WO2021232719A1 - 一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物及其制备方法和应用 - Google Patents

一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物及其制备方法和应用 Download PDF

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WO2021232719A1
WO2021232719A1 PCT/CN2020/130228 CN2020130228W WO2021232719A1 WO 2021232719 A1 WO2021232719 A1 WO 2021232719A1 CN 2020130228 W CN2020130228 W CN 2020130228W WO 2021232719 A1 WO2021232719 A1 WO 2021232719A1
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compound
catalyst
organic solvent
cancer
dichloromethane
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廖国超
刘中秋
杨德盈
练庆海
高玲强
吴鹏
苏诗薇
曾莉茗
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广州中医药大学(广州中医药研究院)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/001172—Sialyl-Thomson-nouvelle antigen [sTn]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/056—Triazole or tetrazole radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • 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

Definitions

  • the invention relates to a conjugate containing an alpha-galactose ceramide analogue and a sugar antigen, and a preparation method and application thereof, and belongs to the technical field of the development of anti-tumor sugar vaccines.
  • Tumor sugar vaccines targeting tumor-associated carbohydrate antigens TACAs
  • TACAs tumor-associated carbohydrate antigens
  • Thomsennouveau (Tn) antigen is abnormally overexpressed on the surface of malignant tumor cells such as breast cancer, prostate cancer, lung cancer and so on. It is an excellent target for carbohydrate antigen tumor vaccine design.
  • Tn carbohydrate antigen is a T cell-independent antigen, it needs the assistance of immunogenic carrier molecules to stimulate T cells and induce a durable antibody response.
  • the traditional strategy is to couple the carbohydrate antigen to the carrier protein (KLH, BSA or CRM197) containing B cell epitopes.
  • the obtained glycoprotein vaccine can produce high-titer and high-affinity antibodies.
  • the KLH-conjugated protein vaccine of Tn has been used. Enter the clinical research stage.
  • glycoprotein vaccines still have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components. In order to avoid these shortcomings, the introduction of fully synthetic carbohydrate antigen vaccines with embedded adjuvants has become a new research strategy.
  • ⁇ -Galactose ceramide analogue (KRN7000), a natural ⁇ -GalGSL analogue isolated from a sponge, is the first lipid that effectively activates iNKT cells.
  • KRN7000 a natural ⁇ -GalGSL analogue isolated from a sponge
  • iNKT cells can directly lyse tumor cells through a perforin-dependent mechanism, and indirectly kill tumor cells by secreting a variety of cytokines to act on T cells and NK cells. This unique property has been used in clinical research, and the adjuvant effect of NKT cells can be used to develop more effective vaccines.
  • the present invention provides a fully synthetic carbohydrate antigen vaccine and its preparation method and application.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a conjugate containing an ⁇ -galactose ceramide analog and a carbohydrate antigen.
  • the technical solution adopted by the present invention is: a conjugate containing an ⁇ -galactose ceramide analog and a carbohydrate antigen, and the conjugate is a compound of general formula (I) or general formula ( I) Isomers, pharmaceutically acceptable salts, hydrates or solvent compounds of the compound;
  • n is an integer of 2-6;
  • n is an integer of 9-25;
  • R is selected from -CH 3 , Any one of the substituents.
  • a potent immunostimulant ⁇ -galactose ceramide analogue (KRN7000) is used as an embedded adjuvant to conjugate the carbohydrate antigen Tn to obtain the conjugate (KRN7000-Tn.) of the present invention, which is a fully synthetic carbohydrate antigen. vaccine.
  • KRN7000 can improve the immunogenicity of Tn carbohydrate antigen, present the Tn carbohydrate antigen to the corresponding immune cells, cause a stronger immune response against carbohydrate antigen Tn, and produce a higher immune response.
  • T cells with high titer, high affinity and memory regulate the immune response and achieve the purpose of killing tumor cells.
  • the conjugate is a compound of structural formula (II) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of a compound of structural formula (II);
  • n is an integer of 2-6.
  • the conjugate is a compound of structural formula (III) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of a compound of structural formula (III);
  • the ⁇ -galactose ceramide analogue is an isomer, a pharmaceutically acceptable salt, a hydrated compound of the general formula (IV) or the general formula (IV) Substance or solvent compound;
  • X is any one of O or N substituents
  • n is an integer of 9-25;
  • R is selected from -CH 3 , Any one of the substituents.
  • Another object of the present invention is to provide a preparation method of the conjugate, which includes the following steps:
  • step (1) Dissolve compound 4 and compound 3 described in step (1) in an organic solvent, and add a catalyst to react to obtain compound 5;
  • step (3) The compound 5 described in step (2) is reacted with thioglycolic acid under the action of a catalyst to obtain compound 6;
  • step (3) The compound 6 described in step (3) is dissolved in an organic solvent, and a catalyst is added to react with acetic anhydride to obtain compound 7;
  • step (4) The compound 7 described in step (4) is dissolved in an organic solvent, and sodium methoxide is added to react to obtain compound 8;
  • step (6) The compound 11 described in step (6) is dissolved in an organic solvent, and boron trifluoride ether is added to react to obtain compound 12;
  • step (9) Dissolve the compound 13 described in step (8) in an organic solvent, add a catalyst, and undergo a debenzylation reaction to obtain the conjugate.
  • n is an integer of 2-6;
  • n is an integer of 9-25;
  • R is selected from -CH 3 , Any one of the substituents in;
  • R 1 is selected from any one substituent selected from -OC(NH)CCl 3 , STol, SPh, Set, Cl, and Br.
  • reaction formula of the preparation method of the present invention is as follows:
  • n is an integer of 2-6;
  • n is an integer of 9-25;
  • R is selected from -CH 3 , Any one of the substituents in;
  • R 1 is selected from any one substituent selected from -OC(NH)CCl 3 , STol, SPh, Set, Cl, and Br.
  • the preparation method of the present invention has a short synthetic route, mild reaction conditions, high yield and convenient operation, and can be used for industrial preparation.
  • the organic solvent is a dichloromethane solution
  • the condensing agent is N,N'-dicyclohexylcarbodiimide (DCC) and A mixture of 1-hydroxybenzotriazole (HOBt).
  • step (2) compound 3 and compound 4 are reacted under the action of a catalyst to obtain coupling product 5.
  • the organic solvent is dichloromethane, diethyl ether or tetrahydrofuran; when the substituent R 1 of compound 4 is -OC(NH)CCl 3 ,
  • the catalyst is selected from any one of boron fluoride ether, trimethylsilyl trifluoromethanesulfonate, and trifluoromethanesulfonic acid; or when the substituent R 1 of compound 4 is STol, SPh or Set,
  • the catalyst is N-iodosuccinimide and any one selected from trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, boron trifluoride ether, and trimethylsilyl trifluoromethanesulfonate;
  • the substituent R 1 of compound 4 is Cl or Br, the catalyst is selected from potassium carbonate, silver carbonate, cesium carbonate, mercury carbonate, silver perchlorate, silver trifluoromethanes
  • the organic solvent is dichloromethane; when the substituent R 1 of compound 4 is -OC(NH)CCl 3 , the catalyst is trimethylsilyl trifluoromethanesulfonate ester.
  • step (3) compound 5 is reacted under the action of a catalyst to obtain compound 6; the catalyst is pyridine, and the volume ratio of the catalyst to the thioglycolic acid is 1. :1.
  • step (4) compound 6 is dissolved in N,N-dimethylformamide, and the protective group is removed under the catalysis of piperidine to obtain an amino group.
  • Compound 7 is obtained under the action of acid anhydride; the volume ratio of the pyridine to the acetic anhydride is 2:1.
  • step (5) compound 7 is dissolved in an organic solvent and reacted under the action of a catalyst to obtain compound 8;
  • the organic solvent is a mixed solution of methanol and dichloromethane The volume ratio of the methanol to the dichloromethane is 1:1;
  • the catalyst is sodium methoxide.
  • step (6) compound 9 and compound 10 are dissolved in an organic solvent, and reacted under the action of a catalyst to obtain compound 11;
  • the organic solvent is dichloromethane and ether.
  • the catalyst is N-iodosuccinimide (NIS) and selected from trimethylsilyl trifluoromethanesulfonate (TMSOTf), boron trifluoride ethyl ether, trifluoromethanesulfonate Any of silver acid and trifluoromethanesulfonic acid.
  • the organic solvent is a mixed solution of dichloromethane and ether
  • the catalyst is N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate A mixture of esters (TMSOTf).
  • step (7) compound 11 is dissolved in an organic solvent, and boron trifluoride ether is added to react to obtain compound 12;
  • the organic solvent is a mixture of acetonitrile and dichloromethane
  • the volume ratio of the acetonitrile to the dichloromethane is 1.5:1.
  • step (8) compound 12 and compound 8 are dissolved in an organic solvent, and reacted under the action of a catalyst to obtain compound 13;
  • the solvent is a mixture of dichloromethane and methanol.
  • the catalyst is a mixture of cuprous iodide, N,N-diisopropylethylamine and glacial acetic acid.
  • step (9) compound 13 is dissolved in an organic solvent and reacted under the action of a catalyst to obtain compound 14;
  • the solvent is a mixed solution of methylene chloride and methanol.
  • the catalyst is a mixture of hydrogen, palladium carbon and palladium hydroxide.
  • Another object of the present invention is to provide the application of the conjugate in the preparation of drugs for the prevention and/or treatment of cancer.
  • the cancer is breast cancer, ovarian cancer, lung cancer, uterine cancer, liver cancer, prostate cancer, melanoma, bowel cancer, renal cell carcinoma, cellular lymphoma, pancreatic cancer , Thyroid cancer, brain cancer, stomach cancer and leukemia.
  • a conjugate containing ⁇ -galactose ceramide analog and carbohydrate antigen provided by the present invention is conjugated with a potent immunostimulant ⁇ -galactose ceramide analog (KRN7000) as an embedded adjuvant Carbohydrate antigen Tn is obtained.
  • KRN7000 can improve the immunogenicity of Tn carbohydrate antigen, present Tn carbohydrate antigen to corresponding immune cells, produce a higher titer specific immune response against tumor carbohydrate antigen Tn, and achieve killing
  • the purpose of dead tumor cells to produce anti-tumor effects is expected to become a new generation of anti-tumor drugs.
  • the preparation method of the conjugate containing ⁇ -galactoseceramide analogue and carbohydrate antigen provided by the present invention has a short synthetic route, mild reaction conditions, high yield and convenient operation, and can be used for industrial preparation.
  • Figure 1 is an evaluation diagram of antibody immune activity of the conjugate (III) prepared in Example 1 of the present invention.
  • Fig. 2 is a flow cytometric evaluation diagram of the antibody serum produced by the conjugate (III) induced by the conjugate prepared in Example 1 of the present invention that specifically recognizes the tumor cell MCF-7;
  • Fig. 3 is a diagram showing the complement-dependent cytotoxicity evaluation of the antibody serum produced by the conjugate (III) induced in mice specifically to kill tumor cells MCF-7 prepared in Example 1 of the present invention.
  • This example is a conjugate containing ⁇ -galactose ceramide analogue and carbohydrate antigen provided by the present invention, and its structural formula is shown in the following formula (III):
  • the preparation method of the above-mentioned conjugate containing ⁇ -galactose ceramide analog and carbohydrate antigen includes the following steps:
  • step (1) dissolving (compound 1) Fmoc-L-threonine (4.5 g, 13.1 mmol) and (compound 2) 2-[2-(2-propyl) in dichloromethane solution (80.0 mL) Alkynyloxy)ethoxy]ethylamine (2.2g, 15.7mmol), add N,N'-dicyclohexylcarbodiimide (3.0g, 14.4mmol) and 1-hydroxybenzotriazole ( 0.7g, 1.3mol), remove the ice bath, return to room temperature, and stir for 4 hours at room temperature; filter through diatomaceous earth, collect the filtrate, and distill the filtrate under reduced pressure to remove the organic solvent to obtain the crude product; silica gel column separation and purification (eluent is acetic acid Ethyl ester/petroleum ether 3: 1) to obtain white solid compound 3 (4.9 g, yield 65.4%).
  • step (1) The compound 4 and the compound 3 described in step (1) are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 5; the reaction formula for obtaining compound 5 is shown in the following formula:
  • step (2) The specific operation of step (2) is as follows: Weigh compound 4 (3.1g, 6.4mmol), compound 3 (2.5g, 5.4mmol) and molecular sieve (7.0g) into the original bottom flask, and add anhydrous grade dichloride The mixture (40.0mL) of methane and redistilled ether (1:3) was dissolved, and stirred at room temperature for 3 hours under nitrogen protection; the reaction solution was cooled to -30°C, and trimethylsilyl trifluoromethanesulfonate (97.0 ⁇ L, 0.5mmol), gradually increase the temperature to -27°C, and stir the reaction at -27°C for 12 hours; dilute the reaction solution with dichloromethane, wash with saturated sodium bicarbonate aqueous solution once, and wash with brine once, and collect the organic layer.
  • step (3) The compound 5 described in step (2) is reacted with thioglycolic acid under the action of a catalyst to obtain compound 6; the reaction formula for obtaining compound 6 is shown in the following formula:
  • step (3) The specific operation of step (3) is: weigh compound 5 (1.5g, 1.9mmol) into a round-bottomed flask, add a mixed solution (4.0mL) of pyridine and thioglycolic acid (1:1), stir at room temperature and react 2 Hours; add saturated sodium bicarbonate aqueous solution and stir until no bubbles are generated under ice bath; dilute the reaction solution with dichloromethane solution, wash twice with saturated sodium bicarbonate aqueous solution and wash twice with brine, collect the organic phase, and anhydrous sodium sulfate After drying, the organic solvent was distilled off under reduced pressure to obtain the crude product; the silica gel column was separated and purified (eluent: methanol/dichloromethane 1:100) to obtain white solid compound 6 (1.1 g, yield 73.9%).
  • step (3) The compound 6 described in step (3) reacts with acetic anhydride under the action of a catalyst to obtain compound 7; the reaction formula for obtaining compound 7 is shown in the following formula:
  • step (4) dry anhydrous grade N,N-dimethylformamide solution (5.0 mL) to dissolve compound 6 (1.1 g, 1.4 mmol), add piperidine (50.0 ⁇ L), and stir at room temperature The reaction was carried out for 4 hours; the organic solvent was distilled off under reduced pressure to obtain the crude product; the silica gel column was separated and purified (methanol/dichloromethane 1:100) to obtain a colorless oily liquid (750.0 mg, yield 92.1%).
  • step (4) The compound 7 described in step (4) is dissolved in an organic solvent, and sodium methoxide is added to react to obtain compound 8; the reaction formula for obtaining compound 8 is shown in the following formula:
  • step (6) takes compound 9 (3.9g, 6.7mmol) and compound 10 (3.0g, 4.2mmol) in a round bottom flask, add molecular sieve (10.0g) that has been dried at high temperature, and protect with nitrogen.
  • step (6) The compound 11 described in step (6) is dissolved in an organic solvent, and boron trifluoride ether is added to react to obtain compound 12; the reaction formula for obtaining compound 12 is shown in the following formula:
  • step (7) dissolve compound 11 (200.0 mg, 144.8 ⁇ mol) in a mixed solution (2.5 mL) of acetonitrile and dichloromethane (1.5:1), and add boron trifluoride ether complex (40.0 ⁇ L, 318.6) ⁇ mol), the reaction was stirred at room temperature for 2 hours; the organic solvent was removed by vacuum distillation to obtain the crude product, and the silica gel column was separated and purified (eluent: ethyl acetate: petroleum ether 1:5) to obtain a white solid compound 12 (122.0 mg, yield 73.1%).
  • step (8) dissolving compound 12 (40.0 mg, 34.7 ⁇ mol), compound 8 (10.0 mg, 20.4 ⁇ mol), cuprous iodide in a mixed solution (3.0 mL) of methanol and dichloromethane (1:2) (195.0mg, 1.0mmol), add N,N-diisopropylethylamine (168.0 ⁇ L, 1.0mmol) and glacial acetic acid (12.0 ⁇ L, 0.2mmol), stir and react at room temperature for 12 hours; diatomite filter to remove insoluble The filtrate was distilled under reduced pressure to remove the solvent to obtain a crude product; the silica gel column was separated and purified to obtain a white solid compound 13 (18.0 mg, yield 53.6%).
  • step (8) Dissolve the compound 13 described in step (8) in an organic solvent, add a catalyst, and undergo a debenzylation reaction to obtain the conjugate.
  • the reaction formula for obtaining the conjugate is shown in the following formula:
  • step (8) dissolve compound 13 (6.0 mg, 3.7 ⁇ mol) in a mixed solvent (10.0 mL) of dichloromethane/methanol (1:1), add palladium on carbon (5.0 mg), pass in hydrogen gas, and seal After stirring for 24 hours, the insoluble matter was filtered through diatomaceous earth, and the filtrate was distilled under reduced pressure to remove the solvent to obtain a white solid compound 14, which is the conjugate Tn-KRN7000 (5.5 mg, 96.1%).
  • the conjugate (fully synthetic sugar vaccine) prepared in example 1 was immunized to mice, and its immune effect was preliminarily evaluated through ELSA experiment, and the fluorescence activated cell sorting (FACS) technology proved that the antibody serum can specifically recognize Tumor cells (MCF-7), and antibody-mediated complementation dependent cytotoxicity (CDC) experiments show that antibody serum has the ability to kill tumor cells under the mediation of complement.
  • FACS fluorescence activated cell sorting
  • mice taken 6 C57BL/6 mice aged 6-8 weeks. After the sugar vaccine is prepared into liposomes, the immunization test is carried out by subcutaneous injection of mice.
  • the vaccines prepared are injected on the 0th, 14th, 21st, and 28th day with one initial immunization and three booster immunization schemes.
  • the injection volume was 0.1mL; on the 38th day, each mouse was taken from 0.1mL to 0.2mL of blood, placed at 0°C for 60 minutes, centrifuged at 4000 rpm for 15 minutes, and the upper clear serum was used for ELISA detection and analysis.
  • Tn-BSA in 0.1M carbonate buffer (pH 9.6), prepare a 2.0 ⁇ g/mL solution, add 100.0 ⁇ L per well to a 96-well plate, and incubate overnight at 4°C; the next day, 37°C incubator Incubate for one hour; wash the plate 3 times (300 ⁇ L/well/time) with PBST (PBS+0.05% Tween-20). After washing the plate, add PBS/1% BSA; add 250.0 ⁇ l to each well; incubate at room temperature for one hour, and wash the plate 3 times with PBST.
  • PBST PBS+0.05% Tween-20
  • the KRN7000-Tn carbohydrate vaccine (conjugate III) synthesized in Example 1 of the present invention can generate specific immune responses more quickly in mice without external adjuvants, and simultaneously activate NKT cells efficiently convert IgM antibody isotype to IgG.
  • MCF-7 is a breast cancer cell that overexpresses Tn antigen
  • MDA-231 tumor cells that do not express Tn antigen are used as a negative control.
  • the fluorescence peak of the antibody serum induced by the KRN7000-Tn carbohydrate vaccine (conjugate III) synthesized in Example 1 of the present invention was significantly shifted to the right. There is no significant difference between serum and antibody serum before MDA-231 immunization.
  • the results show that the antibodies induced by the KRN7000-Tn sugar vaccine can specifically recognize MCF-7 cells expressing Tn antigen.
  • MCF-7 is a breast cancer cell that overexpresses Tn antigen
  • MDA-231 tumor cells that do not express Tn antigen are used as a negative control.
  • the antibody serum induced by the KRN7000-Tn carbohydrate vaccine (conjugate III) synthesized in Example 1 of the present invention has a significantly higher antiserum-mediated MCF-7 cell lysis rate than that of blank serum.
  • the cytotoxicity of MDA-231 cells that do not express Tn antigen is not statistically different from that of blank serum.
  • the results confirm that KRN7000-Tn sugar vaccine has a certain specific anti-tumor effect.
  • the experimental results show that the present invention provides a conjugate (KRN7000-Tn sugar vaccine) containing ⁇ -galactose ceramide analogue and carbohydrate antigen, which is similar to the potent immunostimulant ⁇ -galactose ceramide.
  • KRN7000 was obtained as an embedded adjuvant conjugated with carbohydrate antigen Tn.
  • Tn carbohydrate antigen
  • KRN7000 can improve the immunogenicity of Tn carbohydrate antigen and present Tn carbohydrate antigen to the corresponding immune cells to produce higher titer against tumors.
  • the specific immune response of the carbohydrate antigen Tn achieves the purpose of killing tumor cells and produces anti-tumor effects. It is expected to become a new generation of anti-tumor drugs.

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Abstract

一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物及其制备方法和应用，属于抗肿瘤糖疫苗研制技术领域。提供的一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物，其为通式（Ⅰ）的化合物或通式（Ⅰ）的化合物的异构体、可药用盐、水合物或溶剂化合物；该缀合物能产生具有更高滴度的针对肿瘤糖抗原Tn特异性的免疫反应，达到杀死肿瘤细胞的目的，产生抗肿瘤作用，有望成为新一代的抗肿瘤药物，在（Ⅰ）中：n为2-6的整数；m为9-25的整数；R选自-CH 3、（aa）中的任意一种取代基。

Description

一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物及其制备方法和应用 技术领域
本发明涉及一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物及其制备方法和应用，属于抗肿瘤糖疫苗研制技术领域。
背景技术
癌症防治已成为我国的重要公共卫生问题，严重地威胁人们的生命安全。迫切需要的有效的癌症防治方法，肿瘤疫苗被认为是继手术、化疗和放射之外的第四种治疗方式，而且显示出较好的临床应用前景。以肿瘤细胞表面异常表达的肿瘤相关糖抗原(TACAs)为靶点的肿瘤糖疫苗具有特异性高、副作用小、疗效较好等优点。其中Thomsennouveau(Tn)抗原在乳腺癌、前列腺癌、肺癌等恶性肿瘤细胞表面均异常过量表达，是糖抗原肿瘤疫苗设计的优秀靶点。
由于Tn糖抗原是T细胞非依赖性抗原，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。传统的策略是将糖抗原与含有B细胞表位的载体蛋白(KLH，BSA或者CRM197)偶联，获得的糖蛋白疫苗可产生高滴度高亲和力的抗体，目前Tn的KLH缀合蛋白疫苗已进入临床研究阶段。但糖蛋白疫苗依然存在着偶联位点不确定、偶联率不稳定、组成成分复杂等缺点。为了避免这些缺点，引入内嵌佐剂的全合成糖抗原疫苗成为新的研究策略。
α-半乳糖神经酰胺类似物(KRN7000)，是从一种海绵中分离出的天然α-GalGSL类似物，是第一种有效激活iNKT细胞的脂质。在KRN7000的激活作用下，iNKT细胞可通过穿孔蛋白依赖机制直接裂解肿瘤细胞，还可通过分泌多种细胞因子作用于T细胞和NK细胞间接杀死肿瘤细胞。这种独特的性质已用于临床研究，可利用NKT细胞的佐剂效应来开发更有效的疫苗。
为了解决糖蛋白疫苗存在的缺陷，本发明提供一种全合成糖抗原疫苗及其制备方法和应用。
发明内容
本发明的目的在于克服现有技术的不足，提供一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物。
为实现上述目的，本发明采取的技术方案为：一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物，所述的缀合物为通式(Ⅰ)的化合物或通式(Ⅰ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
Figure PCTCN2020130228-appb-000001
其中：
n为2-6的整数；
m为9-25的整数；
R选自-CH 3、
Figure PCTCN2020130228-appb-000002
中的任意一种取代基。
本发明以强效免疫刺激剂α-半乳糖神经酰胺类似物(KRN7000)作为内嵌佐剂缀合糖抗原Tn得到本发明所述的缀合物(KRN7000-Tn。)，即全合成糖抗原疫苗。在本发明所述的缀合物中，KRN7000能提高Tn糖抗原的免疫原性，将Tn糖抗原提呈到相应的免疫细胞，引起更强的针对糖抗原Tn的免疫反应，产生具有更高滴度、高亲和力和具有记忆力的T细胞调节免疫反应，达到杀死 肿瘤细胞的目的。
作为本发明所述的缀合物的优选实施方式，所述的缀合物为结构式(Ⅱ)的化合物或结构式(Ⅱ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
Figure PCTCN2020130228-appb-000003
其中：
n为2-6的整数。
作为本发明所述的缀合物的优选实施方式，所述的缀合物为结构式(Ⅲ)的化合物或结构式(Ⅲ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
Figure PCTCN2020130228-appb-000004
作为本发明所述的缀合物的优选实施方式，所述的α-半乳糖神经酰胺类似物为通式(Ⅳ)或通式(Ⅳ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
Figure PCTCN2020130228-appb-000005
其中：
X为O或N任意一种取代基；
m为9-25的整数；
R选自-CH 3、
Figure PCTCN2020130228-appb-000006
中的任意一种取代基。
本发明的另一目的是提供所述的缀合物的制备方法，包括如下步骤：
(1)将化合物1和化合物2溶解于有机溶剂中，加入缩合剂反应，得到化合物3；
(2)将化合物4和步骤(1)所述的化合物3溶解于有机溶剂中，加入催化剂反应，得到化合物5；
(3)将步骤(2)所述的化合物5在催化剂的作用下与巯基乙酸反应，得到化合物6；
(4)步骤(3)所述的化合物6溶解于有机溶剂中，加入催化剂，与乙酸酐反应，得到化合物7；
(5)步骤(4)所述的化合物7溶解于有机溶剂中，加入甲醇钠反应，得到化合物8；
(6)取化合物9和化合物10溶解于有机溶剂中，加入催化剂反应，得到化合物11；
(7)取步骤(6)所述的化合物11溶解于有机溶剂中，加入三氟化硼乙醚反应，得到化合物12；
(8)取步骤(5)所述的化合物8与步骤(7)所述的化合物12溶解于有机溶剂中，加入催化剂反应，得到化合物13。
(9)取步骤(8)所述的化合物13溶解于有机溶剂中，加入催化剂，经脱苄基反应，即可获得所述的缀合物。
所述化合物1至所述化合物13的结构式如下所示：
Figure PCTCN2020130228-appb-000007
Figure PCTCN2020130228-appb-000008
Figure PCTCN2020130228-appb-000009
其中：
n为2-6的整数；
m为9-25的整数；
R选自-CH 3、
Figure PCTCN2020130228-appb-000010
中的任意一种取代基；
R 1选自-OC(NH)CCl 3、STol、SPh、Set、Cl、Br中的任意一种取代基。
本发明所述制备方法的反应式如下所示：
Figure PCTCN2020130228-appb-000011
其中：
n为2-6的整数；
m为9-25的整数；
R选自-CH 3、
Figure PCTCN2020130228-appb-000012
中的任意一种取代基；
R 1选自-OC(NH)CCl 3、STol、SPh、Set、Cl、Br中的任意一种取代基。
本发明所述的制备方法，其合成路线简短、反应条件温和、产率高、操作方便，能够用于工业化制备。
作为本发明所述的制备方法的优选实施方式，步骤(1)中，所述有机溶剂为二氯甲烷溶液，所述缩合剂为N,N’-二环己基碳二亚胺(DCC)与1-羟基苯并三唑(HOBt)的混合物。
本发明所述的制备方法，步骤(2)中，化合物3与化合物4在催化剂的作用下反应得到偶联产物5。
作为本发明所述的制备方法的优选实施方式，步骤(2)中，所述有机溶剂为二氯甲烷、乙醚或四氢呋喃；当化合物4的取代基R 1为-OC(NH)CCl 3时，所述催化剂选自氟化硼乙醚、三氟甲磺酸三甲基硅酯、三氟甲磺酸中的任意一种；或者当化合物4的取代基R 1为STol、SPh或Set时，所述催化剂为N-碘代丁二酰亚胺和选自三氟甲磺酸、三氟甲磺酸银、三氟化硼乙醚、三氟甲磺酸三甲基硅酯中的任意一种；或者当化合物4的取代基R 1为Cl或Br时，所述催化剂选自碳酸钾、碳酸银、碳酸铯、碳酸汞、高氯酸银、三氟甲磺酸银、三氟甲磺酸中的任意一种；所述反应的温度为-40～-20℃。
更优选地，步骤(2)中，所述有机溶剂为二氯甲烷；当化合物4的取代基R 1为-OC(NH)CCl 3时，所述催化剂为三氟甲磺酸三甲基硅酯。
作为本发明所述的制备方法的优选实施方式，步骤(3)中，化合物5在催化剂的作用下反应得到化合物6；所述催化剂为吡啶，所述催化剂与所述巯基乙酸的体积比为1:1。
作为本发明所述的制备方法的优选实施方式，步骤(4)中，化合物6溶解于N,N-二甲基甲酰胺，在哌啶的催化下脱去保护集团得到氨基，在吡啶与乙酸酐的作用下得到化合物7；所述吡啶与所述乙酸酐的体积比为2:1。
作为本发明所述的制备方法的优选实施方式，步骤(5)中，化合物7溶于有机溶剂中，在催化剂的作用下反应得到化合物8；所述有机溶剂为甲醇与二氯甲烷的混合溶液，所述甲醇与所述二氯甲烷的体积比为1:1；所述的催化剂为甲醇钠。
作为本发明所述的制备方法的优选实施方式，步骤(6)中，化合物9与化合物10溶于有机溶剂中，在催化剂的作用下反应得到化合物11；所述有机溶剂为二氯甲烷、乙醚或四氢呋喃中任意一种，所述催化剂为N-碘代丁二酰亚胺(NIS)和选自三氟甲磺酸三甲基硅酯(TMSOTf)、三氟化硼乙醚、三氟甲磺酸银、三氟甲磺酸中的任意一种。
更优选地，步骤(6)中，所述有机溶剂为二氯甲烷与乙醚的混合溶液，所述催化剂为N-碘代丁二酰亚胺(NIS)和三氟甲磺酸三甲基硅酯(TMSOTf)的混合物。
作为本发明所述的制备方法的优选实施方式，步骤(7)中，化合物11溶于有机溶剂中，加入三氟化硼乙醚反应，得到化合物12；所述有机溶剂为乙腈与二氯甲烷的混合溶液，所述乙腈与所述二氯甲烷的体积比为1.5:1。
作为本发明所述的制备方法的优选实施方式，步骤(8)中，化合物12与化合物8溶于有机溶剂中，在催化剂的作用下反应得到化合物13；所述溶剂为二氯甲烷与甲醇的混合溶液，所述催化剂为碘化亚铜、N,N-二异丙基乙胺与冰乙酸的混合物。
作为本发明所述的制备方法的优选实施方式，步骤(9)中，化合物13溶于有机溶剂中，在催化剂的作用下反应得到化合物14；所述溶剂二氯甲烷与甲醇的混合溶液，所述催化剂为氢气、钯碳与氢氧化钯的混合物。
本发明的再一目的是提供所述的缀合物在制备预防和/或治疗癌症的药物中的应用。
作为本发明所述的应用的优选实施方式，所述癌症为乳腺癌、卵巢癌、肺癌、子宫癌、肝癌、前列腺癌、黑素瘤、肠癌、肾细胞癌、细胞性淋巴癌、胰腺癌、甲腺癌、脑癌、胃癌和白血病。
与现有技术相比，本发明的有益效果为：
(1)本发明提供的一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物，以强效免疫刺激剂α-半乳糖神经酰胺类似物(KRN7000)作为内嵌佐剂缀合糖抗原Tn获得，其中，KRN7000能提高Tn糖抗原的免疫原性，将Tn糖抗原提呈到相应的免疫细胞，产生具有更高滴度的针对肿瘤糖抗原Tn特异性的免疫反应，达到杀死肿瘤细胞的目的，产生抗肿瘤作用，有望成为新一代的抗肿瘤药物。
(2)本发明提供的含有α-半乳糖神经酰胺类似物与糖抗原的缀合物的制备 方法，其合成路线简短、反应条件温和、产率高、操作方便，能够用于工业化制备。
附图说明
图1为本发明实施例1制备的缀合物(Ⅲ)的抗体免疫活性评价图；
图2为本发明实施例1制备的缀合物(Ⅲ)诱导小鼠产生的抗体血清特异性识别肿瘤细胞MCF-7的流式细胞实验评价图；
图3为本发明实施例1制备的缀合物(Ⅲ)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
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例1
本实施例为本发明提供的一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物，其结构式如下式(Ⅲ)所示：
Figure PCTCN2020130228-appb-000013
上述含有α-半乳糖神经酰胺类似物与糖抗原的缀合物的制备方法，包括如下步骤：
(1)将化合物1和化合物2溶解于有机溶剂中，加入缩合剂反应，得到化合物3；得到化合物3的反应式如下式所示：
Figure PCTCN2020130228-appb-000014
步骤(1)的具体操作为：二氯甲烷溶液(80.0mL)溶解(化合物1)Fmoc-L-苏氨酸(4.5g，13.1mmol)与(化合物2)2-[2-(2-丙炔基氧)乙氧基]乙胺(2.2g，15.7mmol)，冰浴下加入N,N’-二环己基碳二亚胺(3.0g，14.4mmol)与1-羟基苯并三唑(0.7g，1.3mol)，除去冰浴，恢复至室温，室温下搅拌反应4小时；硅藻土过滤，收集滤液，滤液减压蒸馏除去有机溶剂得粗品；硅胶柱分离纯化(洗脱剂为乙酸乙酯/石油醚3：1)得到白色固体化合物3(4.9g，产率为65.4％)。 1H NMR(400MHz,CDCl 3)δ7.76(d,J＝7.5Hz,2H,Ar-H),7.60(d,J＝7.5Hz,2H,Ar-H),7.40(t,J＝7.5Hz,2H,Ar-H),7.31(t,J＝7.4Hz,2H,Ar-H),6.91(d,J＝5.7Hz,1H,-NHCO-),5.89(d,J＝8.0Hz,1H,-NHCO-),4.47(dd,J＝10.5,7.0Hz,1H),4.36(dt,J＝16.5,4.3Hz,2H),4.22(t,J＝7.0Hz,1H),4.18–4.11(m,3H),3.68–3.41(m,8H,-O-CH 2-),2.87(s,1H),2.43(d,J＝2.6Hz,1H,-C≡CH),1.16(d,J＝6.3Hz,3H,-CH 3). 13C NMR(100MHz,CDCl 3)δ170.98,156.80,143.72,143.69,141.32,127.79,127.11,125.09,125.03,120.04,79.45,76.75,74.90,70.00,69.39,69.01,67.18,66.97,58.64,58.38,47.16,39.22,18.31.ESI-TOF HRMS m/z:calcdfor C 26H 30N 2O 6，[M+Na] +:489.1996,found:489.1980.
(2)将化合物4和步骤(1)所述的化合物3溶解于有机溶剂中，加入催化剂反应，得到化合物5；得到化合物5的反应式如下式所示：
Figure PCTCN2020130228-appb-000015
步骤(2)的具体操作为：称取化合物4(3.1g，6.4mmol)、化合物3(2.5g，5.4mmol)和分子筛(7.0g)置于原底烧瓶中，加入无水级别的二氯甲烷和重蒸乙醚(1：3)混合液(40.0mL)溶解，氮气保护下，室温搅拌3小时；反应液冷却至-30℃，加入三氟甲磺酸三甲基硅脂(97.0μL，0.5mmol)，逐渐升温至-27℃，-27℃的温度下搅拌反应12小时；二氯甲烷稀释反应液，依次用饱和碳酸氢钠水溶液洗1次，盐水洗1次，收集有机层，无水硫酸钠干燥后，过滤，滤液减压蒸馏除去有机溶剂得到粗品；硅胶柱分离纯化(洗脱剂为甲醇/二氯甲烷1：100)得到无色油状液体化合物5(1.5g，产率为36.0％)。 1H NMR(400MHz,CDCl 3)δ7.77(d,J＝7.5Hz,2H,Ar-H),7.60(dd,J＝7.5,2.6Hz,2H,Ar-H),7.41(t,J＝7.5Hz,2H,Ar-H),7.33(t,J＝7.4Hz,2H,Ar-H),7.08(d,J＝5.7Hz,1H,-NHCO-),5.92(d,J＝6.8Hz,1H,-NHCO-),5.43(d,J＝3.2Hz,1H),5.28(d,J＝3.6Hz,1H,H-1),4.41(m,J＝7.0,3.4Hz,2H),4.26(m,J＝14.5,7.0Hz,4H),4.16(d,J＝2.4Hz,2H,-O-CH 2-C≡C),4.13–4.05(m,2H),3.90(m,J＝10.9,3.8Hz,1H),3.65(m,J＝5.0,4.1Hz,4H,-O-CH 2-),3.56(m,J＝16.9,11.8,5.7Hz,4H,-O-CH 2-,-N-CH 2-),2.42(t,J＝2.4Hz,1H,-C≡CH),2.17(s,3H,-CO-CH 3),2.08(s,3H,-CO-CH 3),2.04(s,3H,-CO-CH 3),1.18(d,J＝6.4Hz,3H,-CH 3). 13C NMR(100MHz,CDCl 3)δ170.39,170.09,169.84,168.22,156.04,143.73,141.32,127.78,127.12,125.13,125.06,120.06,120.03,97.24,79.58,76.74,74.65,74.44,70.06,69.63,69.49,69.00,67.35,67.26,66.97,61.69,58.47,58.33,57.20,47.14,39.51,29.71,20.73,20.67,16.34.ESI-TOF HRMS m/z:calcdfor C 38H 45N 5O 13,[M+Na] +:802.2906,found:802.2880.
(3)将步骤(2)所述的化合物5在催化剂的作用下与巯基乙酸反应，得到化合物6；得到化合物6的反应式如下式所示：
Figure PCTCN2020130228-appb-000016
步骤(3)的具体操作为：称取化合物5(1.5g，1.9mmol)置于圆底烧瓶中，加入吡啶与巯基乙酸(1：1)的混合溶液(4.0mL)，室温下搅拌反应2小时；冰浴下加入用饱和碳酸氢钠水溶液搅拌至无气泡产生；二氯甲烷溶液稀释反应液，依次用饱和碳酸氢钠水溶液洗2次，盐水洗2次，收集有机相，无水硫酸钠干燥后，减压蒸馏除去有机溶剂得粗品；硅胶柱分离纯化(洗脱剂为甲醇/二氯甲烷1：100)得到白色固体化合物6(1.1g，产率为73.9％)。 1H NMR(400MHz,CDCl 3)δ7.78(dd,J＝7.1,1.2Hz,2H,Ar-H),7.64(d,J＝7.4Hz,2H,Ar-H),7.41(tt,J＝7.5,1.6Hz,2H,Ar-H),7.34(m,J＝6.4,5.2,3.0Hz,2H,Ar-H),6.78(t,1H,-NHCO-),6.51(d,J＝9.1Hz,1H,-NHCO-),5.82(d,J＝8.8Hz,1H,-NHCO-),5.39(d,J＝3.2Hz,1H),5.07(m,J＝11.4,3.2Hz,1H),4.96(s,1H,1-H),4.62–4.40(m,3H),4.26(m,J＝9.5,8.3Hz,4H),4.15(d,J＝2.4Hz,2H,-O-CH 2-),4.08(m,J＝7.1,6.3Hz,2H),3.65(m,J＝7.1,6.5,3.8Hz,4H,-O-CH 2-),3.56(s,2H,-O-CH 2-C≡C),3.46(d,J＝5.9Hz,2H,-N-CH 2-),2.47(t,J＝2.4Hz,1H,-C≡CH),2.16(s,3H,-CO-CH 3),2.03(s,6H,-CO-CH 3),2.00(s,3H,-CO-CH 3),1.27(t,J＝5.5Hz,3H,-CH 3). 13C NMR(100MHz,CDCl 3)δ170.90,170.80,170.40,169.94,156.66,143.71,141.33,127.82,127.80,127.16,125.13,125.06,120.05,120.03,99.87,79.36,77.38,76.75,75.14,69.91,69.25,69.07,68.80,67.35,67.31,62.11,58.53,58.34,47.63,47.20,39.37,23.08,20.84,20.78,20.68,17.64.ESI-TOF HRMS m/z:calcdfor C 40H 49N 3O 14,[M+Na] +:818.3107,found:818.3061.
(4)步骤(3)所述的化合物6在催化剂的作用下与乙酸酐反应，得到化合物7；得到化合物7的反应式如下式所示：
Figure PCTCN2020130228-appb-000017
步骤(4)的具体操作为：干燥无水级别的N,N-二甲基甲酰胺溶液(5.0mL)溶解化合物6(1.1g，1.4mmol)，加入哌啶(50.0μL)，室温下搅拌反应4小时；减压蒸馏除去有机溶剂得粗品；硅胶柱分离纯化(甲醇/二氯甲烷1：100)得到无色油状液体(750.0mg，产率为92.1％)。乙酸酐与吡啶(1：2)的混合溶液(1.5mL)溶解无色油状液体(100.0mg，174.4μmol)，室温下搅拌反应2小时；减压蒸馏除去有机溶剂得粗品；硅胶柱分离纯化(甲醇/乙酸乙酯1：25)得到无色油状液体化合物7(79.0mg，产率为76.2％)。 1H NMR(400MHz,CDCl 3)δ7.07(t,J＝5.2Hz,1H,-NHCO-),6.64(m,J＝9.0,3.5Hz,1H,-NHCO-),6.57(d,J＝9.4Hz,1H,-NHCO-),5.37(m,J＝3.3,1.3Hz,1H),5.08(m,J＝11.3,3.2Hz,1H),4.94(d,J＝3.6Hz,1H,H-1),4.88(s,1H),4.64(dd,J＝8.9,2.5Hz,1H),4.56(m,J＝11.3,9.4,3.6Hz,1H),4.26(m,J＝7.1,5.6,1.3Hz,1H),4.21(d,J＝2.4Hz,2H,-O-CH 2-C≡C),4.17(m,J＝6.3,2.5Hz,1H),4.08(m,J＝11.4,6.5Hz,2H),3.71–3.61(m,4H),3.60–3.50(m,2H),3.44(m,J＝13.9,9.4,7.4,3.1Hz,2H),2.54(t,J＝2.4Hz,1H,-C≡CH),2.17(s,3H,-CO-CH 3),2.13(s,3H,-CO-CH 3),2.03(d,J＝2.5Hz,6H,-CO-CH 3),1.99(s,3H,-CO-CH 3),1.28(d,J＝6.3Hz,3H,-CH 3). 13C NMR(100MHz,CDCl 3)δ170.99,170.86,170.72,170.43,170.39,170.23,100.30,79.37,78.06,77.29,75.13,69.99,69.28,69.08,68.95,67.38,67.22,62.20,58.37,56.47,47.44,39.33,23.26,23.04,20.80,20.75,20.65,18.09.ESI-TOF HRMS m/z:calcdfor C 27H 41N 3O 13,[M+H] +:616.2712,found:616.2717.[M+Na] +:638.2532,found:638.2532.
(5)步骤(4)所述的化合物7溶解于有机溶剂中，加入甲醇钠反应，得到化合物8；得到化合物8的反应式如下式所示：
Figure PCTCN2020130228-appb-000018
步骤(5)的具体操作为：制备甲醇钠溶液：金属钠(5.4mg)，加入到的甲醇溶液(10.0mL)中，冷却至室温待用；甲醇与二氯甲烷(1：1)的混合溶液(3mL)溶解化合物7(79.0mg，128.4mmol)，加入已制备好的甲醇钠溶液(0.1mL)调节pH＝8，持续搅拌反应2小时；加入适量的离子交换树脂调节pH至7终止反应；硅藻土过滤，收集滤液，除去有机溶剂得到白色固体化合物8(54.0mg，产率为86.0％)。 1H NMR(400MHz,MeOD)δ4.75(s,1H,H-1),4.42(d,J＝2.6Hz,1H),4.18–4.08(m,4H),3.79(q,J＝4.7,3.6Hz,2H),3.67–3.55(m,5H),3.52(m,J＝6.0,3.4Hz,2H),3.45–3.35(m,3H),3.24–3.15(m,2H),2.78(t,J＝2.4Hz,1H,-C≡CH),1.99(s,3H,-CO-CH 3),1.97(s,3H,-CO-CH 3),1.17(d,J＝6.4Hz,3H,-CH 3). 13C NMR(100MHz,MeOD)δ172.76,172.27,170.94,99.60,79.18,76.26,74.72,71.52,69.66,69.00,68.97,68.88,68.71,61.30,57.69,57.07,50.02,48.26,48.05,47.83,47.62,47.53,47.41,47.20,46.98,39.09,21.86,21.81,21.15,17.82.ESI-TOF HRMS m/z:calcdfor C 21H 35N 3O 10,[M+H] +:490.2395,found:490.2395.
(6)取化合物9和化合物10溶解于有机溶剂中，加入催化剂反应，得到化合物11；得到化合物11的反应式如下式所示：
Figure PCTCN2020130228-appb-000019
步骤(6)的具体操作为：取化合物9(3.9g，6.7mmol)与化合物10(3.0g，4.2mmol)于圆底烧瓶中，加入已高温干燥过的分子筛(10.0g)，氮气保护，加入无水二氯甲烷/重蒸乙醚(1：3)的混合液(80.0mL)溶解，室温搅拌3小时；反应液冷却至0℃，加入N-碘代丁二酰亚胺(2.9g，12.7mmol)搅拌15分钟后，加入三氟甲磺酸三甲基硅脂(152.0μL，0.8mmol)，0℃下搅拌反应12小时； 用二氯甲烷稀释反应液，依次用饱和的硫代硫酸钠水溶液洗1次，饱和碳酸氢钠水溶液洗2次，盐水洗1次，收集有机层，无水硫酸钠干燥后，过滤，滤液减压蒸馏除去有机溶剂得粗品；硅胶柱分离纯化(洗脱剂为乙酸乙酯/石油醚1：40)得到无色油状液体化合物11(3.0g，产率为50.8％)。 1H NMR(400MHz,CDCl 3)δ7.32(m,J＝13.9,9.1,4.0Hz,15H,Ar-H),5.90(d,J＝7.5Hz,1H,-NHCO-),4.99(d,J＝11.4Hz,1H,Ar-CH 2-O-),4.83(d,J＝2.6Hz,1H,H-1),4.81(d,J＝4.0Hz,1H,Ar-CH 2-O-),4.78(s,1H,Ar-CH 2-O-),4.68(t,J＝12.8Hz,2H,Ar-CH 2-O-),4.58(d,J＝11.4Hz,1H,Ar-CH 2-O-),4.18(m,J＝8.3,4.7,4.2Hz,1H,-N-CH-),4.06–3.96(m,2H,H-2),3.88(m,J＝10.0,2.5Hz,1H,H-3),3.85–3.73(m,4H),3.69–3.62(m,1H),3.49(dd,J＝12.4,7.4Hz,1H,H-6),3.14(m,J＝12.3,5.9Hz,1H,H-6),2.01(t,J＝7.7Hz,2H,-CH 2-CONH-),1.63–1.45(m,4H,-CH 2-),1.13(d,J＝22.6Hz,68H,-CH 2-),0.88(m,J＝7.4,5.6Hz,24H,-CH 3),0.07(d,J＝4.8Hz,12H,Si-CH 3). 13C NMR(100MHz,CDCl 3)δ173.04,138.51,138.43,138.18,128.45,128.38,128.30,127.87,127.84,127.75,127.66,127.45,100.00,79.00,77.36,77.25,76.72,76.42,76.03,75.83,74.89,74.70,73.52,73.32,69.94,69.39,51.62,51.22,36.87,33.43,31.96,29.92,29.76,29.74,29.69,29.64,29.59,29.51,29.48,29.40,26.14,26.07,25.69,22.72,18.35,18.20,14.16,-3.65,-3.93,-4.61,-4.89.ESI-TOF HRMS m/z:calcdfor C 83H 144N 4O 8Si 2,[M+H] +:1382.0595,found:1382.0581.
(7)取步骤(6)所述的化合物11溶解于有机溶剂中，加入三氟化硼乙醚反应，得到化合物12；得到化合物12的反应式如下式所示：
Figure PCTCN2020130228-appb-000020
步骤(7)的具体操作为：乙腈与二氯甲烷(1.5：1)混合溶液(2.5mL)溶解化合物11(200.0mg，144.8μmol)，加入三氟化硼乙醚络合物(40.0μL，318.6μmol)，室温下搅拌反应2小时；减压蒸馏除去有机溶剂得粗品，硅胶柱分离纯化(洗脱剂为乙酸乙酯：石油醚1：5)得到白色固体化合物12(122.0mg，产率为73.1％)。 1H NMR(400MHz,CDCl 3)δ7.43–7.27(m,15H,Ar-H),6.28(d,J＝ 8.5Hz,1H,-NHCO-),4.98(d,J＝11.4Hz,1H),4.92–4.86(m,2H),4.86–4.75(m,2H),4.70(d,J＝11.6Hz,1H),4.59(d,J＝11.4Hz,1H),4.27(m,J＝7.0,3.4Hz,1H),4.05(m,J＝9.9,3.7Hz,1H),3.95–3.79(m,4H),3.73(m,J＝7.4,5.6Hz,1H),3.49(m,J＝12.5,7.3,6.6,2.9Hz,3H),3.03(m,J＝12.5,5.5Hz,1H),2.16(t,J＝7.6Hz,2H,-CO-CH 2-),1.61(q,J＝8.2Hz,4H,-CH 2-),1.38–1.17(m,68H,-CH 2-),0.88(t,J＝6.8Hz,6H,-CH 3). 13C NMR(100MHz,CDCl 3)δ173.17,138.27,138.04,137.75,128.53,128.43,128.41,128.09,128.02,127.97,127.79,127.57,98.70,79.09,77.45,76.33,75.78,74.69,74.61,73.99,73.21,73.07,70.24,69.00,51.07,49.43,36.82,33.86,32.03,29.95,29.93,29.85,29.81,29.79,29.77,29.70,29.58,29.49,29.47,29.44,26.07,25.90,22.80,14.23.ESI-TOF HRMS m/z:calcdfor C 71H 116N 4O 8,[M+H] +:1153.8866,found:1153.8861.
(8)取步骤(5)所述的化合物8与步骤(7)所述的化合物12溶解于有机溶剂中，加入催化剂反应，得到化合物13；得到化合物13的反应式如下式所示：
Figure PCTCN2020130228-appb-000021
步骤(8)的具体操作为：甲醇与二氯甲烷(1：2)混合溶液(3.0mL)溶解化合物12(40.0mg，34.7μmol)、化合物8(10.0mg，20.4μmol)、碘化亚铜(195.0mg，1.0mmol)，加入N,N-二异丙基乙胺(168.0μL，1.0mmol)与冰乙酸(12.0μL，0.2mmol)，室温下搅拌反应12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白色固体化合物13(18.0mg，产率为53.6％)。 1H NMR(400MHz,MeOD/CDCl 3(1:50,v/v,0.6mL))δ7.70(s,1H,-N-CH＝C-),7.45–7.28(m,15H,Ar-H),5.03(d,J＝11.3Hz,1H),4.89(t,J＝4.1Hz,2H,H-1x 2),4.86–4.77(m,3H),4.76(d,J＝3.1Hz,1H),4.66–4.61(m,3H),4.55(d,J＝2.4Hz,1H),4.27(dd,J＝14.1,3.1Hz,1H),4.19(m,J＝8.0,4.8Hz,2H),4.14–4.03(m,3H),3.95(d,J＝7.7Hz,3H),3.87(t,J＝5.5Hz,1H),3.73– 3.66(m,2H),3.66–3.57(m,4H),3.52(d,J＝2.7Hz,2H),3.46(m,J＝7.9,5.9Hz,4H),3.37(m,J＝1.6Hz,1H),3.32(m,J＝12.0,4.1Hz,2H),3.14(q,J＝7.4Hz,2H),2.10(d,J＝3.9Hz,8H,-CO-CH 2-,-CO-CH 3x 2),1.57(q,J＝8.3,6.9Hz,2H,-CH 2-),1.49(m,J＝7.1,3.1Hz,8H,-CH 2-),1.43(d,J＝6.6Hz,6H,-CH 2-),1.25(d,J＝3.8Hz,65H,-CH 2-,-CH 3),0.88(t,J＝6.7Hz,6H,-CH 3). 13C NMR(100MHz,MeOD/CDCl 3(1:50,v/v,0.6mL))δ173.73,171.94,171.09,138.25,137.91,137.59,128.67,128.66,128.35,128.29,128.25,128.00,127.72,99.76,98.25,79.00,77.67,77.55,77.17,77.03,75.92,75.21,74.92,74.88,74.07,73.61,72.70,70.65,70.24,70.02,69.87,69.53,69.44,67.38,63.96,62.10,56.81,54.43,51.47,50.65,49.86,42.60,39.47,36.66,33.33,32.04,29.97,29.92,29.87,29.84,29.81,29.78,29.77,29.73,29.58,29.54,29.48,26.08,25.97,22.80,22.75,22.71,18.55,18.35,17.25,14.16,12.28.ESI-TOF HRMS m/z:calcdfor C 92H 151N 7O 18,[M+H] +:1643.1188,found:1643.1165.
(9)取步骤(8)所述的化合物13溶解于有机溶剂中，加入催化剂，经脱苄基反应，即可获得所述的缀合物。得到所述缀合物的反应式如下式所示：
Figure PCTCN2020130228-appb-000022
步骤(8)的具体操作为：二氯甲烷/甲醇(1:1)的混合溶剂(10.0mL)溶解化合物13(6.0mg，3.7μmol)，加入钯碳(5.0mg)，通入氢气，密封搅拌24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14，即为所述的缀合物Tn-KRN7000(5.5mg，96.1％)。 1H NMR(400MHz,MeOD/CDCl 3(1:10,v/v,0.6mL))δ8.00(s,1H),4.89(d,J＝3.4Hz,3H),4.70–4.52(m,6H),4.23(d,J＝6.3Hz,3H),4.09(s,1H),3.98–3.59(m,15H),3.49(s,7H),3.20(m,J＝7.4Hz,2H),2.25–2.15(m,2H),2.11-2.09(s,6H),1.59(d,J＝13.3Hz,4H),1.42(t,J＝6.6Hz,16H),1.27(s,66H),0.89(t,J＝6.7Hz,6H). 13C NMR(150MHz,MeOD/CDCl 3(1:10,v/v,0.6mL))δ172.95,172.93,172.90, 171.72,100.37,100.23,78.43,77.38,74.69,72.48,71.85,70.69,70.46,70.15,70.09,69.88,69.25,67.68,64.47,62.28,57.69,55.29,51.56,50.90,50.78,49.50,49.38,49.23,49.09,48.95,48.81,48.67,48.52,43.42,39.97,32.57,32.50,32.50,30.40,30.38,30.29,30.22,30.06,29.98,29.94,29.91,26.54,26.49,23.21,22.63,18.82,18.70,17.27,14.29,13.00.ESI-TOF HRMS m/z:calcdfor C 71H 133N 7O 18[M+H] +:1372.9780,found:1372.9766.
实验例1
本实验例1对实施例1制备的缀合物(全合成糖疫苗)进行免疫小鼠，通过ELSA实验初步评价其免疫作用，通过荧光激活细胞分选(FACS)技术证明抗体血清能特异性识别肿瘤细胞(MCF-7)，同时通过抗体介导的互补依赖细胞毒性(CDC)实验表明抗体血清在补体的介导下具有杀死肿瘤细胞的能力。
1.ELISA免疫分析
1)小鼠免疫：
取6-8周龄的C57BL/6小鼠6只。将糖疫苗制备成脂质体后，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方案，分别在第0、14、21、28天注射制备的疫苗，每只每次注射量0.1mL；第38天每只小鼠取血取0.1mL到0.2mL，在0℃放置60分钟，4000转/分钟离心15分钟，取上层清亮血清用于ELISA检测分析。
2)ELISA免疫分析：
0.1M碳酸盐缓冲液(pH 9.6)溶解Tn-BSA，配置成2.0μg/mL溶液，以每孔100.0μL的量加入96孔板，放入4℃孵育过夜；第二天37℃培养箱孵育一小时；用PBST(PBS+0.05％吐温-20)洗板3次(300μL/孔/次)。洗板后，加入PBS/1％BSA；每孔加入250.0μl；常温孵育一小时，用PBST洗板3次。将6只小鼠血清样品等量混合后用PBS稀释300、900、2700、8100、24300、72900、218700与656100倍；将稀释好的血清以每孔100.0μL加入96孔板，每个稀释梯度平行做三个副孔；放置在37℃培养箱孵育两个小时，洗板3次。将HRP(辣根过氧化物酶)标记的 IgG(稀释2000倍)，每孔加入100.0μL，室温孵育一小时；洗板3次。加入TMB溶液，每孔加入100.0μL，室温避光显色20分钟。加入0.5M H 2SO 4溶液，每孔加100.0μL。立即用酶标仪检测吸光度，检测波长为450nm，570nm作为背景波长。
3)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1所示。
4)实验结果：
从图1可看出，本发明实施例1合成的KRN7000-Tn糖疫苗(缀合物Ⅲ)，无需外部佐剂的条件下，在小鼠体内能更快速地产生特异性免疫应答，同时激活NKT细胞，高效地将IgM抗体同种型转换成IgG。
2.流式细胞实验(FACS)
实验方法：取过量表达Tn糖抗原的乳腺癌细胞MCF-7和不表达Tn糖抗原的肿瘤细胞MDA-231分别在含10％胎牛血清(FBS)的MEM培养基中培养(37℃，5％CO 2)；胰酶消化，收集细胞，显微镜下数细胞数，每个试管分装2.0×10 5个细胞，加1mL的含3％的FBS的PBS缓冲液(FACS缓冲液)重悬，离心2分钟，去掉上清液，用FACS缓冲液清洗两次；加入制备好的小鼠血清，在冰中孵育1个小时，FACS缓冲液清洗两次，加入荧光标记的二抗，在冰中避光孵育一个小时，FACS缓冲液清洗两次，重悬于0.8mL的FACS缓冲液中，用流式细胞仪检测。
实验结果：如图2所示，MCF-7是过度表达Tn抗原的乳腺癌细胞，把不表达Tn抗原的MDA-231肿瘤细胞为阴性对照。在MCF7细胞中，与免疫前血清相比，本发明实施例1合成的KRN7000-Tn糖疫苗(缀合物Ⅲ)诱导产生的抗体血清的荧光峰明显向右偏移。在MDA-231免疫前血清与抗体血清无明显差别。结果表明，KRN7000-Tn糖疫苗诱导的抗体能特异性识别表达Tn抗原的MCF-7细胞。
3.抗体介导的互补依赖细胞毒性(CDC)
实验方法：取过量表达Tn糖抗原的乳腺癌细胞MCF-7和不表达Tn糖抗原的肿瘤细胞MDA-231分别在含10％胎牛血清(FBS)的DMEM培养基中培养；将对数生长期的细胞配置成1.0×10 5cell/mL密度的细胞悬液，接种到96孔板中，每孔100μL，约10000个细胞，放置到培养箱中培养过夜。除去培养基，无血清的MEM培养液洗三次，加入MEM稀释的小鼠血清，37℃孵育2小时。无血清的MEM洗三次，加入一定比例(1：10)稀释的补体溶液，在37℃培养1小时。同时设置低参照(仅用无血清培养基)和高参照(5％triton-100处理)组。孵育结束后，离心细胞，取20μL的细胞上清液用PBS稀释至100μL，用100μL的LDH细胞毒性检测试剂显色30分钟。在490nm测量每孔的吸光值，根据低参照和高参照孔，计算细胞的裂解率。
实验结果：如图3所示，MCF-7是过度表达Tn抗原的乳腺癌细胞，把不表达Tn抗原的MDA-231肿瘤细胞为阴性对照。在相同条件下，本发明实施例1合成的KRN7000-Tn糖疫苗(缀合物Ⅲ)诱导产生的抗体血清对小鼠产生的抗血清介导的MCF-7细胞裂解率显著高于空白血清。对不表达Tn抗原的MDA-231细胞的细胞毒性与空白血清的差异无统计学意义，结果证实KRN7000-Tn糖疫苗具有一定的特异性抗肿瘤作用。
综上，实验结果表明，本发明提供的一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物(KRN7000-Tn糖疫苗)，以强效免疫刺激剂α-半乳糖神经酰胺类似物(KRN7000)作为内嵌佐剂缀合糖抗原Tn获得，其中，KRN7000能提高Tn糖抗原的免疫原性，将Tn糖抗原提呈到相应的免疫细胞，产生具有更高滴度的针对肿瘤糖抗原Tn特异性的免疫反应，达到杀死肿瘤细胞的目的，产生抗肿瘤作用，有望成为新一代的抗肿瘤药物。
最后所应当说明的是，上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 一种含有α-半乳糖神经酰胺类似物与糖抗原的缀合物，其特征在于，所述缀合物为通式(Ⅰ)的化合物或通式(Ⅰ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
    Figure PCTCN2020130228-appb-100001
    其中：
    n为2-6的整数；
    m为9-25的整数；
    R选自-CH 3、
    Figure PCTCN2020130228-appb-100002
    中的任意一种取代基。
  2. 如权利要求1所述的缀合物，其特征在于，所述缀合物为结构式(Ⅱ)的化合物或结构式(Ⅱ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
    Figure PCTCN2020130228-appb-100003
    其中：n为2-6的整数。
  3. 如权利要求2所述的缀合物，其特征在于，所述的缀合物为结构式(Ⅲ)的化合物或结构式(Ⅲ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
    Figure PCTCN2020130228-appb-100004
  4. 如权利要求1-3任一所述的缀合物，其特征在于，所述的α-半乳糖神经酰胺类似物为通式(Ⅳ)的化合物或通式(Ⅳ)的化合物的异构体、可药用盐、水合物或溶剂化合物；
    Figure PCTCN2020130228-appb-100005
    其中：
    X为O或N任意一种取代基；
    m为9-25的整数；
    R选自-CH 3、
    Figure PCTCN2020130228-appb-100006
    中的任意一种取代基。
  5. 如权利要求1-4任一所述的缀合物的制备方法，其特征在于，包括如下步骤：
    (1)将化合物1和化合物2溶解于有机溶剂中，加入缩合剂反应，得到化合物3；
    (2)将化合物4和步骤(1)所述的化合物3溶解于有机溶剂中，加入催化剂反应，得到化合物5；
    (3)将步骤(2)所述的化合物5在催化剂的作用下与巯基乙酸反应，得到化合物6；
    (4)步骤(3)所述的化合物6溶解于有机溶剂中，加入催化剂，与乙酸酐反应，得到化合物7；
    (5)步骤(4)所述的化合物7溶解于有机溶剂中，加入甲醇钠反应，得到化合物8；
    (6)取化合物9和化合物10溶解于有机溶剂中，加入催化剂反应，得到化合物11；
    (7)取步骤(6)所述的化合物11溶解于有机溶剂中，加入三氟化硼乙醚反应，得到化合物12；
    (8)取步骤(5)所述的化合物8与步骤(7)所述的化合物12溶解于有机溶剂中，加入催化剂反应，得到化合物13。
    (9)取步骤(8)所述的化合物13溶解于有机溶剂中，加入催化剂，经脱苄基反应，即可获得所述的缀合物。
    所述化合物1至所述化合物13的结构式如下所示：
    Figure PCTCN2020130228-appb-100007
    Figure PCTCN2020130228-appb-100008
    其中：
    n为2-6的整数；
    m为9-25的整数；
    R选自-CH 3、
    Figure PCTCN2020130228-appb-100009
    中的任意一种取代基。
    R 1选自-OC(NH)CCl 3、STol、SPh、Set、Cl、Br中的任意一种取代基。
  6. 如权利要求5所述的制备方法，其特征在于，步骤(1)中，所述有机溶剂为二氯甲烷溶液，所述缩合剂为N,N’-二环己基碳二亚胺与1-羟基苯并三唑的混合物；
    步骤(2)中，所述有机溶剂为二氯甲烷、乙醚或四氢呋喃；当化合物4的取代基R1为-OC(NH)CCl 3时，所述催化剂选自氟化硼乙醚、三氟甲磺酸三甲基硅酯、三氟甲磺酸中的任意一种；或者当化合物4的取代基R 1为STol、SPh或Set时，所述催化剂为N-碘代丁二酰亚胺和选自三氟甲磺酸、三氟甲磺酸银、三氟化硼乙醚、三氟甲磺酸三甲基硅酯中的任意一种；或者当化合物4的取代基R 1为Cl或Br时，所述催化剂选自碳酸钾、碳酸银、碳酸铯、碳酸汞、高氯酸银、三氟甲磺酸银、三氟甲磺酸中的任意一种；所述反应的温度为-40～-20℃；
    步骤(3)中，所述催化剂为吡啶，所述催化剂与所述巯基乙酸的体积比为1:1；
    步骤(4)中，所述有机溶剂为N,N-二甲基甲酰胺，所述催化剂为哌啶，所述催化剂与所述乙酸酐的体积比为2:1；
    步骤(5)中，所述有机溶剂为甲醇与二氯甲烷的混合溶液，所述甲醇与所述二氯甲烷的体积比为1:1；所述催化剂为甲醇钠；
    步骤(6)中，所述有机溶剂选自二氯甲烷、乙醚或四氢呋喃中的任意一种，所述催化剂为N-碘代丁二酰亚胺和选自三氟甲磺酸三甲基硅酯、三氟化硼乙醚、三氟甲磺酸银、三氟甲磺酸中的任意一种；
    步骤(7)中，所述有机溶剂为乙腈与二氯甲烷的混合溶液，所述乙腈与所述二氯甲烷的体积比为1.5:1；
    步骤(8)中，所述溶剂为二氯甲烷与甲醇的混合溶液，所述催化剂为碘化亚铜、N,N-二异丙基乙胺与冰乙酸的混合物；
    步骤(9)中，所述溶剂二氯甲烷与甲醇的混合溶液，所述催化剂为氢气、钯碳与氢氧化钯的混合物。
  7. 如权利要求6所述的制备方法，其特征在于，步骤(2)中，所述有机溶剂为二氯甲烷；当化合物4的取代基R 1为-OC(NH)CCl 3时，所述催化剂为三氟甲磺酸三甲基硅酯；
    步骤(6)中，所述有机溶剂为二氯甲烷与乙醚的混合溶液，所述催化剂为N-碘代丁二酰亚胺和三氟甲磺酸三甲基硅酯的混合物。
  8. 如权利要求1-4任一所述的缀合物在制备预防和/或治疗癌症的药物中的应用。
  9. 如权利要求8所述的应用，所述癌症为乳腺癌、卵巢癌、肺癌、子宫癌、肝癌、前列腺癌、黑素瘤、肠癌、肾细胞癌、细胞性淋巴癌、胰腺癌、甲腺癌、脑癌、胃癌和白血病。
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