WO2021232718A1 - Conjugate and preparation method therefor and use thereof - Google Patents

Conjugate and preparation method therefor and use thereof Download PDF

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WO2021232718A1
WO2021232718A1 PCT/CN2020/130227 CN2020130227W WO2021232718A1 WO 2021232718 A1 WO2021232718 A1 WO 2021232718A1 CN 2020130227 W CN2020130227 W CN 2020130227W WO 2021232718 A1 WO2021232718 A1 WO 2021232718A1
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compound
integer
conjugate
catalyst
organic solvent
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PCT/CN2020/130227
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French (fr)
Chinese (zh)
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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
卢琳琳
王彩艳
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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • 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
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • 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 present invention relates to a conjugate and a preparation method and application thereof, in particular to a conjugate of a dual agonist and carbohydrate antigen Tn, and a preparation method and application thereof.
  • the dual agonist is a TLR4 receptor agonist and NKT Cell agonists belong to the technical field of the development of anti-tumor sugar vaccines.
  • Tumor-associated carbohydrate antigens are overexpressed in a variety of tumor cells, and their glycoprotein vaccines are under clinical research and have good development prospects.
  • 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.
  • Tumor-associated carbohydrate antigens are generally T cell-independent antigens, which only produce low-affinity IgM, and have disadvantages such as poor immunogenicity and immune tolerance.
  • the main challenge currently faced by anti-tumor sugar vaccines is how to overcome the immune tolerance of sugar antigens, enhance their immunogenicity, and enable the body to produce high-affinity IgG antibodies and immune memory, so as to achieve the purpose of killing tumor cells.
  • the classic strategy is to conjugate a carbohydrate antigen with a carrier protein to enhance its immunogenicity, but glycoprotein vaccines have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components.
  • Lipid A of the hydrophobic part of bacterial lipopolysaccharide is an agonist of Toll-like receptor 4 (TLR4), which can target TLR4 and produce an immune response, but its toxicity is too strong.
  • TLR4 Toll-like receptor 4
  • MPLA Monophosphoryl lipid A
  • KRN7000 ⁇ -Galactose ceramide analogue
  • KPN7000 is a natural ⁇ -GalGSL analogue isolated from a sponge, which can activate NKT cells to produce an immune response.
  • KPN7000 has entered phase II clinical research for the comprehensive treatment of patients with non-small cell lung cancer and malignant solid tumors. It is used as a fully synthetic vaccine KRN7000-Tn and KRN7000-sTn conjugated with carbohydrate antigen as an embedded adjuvant without external adjuvant conditions.
  • mice specific immune responses can be generated more quickly, and NKT cells can be activated at the same time, and the IgM antibody isotype can be efficiently converted into IgG (Organic Letters, 2017, 19: 456; Journal of medical chemistry, 2018, 61 :4918.)
  • the present invention selects Tn in TACAs with potential as the research target, selects TLR4 agonist MPLA and NKT cell agonist KRN7000 analogs as endogenous adjuvants, and prepares a TLR4 receptor containing TLR4.
  • a three-component vaccine (MPLA-Tn-KRN7000) conjugated with dual agonists of human and NKT cells (MPLA and KRN7000) and Tn.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a conjugate of a dual agonist and carbohydrate antigen Tn, the dual agonist is TLR4 receptor agonist monophosphorylated lipid A (MPLA) and NKT Cell agonist ⁇ -galactose ceramide analog (KRN7000).
  • MPLA TLR4 receptor agonist monophosphorylated lipid A
  • KRN7000 NKT Cell agonist ⁇ -galactose ceramide analog
  • the technical solution adopted by the present invention is: a conjugate, characterized in that the conjugate contains a dual agonist and a carbohydrate antigen Tn, and the dual agonist is a TLR4 receptor agonist and NKT A cell agonist, the TLR4 receptor agonist is a monophosphorylated lipid A, and the NKT cell agonist is an ⁇ -galactose ceramide analog;
  • MPLA is a monophosphorylated lipid A
  • KRN7000 is an analog of ⁇ -galactose ceramide
  • a is an integer of 1-5, and both b and c are integers of 1-10.
  • TLR4 agonist monophosphorylated lipid A MPLA
  • NKT cell agonist ⁇ -galactose ceramide analogue KRN7000
  • the present invention prepares a monophosphorylated lipid A (MPLA) containing TLR4 agonist and NKT cell agonist ⁇ -Galactose ceramide analog (KRN7000) and a three-component conjugate of carbohydrate antigen Tn (MPLA-Tn-KRN7000).
  • MPLA monophosphorylated lipid A
  • KRN7000 NKT cell agonist ⁇ -Galactose ceramide analog
  • MPLA-Tn-KRN7000 carbohydrate antigen Tn
  • the conjugate includes a compound of general formula (I) or an isomer, pharmaceutically acceptable salt, hydrate, or isomer of a compound of general formula (I).
  • Solvent compound
  • R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
  • R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
  • R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14;
  • a is an integer of 1-5, and both b and c are integers of 1-10;
  • n is an integer of 9-25;
  • R is -CH 3 or
  • 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);
  • a is an integer of 1-5, and b is an integer of 1-10.
  • 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 monophosphorylated lipid A is a compound of general formula (IV) or an isomer, pharmaceutically acceptable salt, hydrated compound of general formula (IV) Substance or solvent compound;
  • R 5 is -(CH 2 )pCH 3 , and p is an integer of 8-12;
  • R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14.
  • the ⁇ -galactoseceramide analogue is a compound of the general formula (V) or an isomer, a pharmaceutically acceptable salt of the compound of the general formula (V), Hydrate or solvent compound;
  • n is any integer from 9-25;
  • R is -H or
  • Another object of the present invention is to provide a preparation method of the conjugate, which includes the following steps:
  • step (3) Dissolve compound 4 and compound 3 described in step (2) in an organic solvent, add a condensing agent, and perform an esterification reaction to obtain compound 5;
  • step (3) The compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst to obtain compound 6;
  • step (6) Dissolve the compound with the compound 7 described in step (5) in an organic solvent, and add a catalyst to react to obtain compound 9;
  • step (6) Dissolve the compound 9 described in step (6) in an organic solvent, add a catalyst, and perform a debenzylation reaction to obtain the conjugate;
  • R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
  • R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
  • R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , r is an integer of 8-14; a is an integer of 1-5, and b and c are both integers of 1-10;
  • n is an integer of 9-25;
  • R is -CH 3 or
  • reaction formula of the preparation method of the present invention is as follows:
  • R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
  • R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
  • R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14;
  • a is an integer of 1-5, and both b and c are integers of 1-10;
  • n is an integer of 9-25;
  • R is -CH 3 or
  • the preparation method provided by the invention has short synthetic route, mild reaction conditions, high yield and convenient operation, and can be widely used in industrial preparation.
  • step (1) compound 1 is dissolved in an organic solvent, and a catalyst is added to obtain compound 2.
  • the solvent is dichloromethane, and the catalyst is zinc powder and A mixture of acetic acid.
  • step (2) the compound 2 described in step (1) is dissolved in an organic solvent and linked with Linker under the action of a catalyst to obtain compound 3;
  • the solvent is dichloromethane
  • the catalyst is N,N-diisopropylethylamine.
  • step (3) compound 4 and compound 3 described in step (2) are dissolved in an organic solvent, and a condensing agent is added to perform an esterification reaction to obtain compound 5.
  • the organic solvent is a dichloromethane solution, and the condensing agent is selected from a mixture of N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt).
  • step (4) the compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst.
  • the organic solvent is a mixed solution of acetonitrile and dichloromethane, the volume ratio of the acetonitrile to the dichloromethane is 1.5:1, and the catalyst is a boron trifluoride ether complex.
  • step (5) the compound 6 described in step (4) is dissolved in an organic solvent, and a catalyst is added to perform a deacetylation reaction to obtain compound 7;
  • the organic solvent is a mixed solution of methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is 2:1; the catalyst is sodium methoxide.
  • step (6) the compound and the compound 7 and compound 8 described in step (5) are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 9;
  • the solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of cuprous iodide, N,N-diisopropylethylamine and glacial acetic acid.
  • step (7) the compound 9 described in step (6) is dissolved in an organic solvent, a catalyst is added, and the debenzylation reaction is carried out to obtain the Conjugate 10;
  • the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of hydrogen, palladium on 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, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, bowel cancer, renal cell carcinoma, cellular lymphoma , Thyroid cancer, brain cancer, stomach cancer or leukemia.
  • TACAs tumor-associated carbohydrate antigens
  • TLR4 agonist monophosphorylated lipid A (MPLA) and NKT cell agonist ⁇ -galactoseramide analog (KRN7000) were selected as endogenous adjuvants to prepare a TLR4 agonist MPLA and NKT cell agonist A three-component conjugate of KRN7000 and carbohydrate antigen Tn (MPLA-Tn-KRN7000).
  • MPLA monophosphorylated lipid A
  • KRN7000 NKT cell agonist ⁇ -galactoseramide analog
  • MPLA-Tn-KRN7000 three-component conjugate of KRN7000 and carbohydrate antigen Tn
  • This conjugate is used as a vaccine to activate TLR4 receptors and NKT cells at the same time to cause a stronger immune response against carbohydrate antigen Tn.
  • T cells with higher titer, high affinity and memory regulate the immune response, so as to achieve the purpose of killing tumor cells specifically.
  • the preparation method of the conjugate provided by the present invention has a short synthetic route, mild reaction conditions, high yield and convenient operation, and can be widely used in industrial preparation.
  • Figure 1 is an evaluation diagram of the immune activity of the MPLA-Tn-KRN7000 three-component sugar vaccine
  • Figure 2 is a flow cytometric evaluation diagram of the antibody serum produced by the MPLA-Tn-KRN7000 three-component carbohydrate vaccine inducing mice to specifically recognize the tumor cell MCF-7;
  • Figure 3 is an evaluation diagram of complement-dependent cytotoxicity of the antibody serum produced by the MPLA-Tn-KRN7000 three-component carbohydrate vaccine inducing mice to specifically kill the tumor cell MCF-7.
  • This embodiment is a conjugate provided by the present invention.
  • 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 preparation method of the above-mentioned conjugate includes the following steps:
  • step (1) dissolve compound 1 (700.0mg, 0.5mmol) and zinc powder (658.0mg, 10.1mmol) in dichloromethane solution (10.0mL), add acetic acid (0.6mL, 10.1mmol), room temperature conditions Stir under the pressure for 12 hours; filter with diatomaceous earth, wash with saturated sodium bicarbonate aqueous solution for 3 times, collect the organic layer, dry with anhydrous sodium sulfate, filter, and distill the filtrate under reduced pressure to remove the organic solvent to obtain the crude product; separate and purify by silica gel column to obtain a colorless oil Liquid compound 2 (503.0 mg, yield 73.2%).
  • step (1) The compound 2 described in step (1) is dissolved in an organic solvent, and linked with Linker under the action of a catalyst to obtain compound 3; the reaction formula for obtaining compound 3 is shown in the following formula:
  • step (3) Dissolve compound 4 and compound 3 described in step (2) in an organic solvent, add a condensing agent, and perform an esterification reaction to obtain compound 5; the reaction formula for obtaining compound 5 is shown in the following formula:
  • step (3) dissolving compound 3 (160.0 mg, 107.9 ⁇ mol), compound 4 (74.0 mg, 129.5 ⁇ mol), 1-(3-dimethylaminopropyl) in dichloromethane solution (3.0 mL) -3-Ethylcarbodiimide hydrochloride (48.0mg, 233.0 ⁇ mol) and Hobt (14.0mg, 103.7 ⁇ mol), stirred at room temperature for 3 hours; diluted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution in turn 2 times, washed with brine once, the water layer was removed and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to remove the organic solvent to obtain a crude product; silica gel column separation and purification to obtain a white solid compound 5 (172.0mg, yield: 78.5 %).
  • step (3) The compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst to obtain compound 6; the reaction formula for obtaining compound 6 is shown in the following formula:
  • step (4) dissolve compound 5 (160.0mg, 69.3 ⁇ mol) in acetonitrile/dichloromethane (1.5:1, 2.5mL), add boron trifluoride ether complex (19.0 ⁇ L, 152.5 ⁇ mol), The reaction was stirred at room temperature for 2 hours; the organic solvent was distilled off under reduced pressure to obtain a crude product, and the silica gel column was separated and purified to obtain compound 6 (107.0 mg, yield 75.3%) as a white solid.
  • step (4) The compound 6 described in step (4) is dissolved in an organic solvent, and a catalyst is added to perform a deacetylation reaction to obtain compound 7; the reaction formula for obtaining compound 7 is shown in the following formula:
  • step (5) The compound and the compound 7 described in step (5) are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 9; the reaction formula for obtaining compound 9 is shown in the following formula:
  • step (6) The specific operation of step (6) is: dissolving compound 8 (52.2 mg, 23.2 ⁇ mol), compound 7 (20.0 mg, 11.9 ⁇ mol), cuprous iodide (113.0 mg, 595.0) in tetrahydrofuran and methanol (1: 2, 3.0 mL) ⁇ mol), add N,N-diisopropylethylamine (97.0 ⁇ L, 595.0 ⁇ mol), stir at room temperature for 12 hours; filter out the insoluble matter with Celite, and distill the filtrate under reduced pressure to remove the solvent to obtain the crude product; silica gel column separation and purification A white solid compound 9 (14.0 mg, yield 30.4%) was obtained.
  • step (6) The compound 9 described in step (6) is dissolved in an organic solvent, and a catalyst is added to carry out the debenzylation reaction to obtain the conjugate;
  • the reaction formula for obtaining the conjugate (V) is as follows Shown:
  • step (6) dissolve compound 9 (8.0 mg, 2.0 ⁇ mol) in dichloromethane/methanol/water (5:5:1, 10.0mL), add palladium hydroxide (5.0mg) and palladium on carbon (5.0 mg), hydrogen gas was introduced, sealed and stirred 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 39, the target product MPLA-Tn-KRN7000 (5.6 mg, 85.8%).
  • mice were immunized with the conjugate (fully synthetic sugar vaccine) prepared in Example 1, and its immune effect was preliminarily evaluated by ELSA experiment.
  • the fluorescence activated cell sorting (FACS) technology proved that antibody serum can specifically recognize tumors.
  • 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.
  • mice C57BL/6 mice aged 6-8 weeks were divided into an immunized group and a control group (PBS), with 6 mice in each group.
  • 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 serum samples of 6 mice in the same group were diluted 300, 900, 2700, 8100, 24300, 72900, 218700 and 656 100 times with PBS; the diluted serum was added to a 96-well plate at 100.0 ⁇ L per well, and each dilution gradient was done in parallel Three secondary wells; placed in a 37°C incubator and incubated for two hours, and the plate was washed 3 times.
  • Add 100.0 ⁇ L of HRP (horseradish peroxidase) labeled IgG (diluted 2000 times) to each well, incubate at room temperature for one hour; wash the plate 3 times.
  • Add TMB solution add 100.0 ⁇ L to each well, and develop color in the dark at room temperature for 20 minutes.
  • Add 0.5M H 2 SO 4 solution add 100.0 ⁇ L to each well.
  • the detection wavelength is 450nm, and 570nm is used as the background wavelength.
  • the MPLA-Tn-KRN7000 three-component sugar vaccine synthesized in Example 1 of the present invention induces the production of high-titer Kappa and IgG antibodies in mice without external adjuvants.
  • the dual adjuvant design of the MPLA-Tn-KRN7000 three-component vaccine can effectively improve the immunogenicity of the Tn carbohydrate 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 fluorescence peak of antibody serum produced by MPLA-Tn-KRN7000 three-component carbohydrate vaccine induced a significant shift to the right.
  • MDA-231 cells there is no significant difference between pre-immune serum and antibody serum. The results show that the antibodies induced by the MPLA-Tn-KRN7000 carbohydrate antigen vaccine prepared in Example 1 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 MPLA-Tn-KRN7000 three-component carbohydrate antigen vaccine produced antiserum-mediated MCF-7 cell lysis rate in mice that was significantly higher than that of blank serum. Under the same conditions, there was no statistically significant difference in the cytotoxicity of antibody-mediated pre-immune serum and antibody serum to MDA-231 cells. The results confirmed that the MPLA-Tn-KRN7000 three-component carbohydrate antigen vaccine prepared in Example 1 has a certain specific anti-cancer effect.
  • the experimental results show that a three-component conjugate (MPLA-Tn-KRN7000) containing the TLR4 agonist MPLA and the NKT cell agonist KRN7000 and the carbohydrate antigen Tn prepared by the present invention is used as a carbohydrate antigen
  • the vaccine by activating TLR4 receptors and NKT cells at the same time, produces a stronger immune response than a single activation of the immune system, causing a stronger immune response against the sugar antigen Tn, and producing T with higher titer, high affinity and memory. Cells regulate the immune response, so as to achieve the purpose of killing tumor cells specifically.

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Abstract

A conjugate and a preparation method therefor. The conjugate comprises a dual agonist and a carbohydrate antigen Tn, wherein the dual agonist is a TLR4 receptor agonist and a NKT cell agonist, the TLR4 receptor agonist being monophosphorylated lipid A, and the NKT cell agonist being an α-galactosyl ceramide analogue. A use of the conjugate in the preparation of drugs for preventing and/or treating cancers. The conjugate is used as a vaccine and can simultaneously activate the TLR4 receptor and NKT cells.

Description

一种缀合物及其制备方法和应用A conjugate and its preparation method and application 技术领域Technical field
本发明涉及一种缀合物及其制备方法和应用，尤其涉及一种双重激动剂与糖抗原Tn的缀合物及其制备方法和应用，所述双重激动剂为TLR4受体激动剂和NKT细胞激动剂，属于抗肿瘤糖疫苗研制技术领域。The present invention relates to a conjugate and a preparation method and application thereof, in particular to a conjugate of a dual agonist and carbohydrate antigen Tn, and a preparation method and application thereof. The dual agonist is a TLR4 receptor agonist and NKT Cell agonists belong to the technical field of the development of anti-tumor sugar vaccines.
背景技术Background technique
肿瘤相关的糖抗原(TACAs)在多种肿瘤细胞中有过量表达，其糖蛋白疫苗正处于临床研究中，具有较好的发展前景。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。肿瘤相关的糖抗原(TACAs)一般属于T细胞非依赖性抗原，只产生低亲和力的IgM，具有免疫原性差和免疫耐受等缺点。因此，目前抗肿瘤糖疫苗面临的主要挑战是如何克服糖抗原的免疫耐受，增强其免疫原性，使机体产生高亲和力的IgG抗体和免疫记忆，从而达到杀死肿瘤细胞的目的。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。Tumor-associated carbohydrate antigens (TACAs) are overexpressed in a variety of tumor cells, and their glycoprotein vaccines are under clinical research and have good development prospects. Among them, 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. Tumor-associated carbohydrate antigens (TACAs) are generally T cell-independent antigens, which only produce low-affinity IgM, and have disadvantages such as poor immunogenicity and immune tolerance. Therefore, the main challenge currently faced by anti-tumor sugar vaccines is how to overcome the immune tolerance of sugar antigens, enhance their immunogenicity, and enable the body to produce high-affinity IgG antibodies and immune memory, so as to achieve the purpose of killing tumor cells. The classic strategy is to conjugate a carbohydrate antigen with a carrier protein to enhance its immunogenicity, but glycoprotein vaccines have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components.
细菌脂多糖(Lipidpolysacchrides，LPS)疏水部分的Lipid A是Toll样受体4(TLR4)的激动剂，能靶向地结合TLR4并产生免疫反应，但其毒性过强。研究发现去掉Lipid A结构中1位磷酸(反应式如下所示)后的Monophosphoryl lipid A(MPLA)依然能靶向地与TLR4结合，毒性明显降低而活性变化不明显(Microbes&Infection,2002,4(9):915-926.)。其作为内嵌佐剂与多种糖抗原结合来制备的抗肿瘤糖缀合物疫苗MPLA-Golob H和MPLA-STn和MPLA-GM2，在无外加佐剂的情况下，都能在小鼠体内更快速地产生高滴度的IgG抗体(Chemical Science,2015,6:7112；Scientific reports,2017,7:11403；Biomolecular Chemistry,2014,12:3238)，Lipid A of the hydrophobic part of bacterial lipopolysaccharide (Lipidpolysacchrides, LPS) is an agonist of Toll-like receptor 4 (TLR4), which can target TLR4 and produce an immune response, but its toxicity is too strong. Studies have found that Monophosphoryl lipid A (MPLA) after removing the phosphate at position 1 in the Lipid A structure (the reaction formula is shown below) can still target TLR4, with significantly reduced toxicity and insignificant activity changes (Microbes&Infection, 2002, 4(9) ):915-926.). It is an anti-tumor glycoconjugate vaccine MPLA-Golob H, MPLA-STn and MPLA-GM2 prepared by combining with a variety of carbohydrate antigens as an embedded adjuvant. It can be used in mice without additional adjuvants. Produce high-titer IgG antibodies more quickly (Chemical Science, 2015, 6: 7112; Scientific reports, 2017, 7: 11403; Biomolecular Chemistry, 2014, 12: 3238),
Figure PCTCN2020130227-appb-000001
Figure PCTCN2020130227-appb-000001
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，能激活NKT细胞产生免疫反应。目前，KPN7000对非小细胞肺癌患者、恶性实体瘤综合治疗均进入Ⅱ期临床研究，其作为内嵌佐剂缀合糖抗原的全合成疫苗KRN7000-Tn，KRN7000-sTn，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(Organic Letters,2017,19:456；Journal of medicinal chemistry,2018,61:4918.)α-Galactose ceramide analogue (KRN7000) is a natural α-GalGSL analogue isolated from a sponge, which can activate NKT cells to produce an immune response. At present, KPN7000 has entered phase II clinical research for the comprehensive treatment of patients with non-small cell lung cancer and malignant solid tumors. It is used as a fully synthetic vaccine KRN7000-Tn and KRN7000-sTn conjugated with carbohydrate antigen as an embedded adjuvant without external adjuvant conditions. In mice, specific immune responses can be generated more quickly, and NKT cells can be activated at the same time, and the IgM antibody isotype can be efficiently converted into IgG (Organic Letters, 2017, 19: 456; Journal of medical chemistry, 2018, 61 :4918.)
Figure PCTCN2020130227-appb-000002
Figure PCTCN2020130227-appb-000002
为了解决上述糖蛋白疫苗的缺点，本发明选择有发展潜力TACAs中的Tn为研究靶点，选用TLR4激动剂MPLA和NKT细胞激动剂KRN7000类似物作为内源性佐剂，制备一种含有TLR4受体和NKT细胞的双重激动剂(MPLA和KRN7000)与Tn缀合的三组分疫苗(MPLA-Tn-KRN7000)。In order to solve the shortcomings of the aforementioned glycoprotein vaccines, the present invention selects Tn in TACAs with potential as the research target, selects TLR4 agonist MPLA and NKT cell agonist KRN7000 analogs as endogenous adjuvants, and prepares a TLR4 receptor containing TLR4. A three-component vaccine (MPLA-Tn-KRN7000) conjugated with dual agonists of human and NKT cells (MPLA and KRN7000) and Tn.
发明内容Summary of the invention
本发明的目的在于克服现有技术的不足，提供一种双重激动剂与糖抗原Tn的缀合物，所述双重激动剂为TLR4受体激动剂单磷酸化的脂质A(MPLA)和NKT细胞激动剂α-半乳糖神经酰胺类似物(KRN7000)。The purpose of the present invention is to overcome the shortcomings of the prior art and provide a conjugate of a dual agonist and carbohydrate antigen Tn, the dual agonist is TLR4 receptor agonist monophosphorylated lipid A (MPLA) and NKT Cell agonist α-galactose ceramide analog (KRN7000).
为实现上述目的，本发明采取的技术方案为：一种缀合物，其特征在于， 所述缀合物含有双重激动剂与糖抗原Tn，所述双重激动剂为TLR4受体激动剂和NKT细胞激动剂，所述TLR4受体激动剂为单磷酸化的脂质A，所述NKT细胞激动剂为α-半乳糖神经酰胺类似物；To achieve the above objective, the technical solution adopted by the present invention is: a conjugate, characterized in that the conjugate contains a dual agonist and a carbohydrate antigen Tn, and the dual agonist is a TLR4 receptor agonist and NKT A cell agonist, the TLR4 receptor agonist is a monophosphorylated lipid A, and the NKT cell agonist is an α-galactose ceramide analog;
所述缀合物的结构通式如下式(A)、式(B)、式(C)或式(D)所示：The general structural formula of the conjugate is shown in the following formula (A), formula (B), formula (C) or formula (D):
Figure PCTCN2020130227-appb-000003
Figure PCTCN2020130227-appb-000003
其中：in:
MPLA为单磷酸化的脂质A；MPLA is a monophosphorylated lipid A;
KRN7000为α-半乳糖神经酰胺类似物；KRN7000 is an analog of α-galactose ceramide;
a为1-5的整数，b与c均为1-10的整数。a is an integer of 1-5, and both b and c are integers of 1-10.
有研究表明，内源性佐剂如TLR4激动剂单磷酸化的脂质A(MPLA)和NKT细胞激动剂α-半乳糖神经酰胺类似物(KRN7000)，可以帮助糖抗原提呈至相应的免疫细胞，从而产生疫苗应答；将其代替载体蛋白，与TACAs糖抗原偶联得到两组分疫苗具有非常好的免疫效果。因此，本发明选择有发展潜力TACAs中的Tn为研究靶点，选用TLR4激动剂MPLA和NKT细胞激动剂KRN7000作为内源性佐剂。为了克服了糖蛋白疫苗偶联位点不确定、偶联率不稳定、组成成分复杂等缺点，本发明制备一种含有TLR4激动剂单磷酸化的脂质A(MPLA)和NKT细胞激动剂α-半乳糖神经酰胺类似物(KRN7000)以及糖抗原Tn三组分的缀合物(MPLA-Tn-KRN7000)。该缀合物作为疫苗，通过同时激活TLR4受体和NKT细胞，以期产生更强的免疫应答，克服Tn免疫原性差的弱点，并且产生具有更高滴度、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。Studies have shown that endogenous adjuvants such as TLR4 agonist monophosphorylated lipid A (MPLA) and NKT cell agonist α-galactose ceramide analogue (KRN7000) can help the presentation of carbohydrate antigens to the corresponding immune system. Cells, thereby generating a vaccine response; replacing the carrier protein with TACAs carbohydrate antigen coupling to obtain a two-component vaccine with a very good immune effect. Therefore, the present invention selects Tn in TACAs with potential as the research target, and selects TLR4 agonist MPLA and NKT cell agonist KRN7000 as endogenous adjuvants. In order to overcome the shortcomings of glycoprotein vaccine coupling site uncertainty, unstable coupling rate, complex composition, etc., the present invention prepares a monophosphorylated lipid A (MPLA) containing TLR4 agonist and NKT cell agonist α -Galactose ceramide analog (KRN7000) and a three-component conjugate of carbohydrate antigen Tn (MPLA-Tn-KRN7000). The conjugate is used as a vaccine to simultaneously activate TLR4 receptors and NKT cells to generate a stronger immune response, overcome the weakness of Tn immunogenicity, and produce T cell regulation with higher titer, high affinity and memory Immune response to achieve the purpose of killing tumor cells.
作为本发明所述的缀合物的优选实施方式，所述的缀合物包括为通式(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；As a preferred embodiment of the conjugate of the present invention, the conjugate includes a compound of general formula (I) or an isomer, pharmaceutically acceptable salt, hydrate, or isomer of a compound of general formula (I). Solvent compound
Figure PCTCN2020130227-appb-000004
Figure PCTCN2020130227-appb-000004
其中：in:
R 1和R 3为-(CH 2)mCH 3，m为10-14的整数； R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
R 2、R 4和R 5为-(CH 2)pCH 3，p为8-12的整数； R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数； R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14;
a为1-5的整数，b与c均为1-10的整数；a is an integer of 1-5, and both b and c are integers of 1-10;
n为9-25的整数；n is an integer of 9-25;
R为-CH 3或
Figure PCTCN2020130227-appb-000005
R is -CH 3 or
Figure PCTCN2020130227-appb-000005
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；As a preferred embodiment of the conjugate of the present invention, 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);
Figure PCTCN2020130227-appb-000006
Figure PCTCN2020130227-appb-000006
其中：a为1-5的整数，b为1-10的整数。Wherein: a is an integer of 1-5, and b is an integer of 1-10.
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；As a preferred embodiment of the conjugate of the present invention, 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);
Figure PCTCN2020130227-appb-000007
Figure PCTCN2020130227-appb-000007
作为本发明所述的缀合物的优选实施方式，所述单磷酸化的脂质A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；As a preferred embodiment of the conjugate of the present invention, the monophosphorylated lipid A is a compound of general formula (IV) or an isomer, pharmaceutically acceptable salt, hydrated compound of general formula (IV) Substance or solvent compound;
Figure PCTCN2020130227-appb-000008
Figure PCTCN2020130227-appb-000008
其中：in:
R 5为-(CH 2)pCH 3，p为8-12的整数； R 5 is -(CH 2 )pCH 3 , and p is an integer of 8-12;
R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数。 R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14.
作为本发明所述的缀合物的优选实施方式，所述α-半乳糖神经酰胺类似物为通式(Ⅴ)的化合物或通式(Ⅴ)的化合物的异构体、可药用盐、水合物或溶剂化合物；As a preferred embodiment of the conjugate of the present invention, the α-galactoseceramide analogue is a compound of the general formula (V) or an isomer, a pharmaceutically acceptable salt of the compound of the general formula (V), Hydrate or solvent compound;
Figure PCTCN2020130227-appb-000009
Figure PCTCN2020130227-appb-000009
其中：in:
n为9-25的任意整数；n is any integer from 9-25;
R为-H或
Figure PCTCN2020130227-appb-000010
R is -H or
Figure PCTCN2020130227-appb-000010
另外，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：In addition, another object of the present invention is to provide a preparation method of the conjugate, which includes the following steps:
(1)取化合物1溶解于有机溶剂中，加入催化剂，得到化合物2；(1) Dissolve compound 1 in an organic solvent and add a catalyst to obtain compound 2;
(2)取步骤(1)所述的化合物2溶解于有机溶剂中，在催化剂的作用下，与Linker链接，得到化合物3；(2) Dissolve compound 2 described in step (1) in an organic solvent, and link with Linker under the action of a catalyst to obtain compound 3;
(3)取化合物4与步骤(2)所述的化合物3溶解于有机溶剂中，加入缩合剂，进行酯化反应，得到化合物5；(3) Dissolve compound 4 and compound 3 described in step (2) in an organic solvent, add a condensing agent, and perform an esterification reaction to obtain compound 5;
(4)取步骤(3)所述的化合物5溶解于有机溶剂中，在催化剂的作用下，脱去保护基团三甲基硅烷，得到化合物6；(4) The compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst to obtain compound 6;
(5)取步骤(4)所述的化合物6溶解于有机溶剂中，加入催化剂，进行脱乙酰化反应，得到化合物7；(5) Dissolve compound 6 described in step (4) in an organic solvent, add a catalyst, and perform a deacetylation reaction to obtain compound 7;
(6)取化合物与步骤(5)所述的化合物7溶解于有机溶剂中，加入催化剂反应，得到化合物9；(6) Dissolve the compound with the compound 7 described in step (5) in an organic solvent, and add a catalyst to react to obtain compound 9;
(7)取步骤(6)所述的化合物9溶解于有机溶剂中，加入催化剂，进行脱苄基反应，即可得到所述的缀合物；(7) Dissolve the compound 9 described in step (6) in an organic solvent, add a catalyst, and perform a debenzylation reaction to obtain the conjugate;
所述化合物1至所述化合物9的结构式如下所示：The structural formulas of the compound 1 to the compound 9 are as follows:
Figure PCTCN2020130227-appb-000011
Figure PCTCN2020130227-appb-000011
Figure PCTCN2020130227-appb-000012
Figure PCTCN2020130227-appb-000012
Figure PCTCN2020130227-appb-000013
Figure PCTCN2020130227-appb-000013
其中，in,
R 1和R 3为-(CH 2)mCH 3，m为10-14的整数； R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
R 2、R 4和R 5为-(CH 2)pCH 3，p为8-12的整数； R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数；a为1-5的整数，b与c均为1-10的整数； R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , r is an integer of 8-14; a is an integer of 1-5, and b and c are both integers of 1-10;
n为9-25的整数；n is an integer of 9-25;
R为-CH 3或
Figure PCTCN2020130227-appb-000014
R is -CH 3 or
Figure PCTCN2020130227-appb-000014
本发明所述的制备方法的反应式如下所示：The reaction formula of the preparation method of the present invention is as follows:
Figure PCTCN2020130227-appb-000015
Figure PCTCN2020130227-appb-000015
Figure PCTCN2020130227-appb-000016
Figure PCTCN2020130227-appb-000016
其中，in,
R 1和R 3为-(CH 2)mCH 3，m为10-14的整数； R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
R 2、R 4和R 5为-(CH 2)pCH 3，p为8-12的整数； R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数； R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14;
a为1-5的整数，b与c均为1-10的整数；a is an integer of 1-5, and both b and c are integers of 1-10;
n为9-25的整数；n is an integer of 9-25;
R为-CH 3或
Figure PCTCN2020130227-appb-000017
R is -CH 3 or
Figure PCTCN2020130227-appb-000017
本发明提供的制备方法，合成路线简短、反应条件温和、产率高、操作方便，能够广泛地应用于工业化制备。The preparation method provided by the invention has short synthetic route, mild reaction conditions, high yield and convenient operation, and can be widely used in industrial preparation.
作为本发明所述的制备方法的优选实施方式，步骤(1)中，取化合物1溶解于有机溶剂中，加入催化剂，得到化合物2；所述溶剂为二氯甲烷，所述催化剂为锌粉与乙酸的混合物。As a preferred embodiment of the preparation method of the present invention, in step (1), compound 1 is dissolved in an organic solvent, and a catalyst is added to obtain compound 2. The solvent is dichloromethane, and the catalyst is zinc powder and A mixture of acetic acid.
作为本发明所述的制备方法的优选实施方式，步骤(2)中，取步骤(1)所述的化合物2溶解于有机溶剂中，在催化剂的作用下，与Linker链接，得到化合物3；所述溶剂为二氯甲烷，所述催化剂为N,N-二异丙基乙胺。As a preferred embodiment of the preparation method of the present invention, in step (2), the compound 2 described in step (1) is dissolved in an organic solvent and linked with Linker under the action of a catalyst to obtain compound 3; The solvent is dichloromethane, and the catalyst is N,N-diisopropylethylamine.
作为本发明所述的制备方法的优选实施方式，步骤(3)中，取化合物4与步骤(2)所述的化合物3溶解于有机溶剂中，加入缩合剂，进行酯化反应，得到化合物5；所述有机溶剂为二氯甲烷溶液，所述缩合剂选自为N,N’-二环己基碳二亚胺(DCC)与1-羟基苯并三唑(HOBt)的混合物。As a preferred embodiment of the preparation method of the present invention, in step (3), compound 4 and compound 3 described in step (2) are dissolved in an organic solvent, and a condensing agent is added to perform an esterification reaction to obtain compound 5. The organic solvent is a dichloromethane solution, and the condensing agent is selected from a mixture of N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt).
作为本发明所述的制备方法的优选实施方式，步骤(4)中，取步骤(3)所述的化合物5溶解于有机溶剂中，在催化剂的作用下，脱去保护基团三甲基硅烷，得到化合物6；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，所述的催化剂为三氟化硼乙醚络合物。As a preferred embodiment of the preparation method of the present invention, in step (4), the compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst. , To obtain compound 6; the organic solvent is a mixed solution of acetonitrile and dichloromethane, the volume ratio of the acetonitrile to the dichloromethane is 1.5:1, and the catalyst is a boron trifluoride ether complex.
作为本发明所述的制备方法的优选实施方式，步骤(5)中，取步骤(4)所述的化合物6溶解于有机溶剂中，加入催化剂，进行脱乙酰化反应，得到化合物7；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2:1；所述的催化剂为甲醇钠。As a preferred embodiment of the preparation method of the present invention, in step (5), the compound 6 described in step (4) is dissolved in an organic solvent, and a catalyst is added to perform a deacetylation reaction to obtain compound 7; The organic solvent is a mixed solution of methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is 2:1; the catalyst is sodium methoxide.
作为本发明所述的制备方法的优选实施方式，步骤(6)中，取化合物与步骤(5)所述的化合物7与化合物8溶解于有机溶剂中，加入催化剂反应，得到化合物9；所述溶剂为二氯甲烷、甲醇与水的混合溶液，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。As a preferred embodiment of the preparation method of the present invention, in step (6), the compound and the compound 7 and compound 8 described in step (5) are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 9; The solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of cuprous iodide, N,N-diisopropylethylamine and glacial acetic acid.
作为本发明所述的制备方法的优选实施方式，步骤(7)中，取步骤(6)所述的化合物9溶解于有机溶剂中，加入催化剂，进行脱苄基反应，即可得到所述的缀合物10；所述有机溶剂为二氯甲烷、甲醇与水的混合溶液，所述催化剂为氢气、钯碳与氢氧化钯的混合物。As a preferred embodiment of the preparation method of the present invention, in step (7), the compound 9 described in step (6) is dissolved in an organic solvent, a catalyst is added, and the debenzylation reaction is carried out to obtain the Conjugate 10; the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of hydrogen, palladium on carbon and palladium hydroxide.
另外，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。In addition, 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.
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、子宫癌、卵巢癌、肺癌、肝癌、前列腺癌、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。As a preferred embodiment of the application of the present invention, the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, bowel cancer, renal cell carcinoma, cellular lymphoma , Thyroid cancer, brain cancer, stomach cancer or leukemia.
与现有技术相比，本发明的有益效果为：Compared with the prior art, the beneficial effects of the present invention are:
(1)为了克服了糖蛋白疫苗偶联位点不确定、偶联率不稳定、组成成分复杂等缺点，本发明选择有发展潜力肿瘤相关的糖抗原(TACAs)中的Tn为研究靶点，选用TLR4激动剂单磷酸化的脂质A(MPLA)和NKT细胞激动剂α-半乳糖神经酰胺类似物(KRN7000)作为内源性佐剂，制备一种含有TLR4激动剂MPLA和NKT细胞激动剂KRN7000以及糖抗原Tn三组分的缀合物(MPLA-Tn-KRN7000)，该缀合物作为疫苗，通过同时激活TLR4受体和NKT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，从而达到特异性杀死肿瘤细胞的目的。(1) In order to overcome the shortcomings of uncertain coupling sites, unstable coupling rates, and complex composition of glycoprotein vaccines, the present invention selects Tn in tumor-associated carbohydrate antigens (TACAs) with development potential as the research target. TLR4 agonist monophosphorylated lipid A (MPLA) and NKT cell agonist α-galactoseramide analog (KRN7000) were selected as endogenous adjuvants to prepare a TLR4 agonist MPLA and NKT cell agonist A three-component conjugate of KRN7000 and carbohydrate antigen Tn (MPLA-Tn-KRN7000). This conjugate is used as a vaccine to activate TLR4 receptors and NKT cells at the same time to cause a stronger immune response against carbohydrate antigen Tn. T cells with higher titer, high affinity and memory regulate the immune response, so as to achieve the purpose of killing tumor cells specifically.
(2)本发明提供的所述缀合物的制备方法，其合成路线简短、反应条件温和、产率高、操作方便，能够广泛地应用于工业化制备。(2) The preparation method of the conjugate provided by the present invention has a short synthetic route, mild reaction conditions, high yield and convenient operation, and can be widely used in industrial preparation.
附图说明Description of the drawings
图1为MPLA-Tn-KRN7000三组分糖疫苗免疫活性评价图；Figure 1 is an evaluation diagram of the immune activity of the MPLA-Tn-KRN7000 three-component sugar vaccine;
图2为MPLA-Tn-KRN7000三组分糖疫苗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；Figure 2 is a flow cytometric evaluation diagram of the antibody serum produced by the MPLA-Tn-KRN7000 three-component carbohydrate vaccine inducing mice to specifically recognize the tumor cell MCF-7;
图3为MPLA-Tn-KRN7000三组分糖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。Figure 3 is an evaluation diagram of complement-dependent cytotoxicity of the antibody serum produced by the MPLA-Tn-KRN7000 three-component carbohydrate vaccine inducing mice to specifically kill the tumor cell MCF-7.
具体实施方式Detailed ways
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。In order to better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
实施例1Example 1
本实施例为本发明提供的一种缀合物，所述缀合物为结构式(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；This embodiment is a conjugate provided by the present invention. 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);
Figure PCTCN2020130227-appb-000018
Figure PCTCN2020130227-appb-000018
上述缀合物的制备方法，包括如下步骤：The preparation method of the above-mentioned conjugate includes the following steps:
(1)取化合物1溶解于有机溶剂中，加入催化剂，得到化合物2；得到化合物2的反应式如下式所示：(1) Dissolve compound 1 in an organic solvent and add a catalyst to obtain compound 2. The reaction formula for obtaining compound 2 is shown in the following formula:
Figure PCTCN2020130227-appb-000019
Figure PCTCN2020130227-appb-000019
步骤(1)的具体操作为：二氯甲烷溶液(10.0mL)溶解化合物1(700.0mg，0.5mmol)与锌粉(658.0mg，10.1mmol)，加入乙酸(0.6mL，10.1mmol)，室温条件下搅拌12小时；硅藻土过滤，饱和碳酸氢钠水溶液洗3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；硅胶柱分离纯化得到无色油状液体化合物2(503.0mg，产率为73.2％)。 1H NMR(400MHz,CDCl 3)δ7.43–7.26(m,15H,Ar-H),6.06(d,J＝7.7Hz,1H,-NHCO-),4.96(d,J＝11.4Hz,1H,Ar-CH 2-O-),4.86(d,J＝3.7Hz,1H,H-1),4.82–4.62(m,5H,Ar-CH 2-O-),4.18(m,J＝8.4,4.2Hz,1H),4.05(m,J＝10.6,9.8,4.0Hz,2H),3.91–3.78(m,4H),3.65(t,J＝6.2Hz,2H),2.93(m,1H,H-6),2.75(m,1H,H-6),2.04(t,J＝7.7Hz,2H,-CH 2-CONH-),1.63–1.44(m,4H,-CH 2-),1.25(d,J＝3.9Hz,68H,-CH 2-),0.89(m, J＝7.9,4.2Hz,24H,-CH 3),0.07(d,J＝5.0Hz,12H,Si-CH 3). 13C NMR(100MHz,CDCl 3)δ173.22,138.57,138.39,138.20,128.56,128.43,128.40,128.02,127.96,127.86,127.78,127.61,127.45,127.38,100.41(C-1),79.38,76.75,76.41,76.20,76.07,74.97,74.55,73.63,73.23,71.34,69.74,51.85,42.02,36.95,33.88,31.96,29.85,29.76,29.74,29.70,29.67,29.64,29.61,29.49,29.40,26.14,26.08,25.73,25.66,22.73,18.31,18.19,14.16,-3.59,-3.86,-4.58,-4.89.ESI-TOF HRMS m/z:calcdfor C 83H 146N 2O 8Si 2,[M+H] +:1356.069,found:1356.0687. The specific operation of step (1) is: dissolve compound 1 (700.0mg, 0.5mmol) and zinc powder (658.0mg, 10.1mmol) in dichloromethane solution (10.0mL), add acetic acid (0.6mL, 10.1mmol), room temperature conditions Stir under the pressure for 12 hours; filter with diatomaceous earth, wash with saturated sodium bicarbonate aqueous solution for 3 times, collect the organic layer, dry with anhydrous sodium sulfate, filter, and distill the filtrate under reduced pressure to remove the organic solvent to obtain the crude product; separate and purify by silica gel column to obtain a colorless oil Liquid compound 2 (503.0 mg, yield 73.2%). 1 H NMR(400MHz,CDCl 3 )δ7.43-7.26(m,15H,Ar-H), 6.06(d,J=7.7Hz,1H,-NHCO-), 4.96(d,J=11.4Hz,1H ,Ar-CH 2 -O-), 4.86(d,J=3.7Hz,1H,H-1), 4.82–4.62(m,5H,Ar-CH 2 -O-), 4.18(m,J=8.4 ,4.2Hz,1H),4.05(m,J=10.6,9.8,4.0Hz,2H),3.91-3.78(m,4H),3.65(t,J=6.2Hz,2H),2.93(m,1H, H-6), 2.75(m,1H,H-6),2.04(t,J=7.7Hz,2H,-CH 2 -CONH-),1.63-1.44(m,4H,-CH 2 -),1.25 (d,J=3.9Hz,68H,-CH 2 -),0.89(m, J=7.9,4.2Hz,24H,-CH 3 ),0.07(d,J=5.0Hz,12H,Si-CH 3 ) . 13 C NMR (100MHz, CDCl 3 ) δ173.22,138.57,138.39,138.20,128.56,128.43,128.40,128.02,127.96,127.86,127.78,127.61,127.45,127.38,100.41(C-1),79.38,76.75,76.41 ,76.20,76.07,74.97,74.55,73.63,73.23,71.34,69.74,51.85,42.02,36.95,33.88,31.96,29.85,29.76,29.74,29.70,29.67,29.64,29.61,29.49,29.40,26.14,26.08, ,25.66,22.73,18.31,18.19,14.16,-3.59,-3.86,-4.58,-4.89.ESI-TOF HRMS m/z:calcdfor C 83 H 146 N 2 O 8 Si 2 ,[M+H] + : 1356.069,found:1356.0687.
(2)取步骤(1)所述的化合物2溶解于有机溶剂中，在催化剂的作用下，与Linker链接，得到化合物3；得到化合物3的反应式如下式所示：(2) The compound 2 described in step (1) is dissolved in an organic solvent, and linked with Linker under the action of a catalyst to obtain compound 3; the reaction formula for obtaining compound 3 is shown in the following formula:
Figure PCTCN2020130227-appb-000020
Figure PCTCN2020130227-appb-000020
步骤(2)的具体操作为：二氯甲烷溶液(6.0mL)溶解化合物2(300.0mg，0.2mmol)与己二酸酐(170.0mg，1.3mmol)，加入N,N-二异丙基乙胺(73.0μL，0.4mmol)调节pH＝8，室温条件下搅拌6小时；二氯甲烷稀释，用饱和碳酸氢钠水溶液洗2次，盐水洗1次，除去水层收集有机层，无水硫酸钠干燥后，过滤，滤液减压蒸馏除去有机溶剂，硅胶柱分离纯化得到无色油状液体化合物3(235.4mg，产率为71.7％)。 1H NMR(400MHz,CDCl 3)δ7.41–7.28(m,15H,Ar-H),6.14(d,J＝7.8Hz,2H,-NHCO-),4.94(d,J＝10.7Hz,1H),4.86–4.58(m,6H,J＝3.7Hz,H-1,Ar-CH 2-O-),4.22(m,1H),4.03(m,J＝10.1,3.6Hz,2H),3.88–3.76(m,4H,H-3,H-5),3.68(d,J＝10.8Hz,3H),3.14(s,1H,H-6),2.33(q,J＝5.5,4.7Hz,2H,-CH 2-COOH-),2.20–1.96(m,4H,-CH 2-CONH-),1.75–1.40(m,8H,-CH 2-),1.25(d,J＝4.8Hz,68H,-CH 2-),0.88(m,J＝9.9,7.5Hz,24H,-CH 3),0.12–0.01(m,12H,Si-CH 3). 13C NMR(100MHz,CDCl 3)δ176.94,173.81,172.80,138.58,138.44,138.34,128.77,128.39,128.36,127.97,127.77,127.75,127.57,127.42,99.70,79.38,76.17,75.91,74.95,74.77,73.61,73.14,69.33,68.53,51.94,39.77,36.93,35.86,33.71,33.52,31.94,29.84,29.74,29.71,29.67,29.62,29.60, 29.48,29.44,29.38,26.11,26.06,25.74,24.85,24.15,22.70,18.28,18.17,14.14,-3.55,-3.89,-4.61,-4.94.ESI-TOF HRMS m/z:calcdfor C 89H 154N 2O 11Si 2，[M+H] +:1484.1164，found 1484.1161. The specific operation of step (2) is: dissolve compound 2 (300.0 mg, 0.2 mmol) and adipic anhydride (170.0 mg, 1.3 mmol) in dichloromethane solution (6.0 mL), and add N,N-diisopropylethylamine (73.0μL, 0.4mmol) adjust pH=8, stir at room temperature for 6 hours; dilute with dichloromethane, wash twice with saturated aqueous sodium bicarbonate solution, wash once with brine, remove the water layer and collect the organic layer, anhydrous sodium sulfate After drying, it was filtered, the filtrate was distilled under reduced pressure to remove the organic solvent, and the silica gel column was separated and purified to obtain compound 3 (235.4 mg, yield 71.7%) as a colorless oily liquid. 1 H NMR(400MHz,CDCl 3 )δ7.41–7.28(m,15H,Ar-H), 6.14(d,J=7.8Hz,2H,-NHCO-), 4.94(d,J=10.7Hz,1H ), 4.86–4.58(m,6H,J=3.7Hz,H-1,Ar-CH 2 -O-),4.22(m,1H),4.03(m,J=10.1,3.6Hz,2H),3.88 –3.76(m,4H,H-3,H-5), 3.68(d,J=10.8Hz,3H), 3.14(s,1H,H-6), 2.33(q,J=5.5,4.7Hz, 2H,-CH 2 -COOH-),2.20–1.96(m,4H,-CH 2 -CONH-),1.75–1.40(m,8H,-CH 2 -),1.25(d,J=4.8Hz,68H ,-CH 2 -), 0.88 (m, J = 9.9, 7.5 Hz, 24H, -CH 3 ), 0.12-0.01 (m, 12H, Si-CH 3 ). 13 C NMR (100MHz, CDCl 3 )δ176. 94,173.81,172.80,138.58,138.44,138.34,128.77,128.39,128.36,127.97,127.77,127.75,127.57,127.42,99.70,79.38,76.17,75.91,74.95,74.77,73.61,73.14,69.33,68.53,51. 36.93, 35.86, 33.71, 33.52, 31.94, 29.84, 29.74, 29.71, 29.67, 29.62, 29.60, 29.48, 29.44, 29.38, 26.11, 26.06, 25.74, 24.85, 24.15, 22.70, 18.28, 18.17, 14.14, -3.55,- 3.89,-4.61,-4.94.ESI-TOF HRMS m/z:calcdfor C 89 H 154 N 2 O 11 Si 2 , [M+H] + :1484.1164, found 1484.1161.
(3)取化合物4与步骤(2)所述的化合物3溶解于有机溶剂中，加入缩合剂，进行酯化反应，得到化合物5；得到化合物5的反应式如下式所示：(3) Dissolve compound 4 and compound 3 described in step (2) in an organic solvent, add a condensing agent, and perform an esterification reaction to obtain compound 5; the reaction formula for obtaining compound 5 is shown in the following formula:
Figure PCTCN2020130227-appb-000021
Figure PCTCN2020130227-appb-000021
步骤(3)的具体操作为：二氯甲烷溶液(3.0mL)溶解化合物3(160.0mg，107.9μmol)、化合物4(74.0mg，129.5μmol)、1-(3-二甲基氨丙基)-3-乙基碳二亚胺盐酸盐(48.0mg，233.0μmol)与Hobt(14.0mg，103.7μmol)，室温下搅拌反应3个小时；二氯甲烷稀释，依次使用饱和碳酸氢钠水溶液洗2次，盐水洗1次，除去水层收集有机层，无水硫酸钠干燥，过滤，滤液减压蒸馏除去有机溶剂得粗品；硅胶柱分离纯化得到白色固体化合物5(172.0mg，产率：78.5％)。 1H NMR(400MHz,CDCl 3)δ7.40–7.28(m,15H,Ar-H),6.88(t,J＝5.4Hz,1H,-NHCO-),6.82(d,J＝8.7Hz,1H,-NHCO-),6.71(d,J＝9.4Hz,1H,-NHCO-),6.28(d,J＝6.8Hz,1H,-NHCO-),6.05(d,J＝7.7Hz,1H,-NHCO-),5.36(d,J＝3.1Hz,1H,Ar-CH 2-O-),5.10(m,J＝11.4,3.2Hz,1H,H-1),4.98–4.96(d,J＝8.2Hz,1H),4.96–4.94(d,J＝3.2Hz,1H,H’-1),4.87–4.63(m,5H,Ar-CH 2-O-),4.57(m,J＝12.9,8.5,4.3Hz,2H),4.30–4.15(m,5H),4.05(m,J＝14.6,9.0,4.6Hz,4H),3.89–3.76(m,4H),3.72–3.59(m,7H),3.52(m,3H),3.40(m,J＝13.0,6.3Hz,1H),3.23–3.11(m,1H),2.52(d,J＝2.4Hz,1H,-C≡CH),2.35(t,2H,-CO-CH 2-),2.16(s,3H,-CO-CH 3),2.13–2.03(m,4H,-CO-CH 2-),2.01(s,6H,-CO-CH 3),1.96(s,3H,-CO-CH 3),1.70–1.46(d,J＝6.2Hz,8H,-CH 2-),1.25(d,J＝4.7Hz,71H,-CH 2-,-CH 3),0.88(m,24H,-CH 3),0.05(d,J＝8.5Hz,12H,Si-CH 3). 13C NMR(100MHz,CDCl 3))δ173.37,173.32,173.04,170.90,170.80,170.41,170.08,138.52,138.45,138.26,128.63,128.55,128.40,128.38,127.99,127.79,127.59,127.34,100.23,100.06,79.43,79.37,77.47,76.12,75.94,75.89,75.48,75.15,74.75,73.64,73.19, 69.94,69.30,69.20,69.10,68.82,67.42,67.18,62.22,58.38,56.41,51.93,47.51,40.10,39.32,36.89,35.85,35.80,33.72,31.93,29.78,29.74,29.71,29.67,29.66,29.62,29.49,29.46,29.37,26.11,26.06,26.01,25.73,25.09,24.78,23.04,22.69,20.82,20.78,20.66,18.29,18.17,18.06,14.14,-3.60,-3.90,-4.60,-4.90.ESI-TOF HRMS m/z:calcdfor C 114H 191N 5O 22Si 2,[M+H] +:2039.3592,found:2039.3583. The specific operation of step (3) is: dissolving compound 3 (160.0 mg, 107.9 μmol), compound 4 (74.0 mg, 129.5 μmol), 1-(3-dimethylaminopropyl) in dichloromethane solution (3.0 mL) -3-Ethylcarbodiimide hydrochloride (48.0mg, 233.0μmol) and Hobt (14.0mg, 103.7μmol), stirred at room temperature for 3 hours; diluted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution in turn 2 times, washed with brine once, the water layer was removed and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to remove the organic solvent to obtain a crude product; silica gel column separation and purification to obtain a white solid compound 5 (172.0mg, yield: 78.5 %). 1 H NMR(400MHz,CDCl 3 )δ7.40–7.28(m,15H,Ar-H), 6.88(t,J=5.4Hz,1H,-NHCO-), 6.82(d,J=8.7Hz,1H ,-NHCO-), 6.71(d,J=9.4Hz,1H,-NHCO-), 6.28(d,J=6.8Hz,1H,-NHCO-),6.05(d,J=7.7Hz,1H,- NHCO-), 5.36 (d, J = 3.1 Hz, 1H, Ar-CH 2 -O-), 5.10 (m, J = 11.4, 3.2 Hz, 1H, H-1), 4.98-4.96 (d, J = 8.2Hz,1H), 4.96–4.94(d,J=3.2Hz,1H,H'-1), 4.87–4.63(m,5H,Ar-CH 2 -O-), 4.57(m,J=12.9, 8.5, 4.3 Hz, 2H), 4.30–4.15 (m, 5H), 4.05 (m, J = 14.6, 9.0, 4.6 Hz, 4H), 3.89–3.76 (m, 4H), 3.72–3.59 (m, 7H) ,3.52(m,3H),3.40(m,J=13.0,6.3Hz,1H),3.23-3.11(m,1H),2.52(d,J=2.4Hz,1H,-C≡CH),2.35( t,2H,-CO-CH 2 -),2.16(s,3H,-CO-CH 3 ),2.13-2.03(m,4H,-CO-CH 2 -),2.01(s,6H,-CO- CH 3 ),1.96(s,3H,-CO-CH 3 ),1.70–1.46(d,J=6.2Hz,8H,-CH 2 -),1.25(d,J=4.7Hz,71H,-CH 2 -,-CH 3 ), 0.88 (m, 24H, -CH 3 ), 0.05 (d, J = 8.5 Hz, 12H, Si-CH 3 ). 13 C NMR (100MHz, CDCl 3 )) δ173.37,173.32,173.04 ,170.90,170.80,170.41,170.08,138.52,138.45,138.26,128.63,128.55,128.40,128.38,127.99,127.79,127.59,127.34,100.23,100.06,79.43,79.37,77.47,76.12,75.94,75,75.15,75.48 ,74.75,73.64,73.19, 69.94,69.30,69.20,69.10,68.82,67.42,67.18,6 2.22,58.38,56.41,51.93,47.51,40.10,39.32,36.89,35.85,35.80,33.72,31.93,29.78,29.74,29.71,29.67,29.66,29.62,29.49,29.46,29.37,26.11,26.06,26.01,25.73 25.09,24.78,23.04,22.69,20.82,20.78,20.66,18.29,18.17,18.06,14.14,-3.60,-3.90,-4.60,-4.90.ESI-TOF HRMS m/z:calcdfor C 114 H 191 N 5 O 22 Si 2 ,[M+H] + :2039.3592,found:2039.3583.
(4)取步骤(3)所述的化合物5溶解于有机溶剂中，在催化剂的作用下，脱去保护基团三甲基硅烷，得到化合物6；得到化合物6的反应式如下式所示：(4) The compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst to obtain compound 6; the reaction formula for obtaining compound 6 is shown in the following formula:
Figure PCTCN2020130227-appb-000022
Figure PCTCN2020130227-appb-000022
步骤(4)的具体操作为：乙腈/二氯甲烷(1.5：1，2.5mL)溶解化合物5(160.0mg，69.3μmol)，加入三氟化硼乙醚络合物(19.0μL，152.5μmol)，室温下搅拌反应2小时；减压蒸馏除去有机溶剂得粗品，硅胶柱分离纯化得到白色固体化合物6(107.0mg，产率为75.3％)。 1H NMR(400MHz,CDCl 3)δ7.43–7.28(m,15H,Ar-H),7.22(d,J＝5.7Hz,1H,-NHCO-),7.09(d,J＝8.8Hz,1H,-NHCO-),6.69(m,J＝9.4,2.7Hz,1H,-NHCO-),6.57(d,J＝8.5Hz,1H,-NHCO-),5.86(s,1H,-NHCO-),5.35(d,J＝3.2Hz,1H),5.08(m,J＝11.3,3.2Hz,1H),4.95(d,J＝7.3Hz,1H,Ar-CH 2-O-),4.94(s,1H,H-1),4.86(d,J＝3.0Hz,1H,H-1),4.84–4.59(m,5H,Ar-CH 2-O-),4.53(m,J＝11.7,9.3,3.3Hz,2H),4.31–4.20(m,3H),4.19(d,J＝2.4Hz,2H),4.14–3.99(m,3H),3.89–3.71(m,5H),3.71–3.58(m,4H),3.58–3.44(m,5H),3.36(m,J＝19.3,14.3,5.0Hz,2H),3.17(m,J＝13.2,8.3,4.7Hz,1H),2.52(t,J＝2.4Hz,1H,-C≡CH),2.32(m,J＝18.5,6.8Hz,2H,-CO-CH 2-),2.21–2.05(m,7H,2x-CO-CH 2-,-CO-CH 3),2.04–1.94(m,9H,3x-CO-CH 3),1.72–1.49(m,8H,4x-CH 2-),1.25(s,71H,34x-CH 2-,-CH 3)0.88(t,J＝6.7Hz,6H,2x-CH 3). 13C NMR(100MHz,CDCl 3)δ173.86,173.39,173.33,170.95,170.82,170.54,170.51,170.36,138.24,138.10,137.77,128.73,128.69,128.53,128.49,128.45,128.06,127.98,127.93,127.74,127.49,100.04,98.75,79.35,77.27,76.17,75.77,75.17,74.54,74.48,73.92,73.15,72.92,69.87,69.31,69.03,68.90,67.48, 67.08,62.28,58.35,56.85,49.50,47.48,40.28,39.27,36.77,35.84,35.63,33.50,31.92,29.84,29.80,29.75,29.73,29.70,29.65,29.62,29.46,29.37,25.87,25.81,25.01,24.87,23.05,22.69,20.82,20.77,20.69,18.27,14.13,-0.00.ESI-TOF HRMS m/z:calcdfor C 102H 163N 5O 22,[M+H] +:1811.1862,found:1811.1831. The specific operation of step (4) is: dissolve compound 5 (160.0mg, 69.3μmol) in acetonitrile/dichloromethane (1.5:1, 2.5mL), add boron trifluoride ether complex (19.0μL, 152.5μmol), The reaction was stirred at room temperature for 2 hours; the organic solvent was distilled off under reduced pressure to obtain a crude product, and the silica gel column was separated and purified to obtain compound 6 (107.0 mg, yield 75.3%) as a white solid. 1 H NMR(400MHz,CDCl 3 )δ7.43-7.28(m,15H,Ar-H), 7.22(d,J=5.7Hz,1H,-NHCO-), 7.09(d,J=8.8Hz,1H ,-NHCO-), 6.69(m,J=9.4,2.7Hz,1H,-NHCO-),6.57(d,J=8.5Hz,1H,-NHCO-),5.86(s,1H,-NHCO-) ,5.35(d,J=3.2Hz,1H),5.08(m,J=11.3,3.2Hz,1H), 4.95(d,J=7.3Hz,1H,Ar-CH 2 -O-), 4.94(s ,1H,H-1), 4.86(d,J=3.0Hz,1H,H-1),4.84-4.59(m,5H,Ar-CH 2 -O-),4.53(m,J=11.7,9.3 ,3.3Hz,2H),4.31–4.20(m,3H), 4.19(d,J=2.4Hz,2H), 4.14–3.99(m,3H), 3.89–3.71(m,5H), 3.71–3.58( m, 4H), 3.58–3.44 (m, 5H), 3.36 (m, J = 19.3, 14.3, 5.0 Hz, 2H), 3.17 (m, J = 13.2, 8.3, 4.7 Hz, 1H), 2.52 (t, J=2.4Hz,1H,-C≡CH), 2.32(m,J=18.5,6.8Hz,2H,-CO-CH 2 -),2.21–2.05(m,7H,2x-CO-CH 2 -, -CO-CH 3 ),2.04-1.94(m,9H,3x-CO-CH 3 ),1.72-1.49(m,8H,4x-CH 2 -),1.25(s,71H,34x-CH 2 -, -CH 3 ) 0.88 (t, J = 6.7 Hz, 6H, 2x-CH 3 ). 13 C NMR (100MHz, CDCl 3 ) δ173.86,173.39,173.33,170.95,170.82,170.54,170.51,170.36,138.24,138.10, 137.77,128.73,128.69,128.53,128.49,128.45,128.06,127.98,127.93,127.74,127.49,100.04,98.75,79.35,77.27,76.17,75.77,75.17,74.54,74.48,73.92,73.15,72.69,69.87, 69.03,68.90,67.48, 67.08,62.28,58.35 ,56.85,49.50,47.48,40.28,39.27,36.77,35.84,35.63,33.50,31.92,29.84,29.80,29.75,29.73,29.70,29.65,29.62,29.46,29.37,25.87,25.81,25.01,24.87,23.05,22.69 ,20.82,20.77,20.69,18.27,14.13,-0.00.ESI-TOF HRMS m/z:calcdfor C 102 H 163 N 5 O 22 ,[M+H] + :1811.1862,found:1811.1831.
(5)取步骤(4)所述的化合物6溶解于有机溶剂中，加入催化剂，进行脱乙酰化反应，得到化合物7；得到化合物7的反应式如下式所示：(5) The compound 6 described in step (4) is dissolved in an organic solvent, and a catalyst is added to perform a deacetylation reaction to obtain compound 7; the reaction formula for obtaining compound 7 is shown in the following formula:
Figure PCTCN2020130227-appb-000023
Figure PCTCN2020130227-appb-000023
步骤(5)的具体操作为：制备甲醇钠溶液：称取金属钠(5.4mg)，加入到的甲醇溶液(10.0mL)中，冷却至室温待用；甲醇/二氯甲烷(2：1，3.0mL)溶解化合物6(105.0mg，58.0μmol)，室温下加入已制备好的甲醇钠溶液(0.1mL)调节pH＝8，搅拌反应3个小时；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7(89.0mg，产率为91.1％)。 1H NMR(400MHz,MeOD/CDCl 3(1:30,v/v,0.6mL))δ7.42–7.29(m,15H,Ar-H),4.96(d,J＝11.3Hz,1H,Ar-CH 2-O-),4.90(d,J＝3.6Hz,1H,H-1),4.86(d,J＝3.8Hz,1H,H-1),4.84–4.58(m,5H,Ar-CH 2-O-),4.53(d,J＝2.3Hz,1H),4.22(m,J＝5.3,2.7Hz,4H),4.15(m,J＝6.5,2.3Hz,1H),4.06–4.00(m,2H),3.94–3.85(m,4H),3.78–3.78–3.72(m,4H),3.69(t,J＝4.3Hz,3H),3.63(m,J＝6.4,3.1Hz,2H),3.55–3.45(m,5H),3.43–3.32(m,2H),3.19(m,J＝13.9,8.3Hz,1H),2.58(q,J＝2.3Hz,1H,-C≡CH),2.39–2.28(m,2H,-CO-CH 2-),2.17(t,J＝7.7Hz,2H,-CO-CH 2-),2.10(d,J＝5.6Hz,5H,-CO-CH 2-,-CO-CH 3),1.72–1.51(m,8H,4x-CH 2-),1.26(d,J＝2.3Hz,71H,34x-CH 2-,-CH 3)0.88(t,J＝6.7Hz,6H,2x-CH 3). 13C NMR(100MHz,MeOD/CDCl 3(1:30,v/v,0.6mL))δ174.53,174.45,174.30,171.15,171.07,138.43,138.26,137.76,128.64,128.60,128.34,128.19,128.13,127.90,127.65,99.72,98.28,79.39,79.29,77.26,76.07,75.58,75.52,75.36,74.90,74.06,73.28,72.66,71.01,70.23,70.07,69.54,69.48,69.24,69.17,67.40,58.56,50.72,49.95,40.52,39.45,36.69,35.78,35.68,33.47,32.07, 30.00,29.96,29.89,29.87,29.84,29.81,29.80,29.76,29.61,29.57,29.51,26.08,26.04,25.34,25.24,22.83,22.70,18.49,14.18.ESI-TOF HRMS m/z:calcdfor C 96H 157N 5O 19,[M+H] +:1685.1546,found:1685.1545. The specific operation of step (5) is: prepare sodium methoxide solution: weigh sodium metal (5.4mg), add to methanol solution (10.0mL), cool to room temperature for later use; methanol/dichloromethane (2:1, 3.0mL) dissolve compound 6 (105.0mg, 58.0μmol), add the prepared sodium methoxide solution (0.1mL) at room temperature to adjust the pH=8, stir the reaction for 3 hours; add an appropriate amount of ion exchange resin to adjust the pH to 7 to stop Reaction; Celite filter, collect the filtrate, remove the organic solvent to obtain white solid compound 7 (89.0mg, yield 91.1%). 1 H NMR (400MHz, MeOD/CDCl 3 (1:30, v/v, 0.6mL)) δ7.42-7.29 (m, 15H, Ar-H), 4.96 (d, J = 11.3Hz, 1H, Ar -CH 2 -O-), 4.90(d,J=3.6Hz,1H,H-1), 4.86(d,J=3.8Hz,1H,H-1), 4.84–4.58(m,5H,Ar- CH 2 -O-), 4.53 (d, J = 2.3 Hz, 1H), 4.22 (m, J = 5.3, 2.7 Hz, 4H), 4.15 (m, J = 6.5, 2.3 Hz, 1H), 4.06–4.00 (m,2H),3.94–3.85(m,4H),3.78–3.78–3.72(m,4H), 3.69(t,J=4.3Hz,3H),3.63(m,J=6.4,3.1Hz,2H ),3.55–3.45(m,5H),3.43–3.32(m,2H),3.19(m,J=13.9,8.3Hz,1H),2.58(q,J=2.3Hz,1H,-C≡CH) ,2.39–2.28(m,2H,-CO-CH 2 -), 2.17(t,J=7.7Hz,2H,-CO-CH 2 -), 2.10(d,J=5.6Hz,5H,-CO- CH 2 -,-CO-CH 3 ),1.72–1.51(m,8H,4x-CH 2 -), 1.26(d,J=2.3Hz,71H,34x-CH 2 -,-CH 3 )0.88(t , J=6.7Hz, 6H, 2x-CH 3 ). 13 C NMR (100MHz, MeOD/CDCl 3 (1:30, v/v, 0.6mL)) δ174.53,174.45,174.30,171.15,171.07,138.43,138.26 ,137.76,128.64,128.60,128.34,128.19,128.13,127.90,127.65,99.72,98.28,79.39,79.29,77.26,76.07,75.58,75.52,75.36,74.90,74.06,73.28,72.66,71.01,70.23,70. ,69.48,69.24,69.17,67.40,58.56,50.72,49.95,40.52,39.45,36.69,35.78,35.68,33.47,32.07,30.00,29.96,29.89,29.87,29.84,29.81,29.80,29.76,29.61,29.57 ,29.51,26.08,26.04,25.34,25.24,22.83,22.70,18.49,14.18.ESI-TOF HRMS m/z:calcdfor C 96 H 157 N 5 O 19 ,[M+H] + :1685.1546,found:1685.1545.
(6)取化合物与步骤(5)所述的化合物7溶解于有机溶剂中，加入催化剂反应，得到化合物9；得到化合物9的反应式如下式所示：(6) The compound and the compound 7 described in step (5) are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 9; the reaction formula for obtaining compound 9 is shown in the following formula:
Figure PCTCN2020130227-appb-000024
Figure PCTCN2020130227-appb-000024
步骤(6)的具体操作为：四氢呋喃与甲醇(1：2，3.0mL)溶解化合物8(52.2mg，23.2μmol)、化合物7(20.0mg，11.9μmol)、碘化亚铜(113.0mg，595.0μmol)，加入N,N-二异丙基乙胺(97.0μL，595.0μmol)，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9(14.0mg，产率为30.4％)。 1H NMR(400MHz,CDCl 3)δ7.93(s,1H),7.44–7.16(m,40H,Ar-H),6.73(s,1H),6.60(s,1H),5.42(t,J＝9.5Hz,1H),5.12(m,J＝25.6,13.8,8.5Hz,3H),5.03–3.97(m,32H),3.97–3.27(m,30H),3.27–3.00(m,2H),2.60–1.94(m,21H,9x-CO-CH 2-,-CO-CH 3),1.75–1.45(m,20H,10x-CH 2-),1.25(s,185H,91x-CH 2-,-CH 3-),0.88(t,J＝6.8Hz,24H,8x-CH 3).ESI-TOF HRMS m/z:calcdfor C 229H 371N 10O 40P,[M+NH4 ++2K +] 2+:1342.8881,found:1342.9264.MOLDI-TOF HRMS m/z:calcdfor C 229H 371N 10O 40P,[M+Na] +:found:3956.819. The specific operation of step (6) is: dissolving compound 8 (52.2 mg, 23.2 μmol), compound 7 (20.0 mg, 11.9 μmol), cuprous iodide (113.0 mg, 595.0) in tetrahydrofuran and methanol (1: 2, 3.0 mL) μmol), add N,N-diisopropylethylamine (97.0μL, 595.0μmol), stir at room temperature for 12 hours; filter out the insoluble matter with Celite, and distill the filtrate under reduced pressure to remove the solvent to obtain the crude product; silica gel column separation and purification A white solid compound 9 (14.0 mg, yield 30.4%) was obtained. 1 H NMR(400MHz,CDCl 3 )δ7.93(s,1H),7.44-7.16(m,40H,Ar-H),6.73(s,1H),6.60(s,1H),5.42(t,J =9.5Hz,1H), 5.12(m,J=25.6,13.8,8.5Hz,3H), 5.03–3.97(m,32H), 3.97–3.27(m,30H), 3.27–3.00(m,2H), 2.60–1.94(m,21H,9x-CO-CH 2 -,-CO-CH 3 ), 1.75–1.45(m,20H,10x-CH 2 -),1.25(s,185H,91x-CH 2 -, -CH 3 -),0.88(t,J=6.8Hz,24H,8x-CH 3 ).ESI-TOF HRMS m/z:calcdfor C 229 H 371 N 10 O 40 P,[M+NH4 + +2K + ] 2+ :1342.8881,found:1342.9264.MOLDI-TOF HRMS m/z:calcdfor C 229 H 371 N 10 O 40 P,[M+Na] + :found:3956.819.
(7)取步骤(6)所述的化合物9溶解于有机溶剂中，加入催化剂，进行脱苄基反应，即可得到所述的缀合物；得到缀合物(Ⅴ)的反应式如下式所示：(7) The compound 9 described in step (6) is dissolved in an organic solvent, and a catalyst is added to carry out the debenzylation reaction to obtain the conjugate; the reaction formula for obtaining the conjugate (Ⅴ) is as follows Shown:
Figure PCTCN2020130227-appb-000025
Figure PCTCN2020130227-appb-000025
步骤(6)的具体操作为：二氯甲烷/甲醇/水(5：5：1，10.0mL)溶解化合物9(8.0mg，2.0μmol)，加入氢氧化钯(5.0mg)与钯碳(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39，目标产物MPLA-Tn-KRN7000(5.6mg，85.8％)。 1H NMR(400MHz,MeOD/CDCl 3/D 2O(20:20:1,v/v/v,0.6mL))δ5.44(s,1H),3.51(s,32H),3.26–2.51(m,36H),2.41–1.81(m,21H-CO-CH 2-,-CO-CH 3),1.61(s,20H,-CH 2-),1.53–1.01(m,179H,-CH 2-),0.90(m,J＝5.8Hz,24H,-CH 3).ESI-TOF HRMS m/z:calcdfor C 173H 323N 10O 40P,[M+2K +] 2+:1645.1274,found:1645.1233. The specific operation of step (6) is: dissolve compound 9 (8.0 mg, 2.0 μmol) in dichloromethane/methanol/water (5:5:1, 10.0mL), add palladium hydroxide (5.0mg) and palladium on carbon (5.0 mg), hydrogen gas was introduced, sealed and stirred 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 39, the target product MPLA-Tn-KRN7000 (5.6 mg, 85.8%). 1 H NMR(400MHz, MeOD/CDCl 3 /D 2 O(20:20:1, v/v/v, 0.6mL)) δ5.44(s,1H), 3.51(s,32H), 3.26-2.51 (m,36H),2.41-1.81(m,21H-CO-CH 2 -,-CO-CH 3 ),1.61(s,20H,-CH 2 -),1.53-1.01(m,179H,-CH 2 -),0.90(m,J=5.8Hz,24H,-CH 3 ).ESI-TOF HRMS m/z:calcdfor C 173 H 323 N 10 O 40 P,[M+2K + ] 2+ :1645.1274,found :1645.1233.
实验例1Experimental example 1
本实验例对实施例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。In this experimental example, mice were immunized with the conjugate (fully synthetic sugar vaccine) prepared in Example 1, and its immune effect was preliminarily evaluated by ELSA experiment. The fluorescence activated cell sorting (FACS) technology proved that antibody serum can specifically recognize tumors. 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.
1.ELISA免疫分析1. ELISA immunoassay
1)小鼠免疫：1) Mouse immunity:
取6-8周龄的C57BL/6小鼠，分成免疫组以及对照组(PBS)，每组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。C57BL/6 mice aged 6-8 weeks were divided into an immunized group and a control group (PBS), with 6 mice in each group. 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.
2)ELISA免疫分析：2) ELISA immunoassay:
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。同组6只小鼠血清样品分别用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。 Dissolve Tn-BSA in 0.1M carbonate buffer (pH 9.6), prepare a 2.0μg/mL solution, add 100.0μL 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 μL/well/time) with PBST (PBS+0.05% Tween-20). After washing the plate, add PBS/1% BSA; add 250.0 μl to each well; incubate at room temperature for one hour, and wash the plate 3 times with PBST. The serum samples of 6 mice in the same group were diluted 300, 900, 2700, 8100, 24300, 72900, 218700 and 656 100 times with PBS; the diluted serum was added to a 96-well plate at 100.0μL per well, and each dilution gradient was done in parallel Three secondary wells; placed in a 37°C incubator and incubated for two hours, and the plate was washed 3 times. Add 100.0 μL of HRP (horseradish peroxidase) labeled IgG (diluted 2000 times) to each well, incubate at room temperature for one hour; wash the plate 3 times. Add TMB solution, add 100.0 μL to each well, and develop color in the dark at room temperature for 20 minutes. Add 0.5M H 2 SO 4 solution, add 100.0 μL to each well. Immediately detect the absorbance with a microplate reader, the detection wavelength is 450nm, and 570nm is used as the background wavelength.
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。3) Plot the absorbance (OD) value against the antiserum dilution value and obtain the best fit line. Use the equation of this line to calculate the dilution value when the OD value reaches 0.2, and calculate the antibody titer based on the reciprocal of the dilution value as shown in Figure 1.
4)实验结果：4) Experimental results:
从图1可看出，本发明实施例1合成的MPLA-Tn-KRN7000三组份糖疫苗，在无外部佐剂的情况下，在小鼠体内诱导产生高滴度的Kappa与IgG抗体，说明MPLA-Tn-KRN7000三组分疫苗的双重佐剂的设计能有效提高Tn糖抗原的免疫原性。It can be seen from Figure 1 that the MPLA-Tn-KRN7000 three-component sugar vaccine synthesized in Example 1 of the present invention induces the production of high-titer Kappa and IgG antibodies in mice without external adjuvants. The dual adjuvant design of the MPLA-Tn-KRN7000 three-component vaccine can effectively improve the immunogenicity of the Tn carbohydrate antigen.
2.流式细胞实验(FACS)2. Flow Cytometry (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MDA231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。 Experimental method: Breast cancer cells MCF-7 overexpressing Tn sugar antigen and tumor cells MDA231 not expressing Tn sugar antigen were cultured in MEM medium containing 10% fetal bovine serum (FBS) (37℃, 5% CO). 2 ); Trypsin digestion, collect cells, count the number of cells under the microscope, aliquot 2.0×10 5 cells into each test tube, add 1 mL of PBS buffer (FACS buffer) containing 3% FBS, resuspend, and centrifuge 2 After 10 minutes, remove the supernatant and wash twice with FACS buffer; add the prepared mouse serum, incubate on ice for 1 hour, wash twice with FACS buffer, add fluorescently labeled secondary antibody, and protect from light on ice Incubate for one hour, wash twice with FACS buffer, resuspend in 0.8mL FACS buffer, and detect with flow cytometer.
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MCF-7细胞中，与免疫前血清相比，MPLA-Tn-KRN7000三组分糖疫苗诱导小鼠产生的抗体血清的荧光峰明显向右偏移。在MDA-231细胞中，免疫前血清与抗体血清无明显差别。结果表明，实施例1制备的MPLA-Tn-KRN7000糖抗原疫苗诱导的抗体能特异性识别表达Tn抗原的MCF-7细胞。Experimental results: As shown in Figure 2, MCF-7 is a breast cancer cell that overexpresses Tn antigen, and MDA-231 tumor cells that do not express Tn antigen are used as a negative control. In MCF-7 cells, compared with pre-immune serum, the fluorescence peak of antibody serum produced by MPLA-Tn-KRN7000 three-component carbohydrate vaccine induced a significant shift to the right. In MDA-231 cells, there is no significant difference between pre-immune serum and antibody serum. The results show that the antibodies induced by the MPLA-Tn-KRN7000 carbohydrate antigen vaccine prepared in Example 1 can specifically recognize MCF-7 cells expressing Tn antigen.
3.抗体介导的互补依赖细胞毒性(CDC)3. Antibody-mediated complementation dependent cytotoxicity (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。 Experimental method: Breast cancer cells MCF-7 overexpressing Tn sugar antigen and tumor cells MDA-231 not expressing Tn sugar antigen were cultured in DMEM medium containing 10% fetal bovine serum (FBS); The cells in the early stage are configured into a cell suspension with a density of 1.0×10 5 cell/mL, inoculated into a 96-well plate, 100 μL per well, about 10,000 cells, placed in an incubator and cultured overnight. The culture medium was removed, the serum-free MEM culture medium was washed three times, mouse serum diluted in MEM was added, and incubated at 37°C for 2 hours. Wash three times in serum-free MEM, add a certain proportion (1:10) of the diluted complement solution, and incubate at 37°C for 1 hour. Both low reference (serum-free medium only) and high reference (5% triton-100 treatment) groups were set up at the same time. After the incubation, the cells were centrifuged, 20 μL of the cell supernatant was diluted to 100 μL with PBS, and the color was developed with 100 μL of LDH cytotoxicity detection reagent for 30 minutes. Measure the absorbance of each well at 490nm, and calculate the cell lysis rate based on the low reference and high reference wells.
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。MPLA-Tn-KRN7000三组分糖抗原疫苗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。而在相同条件下，抗体介导的免疫前血清和抗体血清对MDA-231细胞的细胞毒性差异无统计学意义。结果证实实施例1制备的MPLA-Tn-KRN7000三组分糖抗原疫苗具有一定的特异性抗癌作用。Experimental results: As shown in Figure 3, MCF-7 is a breast cancer cell that overexpresses Tn antigen, and MDA-231 tumor cells that do not express Tn antigen are used as a negative control. The MPLA-Tn-KRN7000 three-component carbohydrate antigen vaccine produced antiserum-mediated MCF-7 cell lysis rate in mice that was significantly higher than that of blank serum. Under the same conditions, there was no statistically significant difference in the cytotoxicity of antibody-mediated pre-immune serum and antibody serum to MDA-231 cells. The results confirmed that the MPLA-Tn-KRN7000 three-component carbohydrate antigen vaccine prepared in Example 1 has a certain specific anti-cancer effect.
综上，实验结果表明，本发明制备的一种含有TLR4激动剂MPLA和NKT细胞激动剂KRN7000以及糖抗原Tn三组分的缀合物(MPLA-Tn-KRN7000)，该缀合物作为糖抗原疫苗，通过同时激活TLR4受体和NKT细胞，比单一激活免疫系统产生更强的免疫应答，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，从而达到特异性杀死肿瘤细胞的目的。In summary, the experimental results show that a three-component conjugate (MPLA-Tn-KRN7000) containing the TLR4 agonist MPLA and the NKT cell agonist KRN7000 and the carbohydrate antigen Tn prepared by the present invention is used as a carbohydrate antigen The vaccine, by activating TLR4 receptors and NKT cells at the same time, produces a stronger immune response than a single activation of the immune system, causing a stronger immune response against the sugar antigen Tn, and producing T with higher titer, high affinity and memory. Cells regulate the immune response, so as to achieve the purpose of killing tumor cells specifically.
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。Finally, it should be noted that the above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes made without departing from the spirit and principle of the present invention , Modification, substitution, combination, and simplification should all be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

  1. 一种缀合物，其特征在于，所述缀合物含有双重激动剂与糖抗原Tn，所述双重激动剂为TLR4受体激动剂和NKT细胞激动剂，所述TLR4受体激动剂为单磷酸化的脂质A，所述NKT细胞激动剂为α-半乳糖神经酰胺类似物；A conjugate, characterized in that the conjugate contains a dual agonist and a carbohydrate antigen Tn, the dual agonist is a TLR4 receptor agonist and an NKT cell agonist, and the TLR4 receptor agonist is a single Phosphorylated lipid A, the NKT cell agonist is an α-galactose ceramide analog;
    所述缀合物的结构通式如下式(A)、式(B)、式(C)或式(D)所示：The general structural formula of the conjugate is shown in the following formula (A), formula (B), formula (C) or formula (D):
    Figure PCTCN2020130227-appb-100001
    Figure PCTCN2020130227-appb-100001
    其中：in:
    MPLA为单磷酸化的脂质A；MPLA is a monophosphorylated lipid A;
    KRN7000为α-半乳糖神经酰胺类似物；KRN7000 is an analog of α-galactose ceramide;
    a为1-5的整数，b与c均为1-10的整数。a is an integer of 1-5, and both b and c are integers of 1-10.
  2. 如权利要求1所述的缀合物，其特征在于，所述的缀合物包括为通式(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；The conjugate according to claim 1, wherein the conjugate comprises a compound of general formula (I) or an isomer, pharmaceutically acceptable salt, or hydrate of a compound of general formula (I) Or solvent compound;
    Figure PCTCN2020130227-appb-100002
    Figure PCTCN2020130227-appb-100002
    其中：in:
    R 1和R 3为-(CH 2)mCH 3，m为10-14的整数； R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
    R 2、R 4和R 5为-(CH 2)pCH 3，p为8-12的整数； R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
    R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数； R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14;
    a为1-5的整数，b与c均为1-10的整数；a is an integer of 1-5, and both b and c are integers of 1-10;
    n为9-25的整数；n is an integer of 9-25;
    R为-CH 3或
    Figure PCTCN2020130227-appb-100003
    R is -CH 3 or
    Figure PCTCN2020130227-appb-100003
  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(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；The conjugate according to claim 2, wherein 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) ；
    Figure PCTCN2020130227-appb-100004
    Figure PCTCN2020130227-appb-100004
    其中：a为1-5的整数，b为1-10的整数。Wherein: a is an integer of 1-5, and b is an integer of 1-10.
  4. 如权利要求1-3任一所述的缀合物，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；The conjugate according to any one of claims 1 to 3, wherein the conjugate is a compound of structural formula (III) or an isomer of a compound of structural formula (III), a pharmaceutically acceptable salt, a hydrate Substance or solvent compound;
    Figure PCTCN2020130227-appb-100005
    Figure PCTCN2020130227-appb-100005
  5. 如权利要求1所述的缀合物，其特征在于，所述单磷酸化的脂质A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；The conjugate according to claim 1, wherein the monophosphorylated lipid A is a compound of general formula (IV) or an isomer of a compound of general formula (IV), a pharmaceutically acceptable salt, Hydrate or solvent compound;
    Figure PCTCN2020130227-appb-100006
    Figure PCTCN2020130227-appb-100006
    其中：in:
    R 5为-(CH 2)pCH 3，p为8-12的整数； R 5 is -(CH 2 )pCH 3 , and p is an integer of 8-12;
    R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数。 R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14.
  6. 如权利要求1所述的缀合物，其特征在于，所述α-半乳糖神经酰胺类似物为通式(Ⅴ)的化合物或通式(Ⅴ)的化合物的异构体、可药用盐、水合物或溶剂化合物；The conjugate according to claim 1, wherein the α-galactose ceramide analog is a compound of general formula (V) or an isomer or pharmaceutically acceptable salt of a compound of general formula (V) , Hydrate or solvent compound;
    Figure PCTCN2020130227-appb-100007
    Figure PCTCN2020130227-appb-100007
    其中：in:
    n为9-25的任意整数；n is any integer from 9-25;
    R为-H或
    Figure PCTCN2020130227-appb-100008
    R is -H or
    Figure PCTCN2020130227-appb-100008
  7. 如权利要求1-6任一所述的缀合物的制备方法，其特征在于，包括如下步骤：The preparation method of the conjugate according to any one of claims 1 to 6, characterized in that it comprises the following steps:
    (1)取化合物1溶解于有机溶剂中，加入催化剂，得到化合物2；(1) Dissolve compound 1 in an organic solvent and add a catalyst to obtain compound 2;
    (2)取步骤(1)所述的化合物2溶解于有机溶剂中，在催化剂的作用下，与Linker链接，得到化合物3；(2) Dissolve compound 2 described in step (1) in an organic solvent, and link with Linker under the action of a catalyst to obtain compound 3;
    (3)取化合物4与步骤(2)所述的化合物3溶解于有机溶剂中，加入缩合剂，进行酯化反应，得到化合物5；(3) Dissolve compound 4 and compound 3 described in step (2) in an organic solvent, add a condensing agent, and perform an esterification reaction to obtain compound 5;
    (4)取步骤(3)所述的化合物5溶解于有机溶剂中，在催化剂的作用下，脱去保护基团三甲基硅烷，得到化合物6；(4) The compound 5 described in step (3) is dissolved in an organic solvent, and the protective group trimethylsilane is removed under the action of a catalyst to obtain compound 6;
    (5)取步骤(4)所述的化合物6溶解于有机溶剂中，加入催化剂，进行脱乙酰化反应，得到化合物7；(5) Dissolve compound 6 described in step (4) in an organic solvent, add a catalyst, and perform a deacetylation reaction to obtain compound 7;
    (6)取化合物与步骤(5)所述的化合物7溶解于有机溶剂中，加入催化剂反应，得到化合物9；(6) Dissolve the compound with the compound 7 described in step (5) in an organic solvent, and add a catalyst to react to obtain compound 9;
    (7)取步骤(6)所述的化合物9溶解于有机溶剂中，加入催化剂，进行脱苄基反应，即可得到所述的缀合物；(7) Dissolve the compound 9 described in step (6) in an organic solvent, add a catalyst, and perform a debenzylation reaction to obtain the conjugate;
    所述化合物1至所述化合物9的结构式如下所示：The structural formulas of the compound 1 to the compound 9 are as follows:
    Figure PCTCN2020130227-appb-100009
    Figure PCTCN2020130227-appb-100009
    Figure PCTCN2020130227-appb-100010
    Figure PCTCN2020130227-appb-100010
    Figure PCTCN2020130227-appb-100011
    Figure PCTCN2020130227-appb-100011
    其中，in,
    R 1和R 3为-(CH 2)mCH 3，m为10-14的整数； R 1 and R 3 are -(CH 2 )mCH 3 , and m is an integer of 10-14;
    R 2、R 4和R 5为-(CH 2)pCH 3，p为8-12的整数； R 2 , R 4 and R 5 are -(CH 2 )pCH 3 , and p is an integer of 8-12;
    R 6为-CO(CH 2)rCH 3或-(CH 2)rCH 3，r为8-14的整数； R 6 is -CO(CH 2 )rCH 3 or -(CH 2 )rCH 3 , and r is an integer of 8-14;
    a为1-5的整数，b与c均为1-10的整数；a is an integer of 1-5, and both b and c are integers of 1-10;
    n为9-25的整数；n is an integer of 9-25;
    R为-CH 3或
    Figure PCTCN2020130227-appb-100012
    R is -CH 3 or
    Figure PCTCN2020130227-appb-100012
  8. 如权利要求7所述的制备方法，其特征在于，步骤(1)中，所述溶剂为二氯甲烷，所述催化剂为锌粉与乙酸的混合物；8. The preparation method according to claim 7, wherein in step (1), the solvent is dichloromethane, and the catalyst is a mixture of zinc powder and acetic acid;
    步骤(2)中，所述溶剂为二氯甲烷，所述催化剂为N,N-二异丙基乙胺；In step (2), the solvent is dichloromethane, and the catalyst is N,N-diisopropylethylamine;
    步骤(3)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；In step (3), the organic solvent is a dichloromethane solution, and the condensing agent is a mixture of N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole;
    步骤(4)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，所述的催化剂为三氟化硼乙醚络合物；In step (4), the organic solvent is a mixed solution of acetonitrile and dichloromethane, the volume ratio of the acetonitrile to the dichloromethane is 1.5:1, and the catalyst is a boron trifluoride ether complex ；
    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2:1，所述的催化剂为甲醇钠；In step (5), the organic solvent is a mixed solution of methanol and dichloromethane, the volume ratio of the methanol to the dichloromethane is 2:1, and the catalyst is sodium methoxide;
    步骤(6)中，所述有机溶剂为二氯甲烷、甲醇与水的混合溶液，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；In step (6), the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of cuprous iodide, N,N-diisopropylethylamine and glacial acetic acid;
    步骤(7)中，所述有机溶剂二氯甲烷、甲醇与水的混合溶液，所述催化剂为氢气、钯碳与氢氧化钯的混合物。In step (7), the organic solvent is a mixed solution of methylene chloride, methanol and water, and the catalyst is a mixture of hydrogen, palladium on carbon and palladium hydroxide.
  9. 如权利要求1-6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。The use of the conjugate according to any one of claims 1 to 6 in the preparation of drugs for the prevention and/or treatment of cancer.
  10. 如权利要求9所述的应用，所述癌症为乳腺癌、子宫癌、卵巢癌、肺癌、肝癌、前列腺癌、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。The application according to claim 9, wherein the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, bowel cancer, renal cell carcinoma, cellular lymphoma, thyroid gland Cancer, brain cancer, stomach cancer or leukemia.
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