, dichloromethane, methanol or dichloromethane/methanol, etc.; preferably dichloromethane/methanol/water.
  • the catalyst for the reaction is cuprous iodide/N,N-diisopropylethylamine or cuprous iodide/N,N-diisopropylethylamine/glacial acetic acid, etc.; Cuprous iodide/N,N-diisopropylethylamine is preferred; the reaction solvent is dichloromethane, dichloromethane/methanol or tetrahydrofuran/methanol, etc.; tetrahydrofuran/methanol is preferred.
  • the catalyst for the reaction is an aqueous solution of boron trifluoride ethyl ether or trifluoroacetic acid, etc., preferably boron trifluoride ethyl ether;
  • the solvent for the reaction is acetonitrile, dichloromethane or tetrahydrofuran, etc., preferably acetonitrile.
  • the condensation reagent in the reaction process can be selected from 4-dimethylaminopyridine (DMAP)/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl Iodonium salt (EDC MEI), N,N'-dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP) or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC. HCl) / 4- dimethylaminopyridine (DMAP) was the like, preferably DMAP / EDC. HCl.
  • DMAP 4-dimethylaminopyridine
  • EDC MEI 4-dimethylaminopyridine
  • EDC MEI 4-dimethylaminopyridine
  • DCC N,N'-dicyclohexylcarbodiimide
  • DMAP 1-(3-dimethylaminopropyl)-3-e
  • the preparation process of compound 7 is as follows:
  • the catalyst for the reaction is triethylsilane/trifluoromethanesulfonic acid or sodium cyanoborohydride/diethyl ether hydrochloride; the reaction is performed in a solvent such as dichloromethane, methanol or tetrahydrofuran.
  • the catalyst for the reaction is sodium cyanoborohydride/diethyl ether hydrochloride; the reaction is carried out in a solvent dichloromethane.
  • the above-mentioned preparation route of the present invention is short, the reaction conditions are mild, the yield is high, and the operation is convenient, and can be used for industrial preparation.
  • the tumor vaccine or anti-tumor drug is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell cancer, cellular lymphoma, thyroid cancer , brain cancer, stomach cancer or leukemia vaccines or drugs.
  • the present invention has the following beneficial effects:
  • the present invention uses potent immunostimulators trehalose derivatives Vizantin and TDE as built-in adjuvants to conjugate sugar antigens (STn, Tn) respectively to obtain the conjugates, wherein trehalose derivatives can improve the immunogenicity of sugar antigens , so that the conjugate can induce T cell-mediated humoral immunity, generate high concentration and high affinity IgG antibody, achieve the purpose of specifically killing tumor cells, and can be prepared as a tumor vaccine or an anti-tumor drug for application.
  • FIG. 1 is a graph showing the evaluation of the immune activity of antibodies produced in mice induced by vaccines prepared with conjugates 1, 2, 3 and 4.
  • FIG. 1 is a graph showing the evaluation of the immune activity of antibodies produced in mice induced by vaccines prepared with conjugates 1, 2, 3 and 4.
  • Figure 2 is a graph showing the evaluation of complement-dependent cytotoxicity of antibody serum produced by vaccine-induced mice produced by conjugates 1, 2, 3 and 4 to specifically kill tumor cell MCF-7.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
  • reaction was carried out at room temperature for 18 h, the reaction solution was cooled to 0 °C, a small amount of methanol solution was dropped to quench the reaction, diluted with ethyl acetate, the mixture was washed with saturated brine, the organic layer was collected, concentrated to obtain the crude product, and purified by silica gel column chromatography to obtain compound 6 as a white solid (14.90 g, 78.4%).
  • Tetrabutylammonium bromide (6eq); reacted at room temperature for 4h, the reaction solution was cooled to 0°C, slowly added dropwise with a small amount of methanol to quench the reaction, diluted with dichloromethane, rinsed with saturated sodium bicarbonate solution and saturated brine successively, and collected organic The phase was concentrated under reduced pressure, and the purified compound 13 (380.01 mg, 97%) was separated and purified by silica gel column.
  • HEPES hydroxyethylpiperazine ethanethiosulfonic acid
  • mice C57BL/6 mice aged 6-8 weeks were taken and divided into four groups with 6 mice in each group.
  • the immunization test was carried out by subcutaneous injection of mice, and the liposomes 1, 2, and 3 prepared in step (1) were injected on the 1st, 14th, 21st, and 28th days respectively with the scheme of one initial immunization and three boosting immunizations. 4.
  • the injection volume of each mouse is 0.1 mL. Blood is collected on the 0, 27, 35, and 49 days, and 0.1 mL to 0.2 mL of blood is collected from each mouse, placed at 4°C for half an hour, centrifuged at 5000 rpm, and separated. Top layer clearing serum.
  • the blank control group pre-immune serum
  • the vaccine titer was measured using the blood collected on the 35th day.
  • ELISA immunoassay Use 0.1M carbonate buffer (pH 9.6) to prepare Tn-HSA or STn-HSA into a 2.0 ⁇ g/mL solution, add 100 ⁇ L per well to a 96-well plate, and put 4 Incubate overnight at °C; incubate at 37 °C for one hour the next day; wash the plate three times with PBST (PBS+0.05% Tween-20), and add 300 ⁇ L of washing solution to each well. After washing the plate, add blocking buffer (PBST/1% BSA); add 250 ⁇ L to each well; incubate at room temperature for one hour, and wash the plate three times with PBST.
  • PBST blocking buffer
  • the sample serum was diluted with PBS from 1:300 to 1:656100 times according to the half-dilution method; the diluted serum was added to 96-well plate at 100 ⁇ L per well, placed in a 37°C incubator for two hours, and washed. plate three times.
  • 100 ⁇ L of HRP (horseradish peroxidase)-labeled Kappa, IgG, and IgM were added to each well, and incubated at room temperature for one hour; the plate was washed 3 times.
  • Add TMB (3,3',5,5'-tetramethylbenzidine) solution add 100 ⁇ L to each well, and develop color at room temperature for 20 min in the dark.
  • 0.5M H 2 SO 4 solution was added to each well 100 ⁇ L. Absorbance was detected with a microplate reader at dual wavelengths of 450-570 nm.
  • the conjugates 1, 2, 3 and 4 of trehalose derivatives and carbohydrate antigens synthesized in Example 1 of the present invention can all induce high titers without adjuvant.
  • the specific IgG antibody titer produced is more than 2 to 3 times that of IgM, indicating that the covalently conjugated carbohydrate antigen vaccine can induce T cell-mediated humoral immunity; among them, the conjugate vaccine 1 double-linked STn antigen and
  • the titers of IgG antibodies elicited by 2 were slightly higher than those of conjugates 3 and 4, which were single-attached to the antigen, indicating that the more complex the space of the antigen, the more epitopes, had a certain promoting effect on the immune response;
  • the fatty acid chain of trehalose derivatives was Conjugate vaccines 1 and 3 with branched CH(C 9 H 19 ) 2 induced slightly higher antibodies than conjugate vaccines 2 and 4 with fatty acid chain (CH 2 ) 20 CH 2
  • CT-26 cells (mouse colon cancer cells) in logarithmic growth phase were trypsinized, seeded in 96-well plates at 1 ⁇ 10 4 cells per well, cultured overnight at 37°C, and cultured with serum-free 1640 The substrate was washed twice. Take the serum samples of 6 mice in the compound 1 group on 38 days, take 7 ⁇ l of each sample, then mix the serum samples of the 6 mice, and dilute 50 times with serum-free 1640 medium to obtain mouse serum Diluent. The mouse serum dilutions of compound group 2, group 3, group 4 and blank control group were prepared according to the above method.
  • Group 1, Group 2, Group 3, Group 4 and blank control group were set as the maximum enzyme activity control group, sample control group and sample treatment group, each group was made 6 replicate wells in parallel, each well 100 ⁇ l of diluted mouse serum solution was added, and the 96-well plate was incubated at 37° C. for 2 h. After washing the plate twice with serum-free 1640 medium, 100 ⁇ l of rabbit complement serum solution was added to each well of the sample treatment group, 100 ⁇ l of LDH release solution was added to each well of the maximum enzyme activity control group, and 100 ⁇ l of LDH release solution was added to each well of the sample control group. Serum in MEM medium and cultured at 37°C for 1 h.
  • Cell lysis rate (%) (absorbance of sample treatment group-absorbance of sample control group)/(absorbance of maximum enzyme activity control group-absorbance of sample control group) ⁇ 100%.

Abstract

本发明涉及化学与医药技术领域，公开了一种海藻糖衍生物与糖抗原的缀合物及其制备方法与应用。本发明所述缀合物以海藻糖作为内嵌佐剂，与肿瘤细胞表面异常表达的肿瘤相关糖抗原（STn或Tn）偶合得到；具有结构明确、合成方法简便、产物质量稳定可控等优点，特别是该疫苗的能克服糖抗原免疫原性弱的缺点，诱导产生高亲和力的特异性IgG抗体，从而达到靶向杀死肿瘤细胞的抗肿瘤效果。

Description

一种海藻糖衍生物与糖抗原的缀合物及其制备方法与应用 技术领域
本发明涉及化学与医药技术领域，尤其涉及一种海藻糖衍生物与糖抗原的缀合物及其制备方法与应用。
背景技术
如今癌症已成为人类的第一杀手并成为全球最大的公共卫生问题，恶性肿瘤死亡率呈明显增长趋势，严重的威胁人类的生活。现治疗癌症的方法有手术、化疗、放疗、免疫疗法，其中肿瘤免疫疗法是研究肿瘤治疗的热门之一。在肿瘤细胞表明异常过量表达的肿瘤相关糖抗原(TACAs)，具有重要的生物学功能，是糖抗原肿瘤疫苗设计的优秀靶点。
但是肿瘤相关糖抗原(TACAs)是T细胞非依赖性抗原，免疫原性较弱，需要在免疫原性载体分子的协助下才能刺激T细胞并诱导持久的抗体反应。如何提高糖抗原的免疫原性，使其更高效地诱导产生高滴度、高亲和力的IgG抗体并特异性地杀死肿瘤细胞，是肿瘤疫苗的研究热点之一。经典的策略是将糖抗原与载体蛋白缀合来增强其免疫原性，但糖蛋白疫苗有偶联位点不确定、偶联率不稳定、组成成分复杂等缺点。因此，肿瘤相关糖抗原疫苗的全合成策略成了新的研究热点之一；全合成糖抗原疫苗具有明确的结构，质量稳定可控等优点，便于各种免疫学和临床研究。近年来，科研工作者发现多种病原菌表面的糖分子可作为全合成糖抗原肿瘤疫苗的内嵌佐剂，其既能克服糖抗原免疫原性弱的缺点，又可以避免蛋白质引起的“表位抑制”效应。
海藻糖-6,6-二霉菌酸酯(TDM，索状因子)是结核分枝杆菌细胞壁中重要的糖脂成分，由海藻糖及2条霉菌酸酯链构成；该糖脂能引起炎症反应，并且对肿瘤细胞有一定的细胞毒性。研究表明，海藻糖-6,6-二霉菌酸酯的衍生物Vizantin和TDE免疫活性高且毒性低，可作为有效佐剂用于疫苗研究。
发明内容
本发明的目的在于提供一种海藻糖衍生物与糖抗原的缀合物。本发明以强效免疫刺激剂海藻糖衍生物Vizantin和TDE作为内嵌佐剂分别缀合糖抗原(STn、Tn)得到所述缀合物，其中海藻糖衍生物能提高糖抗原的免疫原性，所述缀合物能够诱导T细 胞介导体液免疫，产生高浓度的高亲和力的IgG抗体，达到特异性杀死肿瘤细胞的目的。
本发明的另一目的在于提供所述海藻糖衍生物与糖抗原的缀合物的制备方法。
本发明的再一目的在于提供所述海藻糖衍生物与糖抗原的缀合物在制备肿瘤疫苗中的应用。
本发明的还一目的在于提供所述海藻糖衍生物与糖抗原的缀合物在制备抗肿瘤药物中的应用。
本发明的上述目的是通过以下方案予以实现的：
一种海藻糖衍生物与糖抗原的缀合物，所述缀合物结构通式如下式I所示：
[X—L—Y]
式(I)
其中，X表示糖抗原，选自下式中的任意一种糖抗原，或其可要药用盐、水合物或溶剂化物：
Figure PCTCN2020130230-appb-000001
L表示连接体，选自以下
Figure PCTCN2020130230-appb-000002
(CH 2CH 2O) a-CH 2CH 2-C(O)NH-、
Figure PCTCN2020130230-appb-000003
(CH 2CH 2O) a-、-(OCH 2CH 2) a
Figure PCTCN2020130230-appb-000004
-NHC(O)-(CH 2CH 2O) a-CH 2CH 2-C(O)NH-、-NHC(O)-(CH 2CH 2O) a-CH 2CH 2
Figure PCTCN2020130230-appb-000005
-(OCH 2CH 2) a-CH 2CH 2-C(O)NH-、
Figure PCTCN2020130230-appb-000006
(CH2) a-C(O)NH-、-NHC(O)-(CH 2) a-C(O)NH-、-NHC(O)-(CH 2) a
Figure PCTCN2020130230-appb-000007
中的任意一种，a是0到20中的任意一个整数；
Y表示海藻糖衍生物，选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：
Figure PCTCN2020130230-appb-000008
其中，R 1和R 2各自独立地选自氢、-CH 2-CH(OR 3)-(CH 2) m-CH 3，-(CH 2) mCH 3或-(CH 2) mCHR 3 2，R 3为-(CH 2) m-CH 3或-C(O)-(CH 2) m-CH 3，m是选自8-26的整数。
优选地，所述L选自
Figure PCTCN2020130230-appb-000009
(CH 2CH 2O) a-、-(OCH 2CH 2) a
Figure PCTCN2020130230-appb-000010
-NHC(O)-(CH 2CH 2O) a-CH 2CH 2
Figure PCTCN2020130230-appb-000011
(CH2) a-C(O)NH-、-NHC(O)-(CH 2) a
Figure PCTCN2020130230-appb-000012
中的任意一种，a是0到20中的任意一个整数。
优选地，所述L选自
Figure PCTCN2020130230-appb-000013
(CH 2CH 2O) a-、-(OCH 2CH 2) a
Figure PCTCN2020130230-appb-000014
-NHC(O)-(CH 2) a
Figure PCTCN2020130230-appb-000015
中的任意一种，a是0到20中的任意一个整数。
优选地，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：
Figure PCTCN2020130230-appb-000016
其中，R 1和R 2各自独立地选自氢、-(CH 2) mCH 3或-(CH 2) mCHR 3 2，R 3为-(CH 2) m-CH 3或-C(O)-(CH 2) m-CH 3，m是选自8-26的整数。
优选地，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：
Figure PCTCN2020130230-appb-000017
其中，R 1和R 2各自独立地选自氢或-(CH 2) mCH 3，m是选自8-26的整数。
优选地，所述缀合物选自如下任一结构或其可要药用盐、水合物或溶剂化物：
Figure PCTCN2020130230-appb-000018
本发明同时所述海藻糖衍生物与糖抗原的缀合物的制备方法，当Y为
Figure PCTCN2020130230-appb-000019
时，制备过程包括：
S1.化合物7在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物8；
S2.化合物8在三氟乙酸的条件下，脱去PMB基团得到化合物9；
S3.化合物9与相应的脂肪酸链进行酯化缩合得到对应的化合物10或11；
S4.化合物10与化合物Tn或STn、化合物11与化合物STn分别在催化剂的作用 下反应得到化合物19或20、化合物21；
S5.化合物19、20和21分别经脱苄基保护反应，即可得到目标产物；
当Y为
Figure PCTCN2020130230-appb-000020
制备过程包括；
S11.化合物7在2,6-二甲基吡啶的催化下与叔丁基二甲基三氟甲磺酸酯反应得到化合物12；
S21.化合物12在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物化合物13；
S31.化合物13在三氟乙酸的条件下，脱去PMB基团得到化合物14；
S41.化合物14分别与相应的脂肪酸链进行酯化缩合得到对应的化合物15或16；
S51.化合物15或16分别脱去保护基团三甲基硅烷得到相应的化合物17或18；
S61.化合物17或18与三氟化硼***反应得到相应的化合物22或23；
S71.化合物22或23分别经脱苄基保护反应，即可得到目标产物；
其中，化合物7至23的结构如下所示：
Figure PCTCN2020130230-appb-000021
优选地，步骤S5和步骤S71中，脱苄基保护反应的催化剂为氢气/钯碳/氢氧化钯、氢气/钯碳或氢气/氢氧化钯等，优选氢气/钯碳；反应采用的溶剂为二氯甲烷/甲醇/水、二氯甲烷、甲醇或二氯甲烷/甲醇等；优选二氯甲烷/甲醇/水。
优选地，步骤S4和步骤S61中，反应的催化剂为碘化亚铜/N,N-二异丙基乙胺或碘化亚铜/N,N-二异丙基乙胺/冰乙酸等；优选碘化亚铜/N,N-二异丙基乙胺；反应的溶剂为二氯甲烷、二氯甲烷/甲醇或四氢呋喃/甲醇等；优选四氢呋喃/甲醇。
优选地，步骤S51中，反应的催化剂为三氟化硼***或三氟乙酸的水溶液等，优选三氟化硼***；反应的溶剂为乙腈、二氯甲烷或四氢呋喃等，优选为乙腈。
优选地，步骤S3和步骤S41中，反应过程中的缩合试剂可以选择4-二甲氨基吡啶(DMAP)/1-(3-二甲基氨丙基)-3-乙基碳二亚胺甲碘盐(EDC·MEI)，N,N’-二环己基碳二亚胺(DCC)/4-二甲氨基吡啶(DMAP)或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC .HCl)/4-二甲氨基吡啶(DMAP)等，优选DMAP/EDC .HCl。
优选地，化合物7的制备过程如下：
S31.以海藻糖为起始原料，在对甲苯磺酸的作用下，4-甲氧基苯甲醛二甲缩醛对原料进行4,6位羟基选择性保护，得到化合物5，用溴化卞对裸露的羟基进行全苄基化得化合物6；
S32.化合物6选择性还原4,4’位糖羟基得到化合物7；
其中化合物5和6的结构如下：
Figure PCTCN2020130230-appb-000022
优选地，步骤S32中，反应的催化剂为三乙基硅烷/三氟甲磺酸或氰基硼氢化钠/盐酸***；反应在溶剂为二氯甲烷、甲醇或四氢呋喃中进行。
更优选地，步骤S32中，反应的催化剂为氰基硼氢化钠/盐酸***；反应在溶剂二氯甲烷中进行。
本发明上述制备路线简短、反应条件温和、产率高、操作方便，能够用于工业化制备。
所述海藻糖衍生物与糖抗原的缀合物在制备肿瘤疫苗中的应用也在本发明的保护范围之内。
所述海藻糖衍生物与糖抗原的缀合物在制备抗肿瘤药物中的应用也在本发明的保护范围之内。
优选地，所述肿瘤疫苗或抗肿瘤药物为乳腺癌、子宫癌、卵巢癌、肺癌、肝癌、***癌、黑素瘤、胰腺癌、肠癌、肾细胞癌、细胞性淋巴癌、甲腺癌、脑癌、胃癌或白血病的疫苗或药物。
与现有技术相比，本发明具有以下有益效果：
本发明以强效免疫刺激剂海藻糖衍生物Vizantin和TDE作为内嵌佐剂分别缀合糖抗原(STn、Tn)得到所述缀合物，其中海藻糖衍生物能提高糖抗原的免疫原性，使得所述缀合物能够诱导T细胞介导体液免疫，产生高浓度的高亲和力的IgG抗体，达到特异性杀死肿瘤细胞的目的，可制备成为肿瘤疫苗或抗肿瘤药物进行应用。
附图说明
图1为缀合物1，2，3与4制备的疫苗诱导小鼠产生的抗体免疫活性评价图。
图2为缀合物1，2，3与4制备的疫苗诱导小鼠产生的抗体血清特异性杀死肿瘤细胞MCF-7的补体依赖性细胞毒性评价图。
具体实施方式
下面结合具体实施例对本发明做出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1海藻糖衍生物与糖抗原缀合物1、2、3、4与L2-3的制备
1)化合物5的合成
Figure PCTCN2020130230-appb-000023
将海藻糖(10.00g,0.03mol)和对甲苯磺酸一水化合物(1.10g,5.84mol)溶于二甲基甲酰胺中，在氮气保护下滴加茴香醛二甲缩醛(20mL,0.117mol)，室温搅拌反应，反应24小时后，乙酸乙酯稀释，饱和碳酸氢钠溶液，静置，有白色固体析出，抽滤，得化合物5(11.90g,70％)。 1H NMR(400MHz，CD 3OD)δ7.41-7.39(d,J＝8.8Hz,4H),6.88-6.68(d,J＝8.8Hz,4H),5.51(s,2H,PhCH),5.11(d,J＝3.6Hz,2H,H-1&H-1′),4.19(dt,J＝10.0,4.8Hz,2H,H5&H5′),4.12-4.06(m,2H,H3&H3′),4.03-3.98(t,J＝9.6,2H,H4&H4′),3.77(s，6H),3.72-3.67(t,J＝10.4Hz,2H),3.61(dd,J 1＝9.2Hz,J 2＝4.0Hz,2H),3.45(t,J＝9.6,2H),3.31-3.29(m,2H)；HR-ESI-MS(m/z):calcd for C 28H 34O 13[M+H] +579.2072 found,579.2067.
2)化合物6的制备
Figure PCTCN2020130230-appb-000024
化合物5(2.98g，5.75mmol)溶于N-N-二甲基甲酰胺中，在氮气的保护下，加入 氢化钠(60％in oil；2.30g，57.5mmol)，反应液降温到0℃，依次加入溴化卞(5.5mL，45.98mmol)、四丁基碘化铵(425.01mg，1.15mmol)。室温反应18h，反应液降温至0℃，滴少量甲醇溶液进行反应的淬灭，乙酸乙酯稀释，饱和食盐水冲洗混合液，收集有机层，浓缩得粗品，硅胶柱色谱纯化得白色固体化合物6(14.90g,78.4％)。 1H NMR(400MHz，CDCl 3)δ7.34-7.24(m,24H),6.92-6.90(d,J＝8.8Hz，4H)5.51(s,2H,PhCH)，5.11(d,J＝3.8Hz，2H,H-1&H-1′),4.96(d,J＝10.8Hz,2H,PhCH 2O),4.85-4.81(dd,J＝10.8Hz,4H),4.72(d,J＝10.8Hz,2H,PhCH 2O),4.27(dt,J＝10.0,4.8Hz,2H,H5&H5′),4.14(t,J＝9.4Hz,2H,H3&H3′),4.12-4.10(m,2H,H4&H4′),3.83(S,6H),3.69-3.59(m,6H)；HR-ESI-MS(m/z):calcd for C 54H 58O 11[M+H] +939.395 found，939.3933.
3)化合物7的制备
Figure PCTCN2020130230-appb-000025
将6(2.00g，2.11mol)溶于四氢呋喃中，依次加入氰基硼氢化钠、甲基橙指示剂，冰浴下，缓慢滴加盐酸***溶液，滴加至反应液的颜色由橙黄色渐变为粉红色，且半分钟内不变化，然后，缓慢升至室温，继续反应8小时，加乙酸乙酯溶液稀释，饱和食盐水洗3次，有机层减压浓缩，得黄色油状液体粗品。硅胶柱色谱纯化得呈白色油状液体化合物7(1.31g,68.2％)。 1H NMR(400MHz，CDCl 3)δ7.37-7.23(m,20H),7.19-7.16(d,J＝6.4Hz,4H),6.83-6.80(d,J＝6.4Hz,4H),5.20(d,J＝3.6Hz,2H),5.00(d,J＝11.2Hz,2H),4.80(d,J＝11.2Hz,2H),4.70-4.60(q,J＝12.0Hz,4H),4.40(q,J＝12.0Hz,4H),4.73-4.63(m,6H),4.12-4.08(dt,J 1＝13.6Hz,J 2＝7.6Hz,2H),3.89-3.87(t,J＝9.2Hz,2H),3.77(s,6H),3.67-3.65(t,J＝9.2Hz,2H)，3.53(dd,J＝9.6,3.6Hz,2H),3.51-3.43(qd,J＝10.4,4.0Hz,4H)；HR-ESI-MS(m/z):calcd for C 56H 62O 13[M+COOH] -987.4172 found,987.4177
4)化合物8的合成
Figure PCTCN2020130230-appb-000026
N-N'-二甲基甲酰胺溶解化合物7(1.01g,1.06mmol)，加入氢化钠(60％in oil；2.31g,57.5mmol)，反应液降温至0℃，依次加入溴丙炔(5.5mL，45.98mmol)、四丁基溴化铵(425.10mg,1.15mmol)。室温搅拌反应4h，反应液的颜色由乳白变为浊黄色，滴少量甲醇溶液进行反应的淬灭，乙酸乙酯稀释反应液，饱和食盐水冲洗混合液，有机层浓缩的粗品，硅胶柱色谱纯化得白色油状化合物8(0.45g,42.3％)。 1H NMR(400MHz,CDCl 3)δ7.40-7.27(m,20H),7.21-7.18(d,J＝8.4Hz,4H),6.84-6.81(d,J＝8.4Hz,4H),5.15-5.14(d,J＝3.6Hz,2H),4.98-4.83(q,J＝10.4Hz,4H),4.64(s,4H),4.48-4.45(d,J＝11.6Hz,4H),4.39-4.33(m,4H),4.16-4.11(m,2H),3.98-3.96(t,J＝9.2Hz,2H),3.79(s,6H)，3.53-3.48(m,6H),3.40-3.37(m,4H),2.41(t,J＝2.4Hz,2H)；HR-ESI-MS(m/z):calcd for C 62H 66O 13[M+Na] +1041.4396 found,1041.4395.
5)化合物9的合成
Figure PCTCN2020130230-appb-000027
二氯甲烷溶解化合物8(50.20mg,0.0491mmol)，缓慢滴加5％TFA(0.15mL)，反应2h，二氯甲烷稀释，饱和碳酸氢钠冲洗，有机相减压浓缩得粗品，柱层析分离纯化得透明无色油状化合物9(33.20mg,85.7％)。 1H NMR(400MHz,CDCl 3)δ7.39-7.28(m,20H),5.12-5.11(d,J＝3.6Hz,2H),5.01-4.98(d,J＝10.8Hz,2H),4.86-4.83(d,J＝10.8Hz,2H),4.72-4.65(m,4H),4.46-4.37(m,4H),4.04-4.00(m,4H),3.72-3.60(m,4H),3.54-3.49(m,4H),2.49-2.48(t,J＝2.0Hz,2H)；HR-ESI-MS(m/z):calcd for C 46H 50O 11[M+COOH] -823.3335 found,823.3311.
6)化合物10的合成
Figure PCTCN2020130230-appb-000028
二氯甲烷溶解化合物9(21.03mg)，自制脂肪酸链(22.00mg)，EDC/HCl(4eq)和DMAP(1eq)，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物10(20.36mg,54％)。 1H NMR(400MHz in CDCl 3)δ7.40-7.27(20H,m),5.15-5.14(2H,d,J＝3.2Hz),5.00-4.98(2H,d,J＝10.8Hz),4.86-4.84(2H,d,J＝10.8Hz),4.72-4.66(2H,q,J＝12.4Hz),4.46-4.42(2H,dd,J＝12.4Hz),4.32-4.28(2H,dd,J 1＝15.2Hz,J 2＝2.4Hz),4.19(2H,m),4.14(4H,m),4.01(2H,t,J＝9.2Hz),3.52(2H,dd,J 1＝9.6Hz,J 2＝3.2Hz),3.45(2H,t,J＝9.2Hz),2.44(2H,s),2.19(4H,d,J＝6.8Hz),1.80(2H,m),1.24(64H,m),0.87(12H,t,J＝6.4Hz). 13C NMR(100MHz in CDCl 3)δ14.15,22.71,26.56,26.51,29.37,29.64,29.67,29.95,31.93,33.71,33.78,34.86,39.08,60.14,62.59,68.89,72.95,74.62,75.75,77.25,79.26,79.70,81.33,94.02,127.44,127.58,127.77,127.92,128.11,128.48,128.55,137.78,137.71,138.45,173.24.HR-ESI-MS(m/z):calcd for C 88H 130O 13[M+Na] +1417.9404 found,1417.9404.
7)化合物11的制备
Figure PCTCN2020130230-appb-000029
二氯甲烷溶解化合物9(40.20mg)，Behenic Acid(52.53mg)，EDC/HCl和DMAP，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物11(46.80mg,63％)。 1H NMR(400MHz in CDCl 3)δ7.37-7.25(20H,m),5.15-5.14(2H,d,J＝3.6Hz),5.00-4.98(2H,d,J＝10.8Hz),4.86-4.84(2H,d,J＝10.8Hz),4.69(4H,m),4.37-4.33(2H,dd,J 1＝15.6Hz,J 2＝2.4Hz),4.32-4.28(2H,dd,J 1＝15.6Hz,J 2＝2.4Hz),4.15-4.03(6H,m),3.96 (2H,t,J＝9.2Hz),3.49-3.44(2H,dd,J 1＝10.0Hz,J 2＝3.6Hz),3.41-3.39(4H,t,J＝9.2Hz,2H),2.37(2H,t,J＝2.4Hz),2.25(2H,t,J＝7.6Hz),1.48(4H,m),1.20(76H,m),0.82(6H,t,J＝6.8Hz). 13C NMR(100MHz in CDCl 3)δ14.17,22.73,24.88,29.19,29.30,29.40,29.52,29.65,29.70,29.74,31.96,34.16,60.11,62.73,68.90,72.95,74.63,75.76,77.27,79.18,79.72,81.36,94.04,127.44,127.59,127.81,127.95,128.13,128.51,128.57,137.78,137.71,138.41,173.51.HR-ESI-MS(m/z):calcd for C 90H 134O 13，[M+Na] +1445.9710 found,1445.9730.
8)化合物12的合成
Figure PCTCN2020130230-appb-000030
二氯甲烷溶解化合物7(270.21mg,0.29mmol)和2,6-二甲基吡啶(0.15mL)，0℃下缓慢加入叔丁基二甲基三氟甲磺酸酯(0.14mL)，室温下搅拌反应30min，加水淬灭，二氯甲烷萃取，有机相减压浓缩，粗品过硅胶柱，得淡黄色油状化合物12(171.14mg,56.5％)。 1H NMR(400MHz,CDCl 3)δ7.56-7.09(m,26H),6.84-6.82(d,J＝6.0Hz,4H),5.32-5.26(d,J＝3.6Hz,2H),5.10-5.01(dd,J 1＝28.4Hz,J 2＝11.6Hz,2H),4.85(d,J＝11.2Hz,1H),4.76-4.75(m,2H),4.63(d,J＝12.0Hz,2H),4.55-4.52(d,J＝12.0Hz,1H),4.45-4.32(m,4H),3.92-3.81(m,2H),3.80(s,6H),3.67-3.58(m,4H),3.51-3.43(m,4H)；HR-ESI-MS(m/z):calcd for C 62H 76O 13Si，[M+Na] +1079.4947 found,1079.4951.
9)化合物13的合成
Figure PCTCN2020130230-appb-000031
-N'-二甲基甲酰胺溶解化合物12(366.1mg,1eq)，在氮气的保护下，加入氢化钠(60％in oil；6eq)，0℃条件下依次加入溴丙炔(6eq)与四丁基溴化铵(6eq)；室温反应4h，反应液降温至0℃，缓慢滴加少量甲醇淬灭反应，二氯甲烷稀释，依次用 饱和碳酸氢钠溶液、饱和食盐水冲洗，收集有机相，减压浓缩，过硅胶柱分离纯化的化合物13(380.01mg,97％). 1H NMR(400MHz,CDCl 3)δ7.40-7.27(m,20H),7.21-7.18(m,4H),6.84-6.81(m,4H),5.26(dd,J 1＝22.4Hz,J 2＝2.8Hz,2H),5.12(d,J＝11.6Hz,1H),4.87-4.84(d,J＝11.6Hz,2H),4.64(s,4H),4.39-4.33(m,4H),4.16-4.11(m,2H),3.79(m,6H),3.53-3.48(m,6H),3.40-3.37(m,4H),2.41(m,2H),2.43(s,1H),0.85(s,9H),0.01(dd,J＝3.2Hz,6H)；HR-ESI-MS(m/z):calcd for C 65H 78O 13Si，[M+Na] +1117.5104found,1117.5107.
10)化合物14的合成
Figure PCTCN2020130230-appb-000032
二氯甲烷溶解化合物13(50.20mg,0.049mmol)，缓慢滴加5％TFA(0.15mL)，反应2h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩，所得粗品经柱层析分离纯化，得透明无色油状化合物14(33.10mg，87.5％)； 1H NMR(400MHz,CDCl 3)δ7.32-7.12(m,20H),5.13-5.08(dd,J 1＝21.6Hz,J 2＝2.8Hz,2H),5.01-4.98(d,J＝11.6Hz,1H),5.01-4.98(d,J＝11.6Hz,1H),4.94-4.91(d,J＝11.6Hz,1H),4.82-4.80(d,J＝11.6Hz,1H),4.73-4.70(d,J＝11.6Hz,1H),4.66-4.59(m,2H),4.56-4.50(m,2H),4.38-4.30(m,2H),4.03-4.01(t,J＝9.2Hz,2H),3.93-3.83(m,2H),3.76-3.70(m,2H),3.62-3.52(m,4H),3.49-3.43(m,5H),2.47(s,1H),0.87(s,9H),0.01(d,J＝24.0Hz,6H)； 13C NMR(100MHz,CDCl 3)δ139.09,138.53,137.97,137.86,128.52,128.48,128.46,128.17,128.15,127.79,127.76,127.63,127.43,127.08,93.54,81.16,81.08,80.32,79.96,79.85,77.43,77.32,76.80,75.52,74.91,74.54,73.13,72.93,72.85,71.13,70.38,61.38,59.97,26.08,18.17,-3.66,-4.88.HR-ESI-MS(m/z):calcd for C 49H 62O 11Si,[M+COOH] -899.4043 found,899.4034.
10)化合物15的合成
Figure PCTCN2020130230-appb-000033
二氯甲烷溶解化合物14(140.01mg)，自制脂肪酸链(213.77mg)，EDC/HCl和DMAP，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物15(169.50mg,70.3％)； 1H NMR(400MHz,CDCl 3)δ7.37-7.18(m,20H),5.22-5.18(dd,J 1＝16.8Hz,J 2＝3.2Hz,2H),5.11-5.08(d,J＝11.6Hz,1H),5.00-4.97(d,J＝11.6Hz,1H),4.88-4.86(d,J＝10.4Hz 1H),4.77-4.63(m,4H),4.57-4.54(d,J＝11.6Hz,1H),4.44-4.27(dq,J 1＝15.2Hz,J 2＝2.0Hz,2H),4.18-4.04(m,6H),3.89(dd,J 1＝12.0Hz,J 2＝3.6Hz,1H),3.79(t,J＝8.8Hz,1H),3.65-3.63(t,J＝9.6Hz,1H),3.57-3.53(td,J 1＝9.2Hz,J 2＝3.2Hz,2H),3.46(t,J＝9.2Hz,1H),2.44(s,1H),2.18(m,4H),1.78(s,2H),1.26(m,64H),0.89-0.85(m,21H),0.01-0.00(d,J＝2.4Hz,6H). 13C NMR(100MHz,CDCl 3)δ14.15,22.71,26.56,26.51,29.37,29.64,29.67,29.95,31.93,33.71,33.78,34.86,39.08,60.14,62.59,68.89,72.95,74.62,75.75,77.25,79.26,79.70,81.33,94.02,127.44,127.58,127.77,127.92,128.11,128.48,128.55,137.78,137.71,138.45,173.24.HR-ESI-MS(m/z):calcd for C 91H 142O 13Si[M+Na] +1494.0112 found,1494.0111.
11)化合物16的合成
Figure PCTCN2020130230-appb-000034
二氯甲烷溶解化合物14(250.00mg)，Behenic Acid(398.80mg)，EDC/HCl(223.47mg)和DMAP(35.75mg)，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物16(334.51mg,76.2％)； 1H NMR(400MHz,CDCl 3)δ7.36-7.21(m,20H),5.22-5.18(dd,J 1＝16.8Hz,J 2＝3.2Hz,2H),5.11-5.08(d,J＝11.6Hz,1H),5.00-4.97(d,J＝11.6Hz, 1H),4.88-4.86(d,J＝10.4Hz 1H),4.75-4.56(m,5H),4.44-4.20(m,2H),4.09(m,6H),3.93-3.76(m,2H),3.66-3.45(m,4H),2.43(d,J＝4Hz,1H),2.2(m,4H),1.62(s,2H),1.25(m,76H),0.88-0.85(m,15H),0.01-0.00(s,6H). 13C NMR(100MHz,CDCl 3)δ-5.02,-3.62,1.03,14.14,18.13,22.71,24.79,24.87,26.01,29.18,29.29,29.38,29.50,29.65,29.72,31.94,34.11,34.15,60.06,62.79,68.82,70.57,70.60,72.74,73.09,74.59,74.80,75.58,79.73,79.89,80.89,81.10,93.38,127.03,127.32,127.58,127.81,127.82,127.87,128.11,128.20,128.47,128.57,137.61,137.75,138.37,138.99,173.47.HR-ESI-MS(m/z)calcd for C 96H 138O 13Na,(M+Na) +1522.0036,found 1522.0020.
12)化合物17与18的合成
Figure PCTCN2020130230-appb-000035
乙腈溶解化合物16(220.01mg)，0℃下缓慢滴加三氟化硼***(40μL)，反应液逐渐变黄色，反应2小时后，用饱和碳酸氢钠溶液终止中和反应，二氯甲烷溶液稀释萃取，收集有机相，减压浓缩，所得粗品经柱层析分离纯化，得透明淡黄色油状液体化合物18(140.00mg,68.9％)。 1H NMR(400MHz,CDCl 3)δ7.36-7.21(m,20H),5.17-5.13(d,J＝3.2Hz,2H),5.01-4.83(dq,J 1＝10.8Hz,J 2＝6.0Hz,4H),4.73-4.66(m,4H),4.44-4.40(dd,J 1＝15.2Hz,J 2＝2.4Hz,1H),4.34-4.28(m,2H),4.23-4.11(m,4H),4.04-4.00(t,J＝9.6Hz,2H),3.90-3.85(m,2H),3.56-3.40(m,4H),2.43(t,J＝2.4Hz,1H),2.29-2.24(m,4H),1.56(m,4H),1.25(m,76H),0.88-0.85(t,J＝2.4Hz,6H).HR-ESI-MS(m/z)calcd for C 87H 132O 13,(M+Na) +1407.9578,found 1407.9567.
Figure PCTCN2020130230-appb-000036
同方法制得白色固体化合物17(41.76mg，60.4％)。 1H NMR(400MHz,CDCl 3)δ7.36-7.30(m,20H),5.18(d,J＝3.2Hz,2H),5.01-4.83(dq,J 1＝10.8Hz,J 2＝6.0Hz,4H), 4.75-4.56(m,4H),4.45-4.41(dd,J 1＝15.2Hz,J 2＝2.4Hz,1H),4.32-4.28(m,2H),4.23-4.11(m,4H),4.04-3.99(t,J＝9.6Hz,2H),3.89-3.85(m,2H),3.54-3.40(m,4H),2.45(s,1H),2.21-2.18(m,4H),1.80(m,2H),1.25(s,64H),0.88-0.85(t,J＝6.4Hz,12H), 13C NMR(100MHz,CDCl 3)δ14.15,22.71,26.52,26.57,29.38,29.65,29.67,29.94,31.93,33.71,33.75,33.77,33.79,34.88,34.94,39.04,39.10,60.14,62.55,68.93,69.92,70.16,72.87,73.05,74.66,75.53,75.76,78.91,79.40,79.68,80.61,81.40,94.04,94.37,127.45,127.80,127.83,127.93,128.05,128.10,128.50,128.55,137.73,137.75,138.39,138.66,173.27,174.23.HR-ESI-MS(m/z)calcd for C 85H 128O 13Na,(M+Na) +1379.9247,found 1379.9206.
13)化合物19的合成
Figure PCTCN2020130230-appb-000037
四氢呋喃与甲醇(1：1)溶解化合物10(40.00mg,0.028mmol)、Tn(20.00mg,0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物19(24.35mg,43％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ7.83(s,2H),7.21-7.16(m,20H),5.11(s,2H),4.86(m,4H),4.62(m,20H),4.03(m,14H),3.70(s,4H),3.49(m,13H),2.1(s,4H),1.83(s,6H),1.68(s,64H),0.78(m,12H)；HR-ESI-MS(m/z):calcd for C 108H 166N 8O 25,[M+H] +1976.2037 found,1976.2007
14)化合物20的合成
Figure PCTCN2020130230-appb-000038
四氢呋喃与甲醇(1：1)溶解化合物10(40.01mg，0.028mmol)、STn(20mg，0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物20(38mg,51.9％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ7.63(s,1H),7.38-7.24(m,20H),5.19(s,2H),5.00-4.96(d,J＝12.8Hz,4H),4.86-4.82(d,J＝8.0Hz,2H),4.76-4.69(m,8H),3.56(m,5H),4.51(s,4H),4.13(m,4H),3.97-3.89(m,5H),3.90-3.63(m,24H),3.58-3.47(m,10H),2.75-2.69(dd,J 1＝13.2Hz,J 2＝3.6Hz,4H),2.24-2.21(d,J＝6.8Hz,5H),2.03-20.1(d,J＝9.2Hz,12H),1.84(m,4H),1.26(s,64H),0.88(t,J＝6.4Hz,12H)；HR-ESI-MS(m/z):calcd for C 130H 200N 10O 41,[M+2H] +21279.7009 found,1279.7037.
15)化合物21的合成
Figure PCTCN2020130230-appb-000039
四氢呋喃与甲醇(1：1)溶解化合物11(30.01mg,0.021mmol)、STn(29.01mg，0.050mmol)、碘化亚铜(79.80mg,0.42mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶 柱分离纯化得到化合物21(22.31mg,43％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ7.74-7.67(d,J＝26.4Hz,1H),7.36-7.07(m,20H),5.19(d,J＝5.2Hz,2H),5.07-4.93(d,1H),4.90-4.82(d,J＝5.2Hz,1H),4.76-4.63(m,4H),4.60-4.54(m,2H),4.41-4.09(m,20H),3.92-3.65(m,14H),3.58-3.48(m,8H),3.26-3.25(d,J＝4.8Hz,2H),2.56-2.52(m,2H),2.34-2.27(m,4H),2.08-1.98(m,12H),1.80-1.62(m,2H),1.60-1.50(m,4H),1.26(s,76H),0.88(m,6H).HR-ESI-MS(m/z):calcd for C 132H 204N 10O 41,[M+2H] +2 1293.7165 found,1293.7167
16)化合物22的合成
Figure PCTCN2020130230-appb-000040
四氢呋喃与甲醇(1：1)溶解化合物17(35.01mg，0.026mmol)、STn(20mg，0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物22(14.5mg，31.1％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ7.77-7.71(s,1H),7.40-7.24(m,20H),5.21-5.20(dd,J 1＝6.8Hz,J 2＝3.2Hz,2H),5.01-4.87(m,5H),4.78-4.75(d,J＝11.6Hz,2H),4.71-4.47(m,8H),4.23-4.16(m,5H),4.16-4.01(m,7H),4.13(m,4H),3.97-3.44(m,21H),2.75-2.71(m,1H),2.24-2.20(m,4H),2.05(s,6H),1.84-1.77(m,4H),1.62(m,1H),1.46(m,4H),1.26(s,64H),0.88(t,J＝5.6Hz,12H)；HR-ESI-MS(m/z):calcd for C 106H 163N 5O 27,[M+H] +1939.1608 found,1939.1629
17)化合物23的合成
Figure PCTCN2020130230-appb-000041
四氢呋喃与甲醇(1：1)溶解化合物18(36.01mg,0.026mmol)、STn(20mg，0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物23(22.80mg,44.5％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ7.65(s,1H),7.38-7.28(m,20H),5.22-5.20(d,J＝7.6Hz,2H),5.01-4.90(m,5H),4.78-4.71(m,6H),4.51-4.48(m,3H),4.27-3.99(m,11H),3.60-3.43(m,7H),2.74-2.70(m,1H),2.27(s,4H),2.04-2.00(d,J＝12.0Hz,6H),1.80-1.75(m,1H),1.57s,5H),1.26(s,64H),0.88(t,J＝5.6Hz,12H)；HR-ESI-MS(m/z):calcd for C 108H 167N 5O 27,[M+H] +1967.1921 found,1967.1908
18)化合物L2-3的合成
Figure PCTCN2020130230-appb-000042
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物19(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物L2-3(4.81mg，56.7％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ8.01(s,2H),4.92-4.90(m,2H),4.88(s,2H),4.78-4.50(m,14H),4.21(m,2H),4.23-4.17(m,4H),4.01-3.92(m,4H),3.86-3.82(t,J＝9.2Hz,2H),3.72-3.68(m,4H),3.64-3.48(m, 7H),3.41-3.38(dd,J 1＝9.2Hz,J 2＝2.8Hz,2H),3.39-3.23(t,J＝9.6Hz,4H),2.97-2.92(q,J＝7.6Hz,1H),2.61(s,1H),2.20-2.18(d,J＝6.8Hz,3H),1.88(s,6H),1.75(s,2H),1.19(s,64H),0.88(t,J＝5.6Hz,12H).HR-ESI-MS(m/z):calcd for C 80H 142N 8O 25[M+H] +1616.0159 found,1616.0162.
19)化合物1的合成
Figure PCTCN2020130230-appb-000043
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物20(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物1(4.72mg，53.8％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ8.0(s,2H),4.96(s,2H),4.90-4.87(m,3H),4.6-4.53(m,5H),4.22-4.11(m,7H),3.98-3.86(m,6H),3.80-3.69(m,10H),3.65-3.47(m,10H),3.43-3.31(m,8H),2.94-2.91(dd,J 1＝11.2Hz,J 2＝4.8Hz,2H),2.60(m,2H),2.21-2.19(d,J＝6.4Hz,4H),1.93-1.75(m,12H),1.69-1.62(m,2H),1.55-1.48(m,2H),1.21(s,64H),0.88(t,J＝6.0Hz,12H).HR-ESI-MS(m/z):calcd for C 102H 176N 10O 41[M+Na] +2220.1887 found,2220.1807.
20)化合物2的合成
Figure PCTCN2020130230-appb-000044
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物21(10.05mg)，加入钯碳(15.64 mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物2(6.10mg,54％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ8.09(s,2H),5.1(s,2H),4.97-4.96(d,J＝1.2Hz,2H),4.85-4.74(m,5H),4.43-4.13(m,10H),4.10-3.96(m,9H),3.91-3.68(m,25H),3.62-3.41(m,11H),3.05-3.00(dd,J 1＝14.4Hz,J 2＝7.2Hz,2H),2.75-2.70(m,2H),2.41-2.37(t,J＝7.2Hz,4H),2.05(s,12H),1.81(m,2H),1.66-1.62(m,4H),1.27(s,76H),0.88(t,J＝6.0Hz,6H).HR-ESI-MS(m/z):calcd for C 104H 180N 10O 41,[M+2H] +2 1113.6226 found,1113.6251.
21)化合物3的合成
Figure PCTCN2020130230-appb-000045
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物22(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物3(6.01mg,74.1％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ8.12(s,1H),5.1(s,2H),5.00-4.96(m,2H),4.66-4.61(m,4H),4.36-4.30(m,2H),4.29-4.16(m,4H),4.09-3.97(m,5H),3.9-3.42(m,22H),3.23-3.1(m,2H),3.05-3.00(dd,J 1＝14.8Hz,J 2＝7.2Hz,2H),2.78-2.67(m,4H),2.32-2.28(m,4H),2.3(m,4H),2.05(s,6H),1.85(s,2H),1.66-1.62(m,1H),1.28(s,64H),0.88(t,J＝5.6Hz,6H).HR-ESI-MS(m/z):calcd for C 78H 139N 5O 27,[M+H] +1578.9730 found,1578.9726
22)化合物4的合成
Figure PCTCN2020130230-appb-000046
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物23(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物4(5.9mg，66.5％)。 1H NMR(400MHz,CD 3OD/CDCl 3)δ8.12(s,1H),5.11-5.10(d,J＝5.4Hz,2H),5.00-4.95(m,2H),4.38-4.21(m,5H),4.01-4.67(m,16H),3.63-3.45(m,6H),3.1-3.0(m,2H),2.72-2.70(d,J＝21.6Hz,1H),2.41-2.38(td,J 1＝17.2Hz,J 2＝7.6Hz,4H),2.07(s,6H),1.81-1.78(m,1H),1.66-1.62(m,4H),1.26(s,76H),0.89(t,J＝5.6Hz,6H).HR-ESI-MS(m/z):calcd for C 80H 143N 5O 27,[M+H] +1607.0043 found,1607.0044.
实施例2：ELISA免疫分析
(1)将制备好的化合物1至4分别作为疫苗分子1，2，3与4，按照疫苗分子：二硬脂酰基磷脂酰胆碱把(DSPC)：胆固醇＝1:6.5:5溶解于DCM-MeOH(1:1，v/v，2mL)混合液中，旋干溶剂，使瓶壁上形成一层薄的脂质膜。加入2.0mL羟乙基哌嗪乙硫磺酸(HEPES)缓冲液(20Mm,pH＝7.5)，超声10～20min得脂质体1，2，3与4。
(2)小鼠的免疫：取6-8周龄的C57BL/6小鼠，分成四组，每组6只。通过小鼠皮下注射的方式进行免疫试验，采用一次初始免疫和三次增强免疫的方案，分别在第1,14,21,28天注射步骤(1)中制备的脂质体1，2，3与4，每只每次注射量0.1mL，在第0,27,35,49天采血，每只小鼠取血0.1mL～0.2mL，4℃放置半个小时，离心，5000转/分钟，分离上层清亮血清。在本实验中，空白对照组(为免疫前血清)用到的是第一次取血分离出的血清，测定疫苗滴度用到的是第35天采血分离出的血清。
(3)ELISA免疫分析：用0.1M碳酸盐缓冲液(pH 9.6)，将Tn-HSA或STn-HSA配置成2.0μg/mL溶液，以每孔100μL的量加入96孔板，放入4℃孵育过夜；第二 天放入37℃培养箱孵育一小时；用PBST(PBS+0.05％吐温-20)洗板3次，每孔加入300μL洗液。洗板后，加入blocking buffer(PBST/1％BSA)；每孔加入250μL；常温孵育一小时，用PBST洗板3次。样品血清稀释，用PBS按照对半数稀释法从1：300倍稀释至1：656100倍；将稀释好的血清以每孔100μL加入96孔板中，放置在37℃培养箱孵育两个小时，洗板三次。分别在每孔加入100μL的HRP(辣根过氧化物酶)标记的Kappa,IgG,IgM，室温孵育一个小时；洗板3次。加入TMB(3,3',5,5'-四甲基联苯胺)溶液，每孔加入100μL，室温避光显色20min。加入0.5M H 2SO 4溶液，每孔加100μL。在450-570nm双波长处，用酶标仪检测吸光度。
4)将吸光度(OD)值相对于抗血清稀释值作图，并获得最佳拟合线。使用该线的方程式来计算OD值达到0.2时的稀释度值，并且根据稀释值的倒数计算抗体滴度如图6所示。
从图1可看出，本发明实施例1合成的海藻糖衍生物与糖抗原的缀合物1，2，3与4，在无外加佐剂的情况下，均能诱导产生高滴度的抗体，产生的特异性IgG抗体滴度是IgM的2～3倍多，表明共价偶联的糖抗原疫苗能诱导T细胞介导的体液免疫；其中双接STn抗原的缀合物疫苗1与2引发的IgG抗体滴度均略高于单接抗原的缀合物3与4，说明抗原的空间越复杂，表位数多，对免疫反应具有一定的促进作用；海藻糖衍生物脂肪酸链为具有支链的CH(C 9H 19) 2的缀合物疫苗1与3诱导产生的抗体分别略高于脂肪酸链为酯链(CH 2) 20CH 2的缀合物疫苗2与4，说明海藻糖衍生物脂肪酸链的个数与长短对活性有一定影响。
实施例3：抗体介导的互补依赖细胞毒性(CDC)
用胰酶消化处于对数生长期的CT-26细胞(小鼠结肠癌细胞)，以每孔1×10 4个细胞种于96孔板，在37℃培养过夜，用不含血清的1640培养基洗板两次。取化合物1组在38天取血的6只小鼠血清样品，每个样品取7μl，然后将6只小鼠的血清样品混合，用不含血清的1640培养基稀释50倍，得小鼠血清稀释液。化合物2组、3组、4组以及空白对照组的小鼠血清稀释液按上述方法制备。1组、2组、3组、4组以及空白对照组(为免疫前血清)均设置样品最大酶活性对照组，样品对照组以及样品处理组，每个组平行做6个复孔，每孔加入100μl稀释后的小鼠血清溶液，将96孔板置于37℃培养2h。用不含血清的1640培养基洗板两次后，在样品处理组每孔加入100μl兔补体血清溶液，最大酶活性对照组每孔加入100μl的LDH释放液，样品对照组每孔 加入100μl不含血清的MEM培养基，置于37℃培养1h。
在新的96孔板每孔加入100μl的PBS溶液，小心吸取40μl上述96孔板的细胞上清液加至此新的96孔板中，每孔加入60μl的LDH检测液，避光孵育30min。用酶标仪在490nm处检测。
细胞裂解率(％)＝(样品处理组吸光度-样品对照组吸光度)/(最大酶活性对照组吸光度-样品对照组吸光度)×100％。
结果如图2所示，本发明实施例1合成的1，2，3与4糖疫苗诱导产生的抗血清均能介导CT-26细胞裂解，细胞裂解率均显著高于空白对照组(P<0.001)，说明糖疫苗1、2、3与4所诱导产生的抗体都有杀死肿瘤细胞能力。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种海藻糖衍生物与糖抗原的缀合物，其特征在于，所述缀合物结构通式如下式I所示：
    [X—L—Y]
    式(I)
    其中，X表示糖抗原，选自下式中的任意一种糖抗原，或其可要药用盐、水合物或溶剂化物：
    Figure PCTCN2020130230-appb-100001
    L表示连接体，选自以下
    Figure PCTCN2020130230-appb-100002
    (CH 2CH 2O) a-CH 2CH 2-C(O)NH-、
    Figure PCTCN2020130230-appb-100003
    (CH 2CH 2O) a-、-(OCH 2CH 2) a
    Figure PCTCN2020130230-appb-100004
    -NHC(O)-(CH 2CH 2O) a-CH 2CH 2-C(O)NH-、-NHC(O)-(CH 2CH 2O) a-CH 2CH 2
    Figure PCTCN2020130230-appb-100005
    -(OCH 2CH 2) a-CH 2CH 2-C(O)NH-、
    Figure PCTCN2020130230-appb-100006
    (CH2) a-C(O)NH-、-NHC(O)-(CH 2) a-C(O)NH-、-NHC(O)-(CH 2) a
    Figure PCTCN2020130230-appb-100007
    中的任意一种，a是0到20中的任意一个整数；
    Y表示海藻糖衍生物，选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：
    Figure PCTCN2020130230-appb-100008
    其中，R 1和R 2各自独立地选自氢、-CH 2-CH(OR 3)-(CH 2) m-CH 3，-(CH 2) mCH 3或-(CH 2) mCHR 3 2，R 3为-(CH 2) m-CH 3或-C(O)-(CH 2) m-CH 3，m是选自8-26的整数。
  2. 根据权利要求1所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述L选自
    Figure PCTCN2020130230-appb-100009
    (CH 2CH 2O) a-、-(OCH 2CH 2) a
    Figure PCTCN2020130230-appb-100010
    -NHC(O)-(CH 2CH 2O) a-CH 2CH 2
    Figure PCTCN2020130230-appb-100011
    Figure PCTCN2020130230-appb-100012
    (CH2) a-C(O)NH-、-NHC(O)-(CH 2) a
    Figure PCTCN2020130230-appb-100013
    中的任意一种，a是0到20 中的任意一个整数。
  3. 根据权利要求2所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述L选自
    Figure PCTCN2020130230-appb-100014
    (CH 2CH 2O) a-、-(OCH 2CH 2) a
    Figure PCTCN2020130230-appb-100015
    -NHC(O)-(CH 2) a
    Figure PCTCN2020130230-appb-100016
    中的任意一种，a是0到20中的任意一个整数。
  4. 根据权利要求1所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：
    Figure PCTCN2020130230-appb-100017
    其中，R 1和R 2各自独立地选自氢、-(CH 2) mCH 3或-(CH 2) mCHR 3 2，R 3为-(CH 2) m-CH 3或-C(O)-(CH 2) m-CH 3，m是选自8-26的整数。
  5. 根据权利要求4所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：
    Figure PCTCN2020130230-appb-100018
    其中，R 1和R 2各自独立地选自氢或-(CH 2) mCH 3，m是选自8-26的整数。
  6. 根据权利要求1所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述缀合物选自如下任一结构或其可要药用盐、水合物或溶剂化物：
    Figure PCTCN2020130230-appb-100019
    Figure PCTCN2020130230-appb-100020
  7. 权利要求1至6任一所述海藻糖衍生物与糖抗原的缀合物的制备方法，其特征在于，当Y为
    Figure PCTCN2020130230-appb-100021
    时，制备过程包括：
    S1.化合物7在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物8；
    S2.化合物8在三氟乙酸的条件下，脱去PMB基团得到化合物9；
    S3.化合物9与相应的脂肪酸链进行酯化缩合得到对应的化合物10或11；
    S4.化合物10与化合物Tn或STn、化合物11与化合物STn分别在催化剂的作用下反应得到化合物19或20、化合物21；
    S5.化合物19、20和21分别经脱苄基保护反应，即可得到目标产物；
    当Y为
    Figure PCTCN2020130230-appb-100022
    制备过程包括；
    S11.化合物7在2,6-二甲基吡啶的催化下与叔丁基二甲基三氟甲磺酸酯反应得到 化合物12；
    S21.化合物12在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物化合物13；
    S31.化合物13在三氟乙酸的条件下，脱去PMB基团得到化合物14；
    S41.化合物14分别与相应的脂肪酸链进行酯化缩合得到对应的化合物15或16；
    S51.化合物15或16分别脱去保护基团三甲基硅烷得到相应的化合物17或18；
    S61.化合物17或18与三氟化硼***反应得到相应的化合物22或23；
    S71.化合物22或23分别经脱苄基保护反应，即可得到目标产物；
    其中，化合物7至23的结构如下所示：
    Figure PCTCN2020130230-appb-100023
  8. 根据权利要求7所述海藻糖衍生物与糖抗原的缀合物的制备方法，其特征在于，化合物7的制备过程如下：
    S31.以海藻糖为起始原料，在对甲苯磺酸的作用下，4-甲氧基苯甲醛二甲缩醛对原料进行4,6位羟基选择性保护，得到化合物5，用溴化卞对裸露的羟基进行全苄基 化得化合物6；
    S32.化合物6选择性还原4,4’位糖羟基得到化合物7；
    其中化合物5和6的结构如下：
    Figure PCTCN2020130230-appb-100024
  9. 权利要求1至6任一所述海藻糖衍生物与糖抗原的缀合物在制备肿瘤疫苗中的应用。
  10. 权利要求1至6任一所述海藻糖衍生物与糖抗原的缀合物在制备抗肿瘤药物中的应用。
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