Conjugate containing trehalose derivative and carbohydrate antigen as well as preparation method and application thereof

Abstract

The invention discloses a conjugate containing a trehalose derivative and a carbohydrate antigen as well as a preparation method and application of the conjugate, and belongs to the technical field of anti-tumor carbohydrate vaccines. The conjugate is obtained by taking an immunologic stimulant trehalose derivative of a mincle receptor as an embedded adjuvant to be conjugated with a sugar antigen TF, the trehalose derivative can improve the immunogenicity of the TF sugar antigen, the TF sugar antigen is presented to a corresponding immune cell, and a higher-titer immune response aiming at tumor sugar antigen TF specificity is generated. In addition, the compound also has the advantages of clear structure, simple and convenient synthesis method, stable and controllable product quality and the like, and can overcome the defect of weak immunogenicity of sugar antigens; meanwhile, under the action of an embedded adjuvant trehalose derivative, the vaccine can also generate immune memory.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor sugar vaccines, and more specifically, to a conjugate containing a trehalose derivative and a sugar antigen, and a preparation method and application thereof. Background Art

[0002] Surgery, radiotherapy and chemotherapy are traditional treatments for cancer, but surgical resection is usually imprecise and recovery is slow, while radiotherapy and chemotherapy have the disadvantages of poor targeting and serious side effects. With the rapid development of biology and technology, immunotherapy has become a new therapy favored in tumor prevention and treatment. Tumor sugar vaccines targeting tumor-associated carbohydrate antigens (TACAs) abnormally expressed on the surface of tumor cells have the advantages of strong specificity, low toxicity, and long-lasting efficacy, showing good prospects in tumor prevention and treatment.

[0003] TACAs are products of abnormal glycosylation of tumor cells, and are divided into four categories: Mucin glycoprotein antigen, ganglioside antigen, glycosphingolipid glycogen antigen, and Lewis blood group-related glycogen antigen. Among them, Thomson-Friedenreich antigen (TF) in Mucin glycoprotein antigen is a disaccharide antigen extended from Tn antigen; TF is highly expressed in more than 80% of human cancers, especially in epithelial tumors such as ovarian cancer, breast cancer, gastric cancer, and prostate cancer; and is almost not expressed in normal cells. In addition, TF participates in tumor cell proliferation by interacting with specific galactose lectins, and is closely related to tumor growth and metastasis; therefore, TF is selected as the target antigen of tumor sugar vaccine. However, TF sugar antigen is a T cell-independent antigen and cannot produce immune memory cells. It needs the assistance of immunogenic carrier molecules or embedded adjuvants to stimulate T cells and induce lasting antibody responses. The traditional strategy is to couple sugar antigens with carrier proteins containing B cell epitopes (KLH, TTox or CRM197). The resulting glycoprotein vaccines can produce high-titer and high-affinity antibodies. However, glycoprotein vaccines still have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components. In order to avoid these disadvantages, the introduction of fully synthetic sugar antigen vaccines with embedded adjuvants has become a new research strategy.

[0004] Trehalose-6,6-dimycolate (TDM, cord factor) is an important glycolipid component in the cell wall of Mycobacterium tuberculosis, composed of trehalose and two mycolate chains; it is an agonist of the C-type lectin receptor (mincle). Studies have shown that when vizantin and TDB, derivatives of trehalose-6,6-dimycolate, bind to the macrophage-induced C-type lectin receptor (mincle), they can promote the maturation of antigen-presenting cells and activate Th1 and Th17 responses, and have adjuvant properties. Vizantin and TDB not only have high immunological activity but also low toxicity; therefore, they can be used as embedded adjuvants in vaccine research and development.

[0005] Patent CN111875649A proposes to use potent immunostimulants, trehalose derivatives, Vizantin and TDE as embedded adjuvants to conjugate sugar antigens (STn, Tn) to obtain the conjugate, wherein the trehalose derivative can improve the immunogenicity of the sugar antigen, so that the conjugate can induce T cell-mediated humoral immunity, produce high concentrations of high-affinity IgG antibodies, and achieve the purpose of specifically killing tumor cells. However, there are no reports of fully synthetic sugar antigen vaccines that use trehalose derivatives, an immune agonist of mincle receptors, as embedded adjuvants to conjugate TF. Summary of the invention

[0006] The purpose of the present invention is to provide a conjugate of a trehalose derivative and a sugar antigen. The present invention uses a potent immunostimulant trehalose derivative (vizantin or TDB) as an embedded adjuvant to conjugate the sugar antigen TF to obtain the conjugate (vizantin-TF or vizantin-TDB) of the present invention, that is, a fully synthetic sugar antigen vaccine. The trehalose derivative can improve the immunogenicity of the TF sugar antigen, present the TF sugar antigen to the corresponding immune cells, induce a stronger immune response against the sugar antigen TF, and produce IgG antibodies with higher titer, high affinity and.

[0007] Another object of the present invention is to provide a method for preparing a conjugate of a trehalose derivative and a sugar antigen.

[0008] Another object of the present invention is to provide an anti-tumor sugar vaccine containing the above conjugate.

[0009] Another object of the present invention is to provide a use of a conjugate of a trehalose derivative and a sugar antigen in the preparation of a drug for preventing and / or treating cancer.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention protects a conjugate, which is a compound of the general formula (I) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of the general formula (I);

[0012]

[0013] Wherein, L represents a linker, and L is selected from R'-C(O)NH-R”-C(O)NH-R”’-, R"'-R"-C(O)NH-, R*-C(O)NH-, R”'-, -NHC(O)-R”’-R”-C(O)NH-, -R”’-R”-C(O)NH-, -NHC(O)-R*-C(O)NH- or -NHC(O)-R*

[0014] R' is selected from C1-C4 alkyl; R" is selected from C1-C4 alkyl; R"' is selected from (CH2CH2O) a , a is selected from any integer between 0 and 20; R* is selected from C0-C 20 alkyl;

[0015] X is selected from O or N;

[0016] R is selected from -R**-CH(OR 3 )-R 4 , -R 4 、-(CH2) x CHR 3 2 or -R 4 R 3 2; x is an integer selected from 1-5;

[0017] R** is selected from C1-C4 alkyl, R 4 For C9-C 27 Alkyl, R 3 Selected from -(CH2) m -CH3 or -C(O)-R 4 ; m is selected from an integer of 8-26.

[0018] In some embodiments, L is selected from

[0019] CH2-C(O)NH-CH2CH2-C(O)NH-(CH2CH2O) a -、

[0020] (CH2CH2O) a -CH2CH2-C(O)NH-,

[0021] (CH2) a -C(O)NH-, (CH2CH2O) a -、-NHC(O)-(CH2CH2O) a -CH2CH2-C(O)NH-, -(OCH2CH2) a -CH2CH2-C(O)NH-, -NHC(O)-(CH2) a -C(O)NH- or -NHC(O)-(CH2) a a is selected from any integer between 0 and 20.

[0022] In some embodiments, the conjugate is a compound of formula (II) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of formula (II);

[0023]

[0024] n is an integer from 1 to 7;

[0025] R is selected from -R**-CH(OR 3 )-R 4 , -R 4 、-(CH2) x CHR 3 2 or -R 4 R 3 2; x is an integer selected from 1-5;

[0026] R** is selected from C1-C4 alkyl, R 4 For C9-C 27 Alkyl, R 3 Selected from -(CH2) m -CH3 or -C(O)-R 4 ; m is selected from an integer of 8-26.

[0027] In some embodiments, R is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH 3、 -(CH2) x CHR 3 2 or -(CH2) m CHR 3 2;

[0028] R 3 Selected from -(CH2) m -CH3 and -C(O)-(CH2) m -CH3, m is selected from an integer of 8-26, and x is selected from an integer of 1-5.

[0029] In some embodiments, the conjugate is a compound of formula (III) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of formula (III);

[0030]

[0031] A conjugate formed by the reaction of a trehalose compound and a sugar antigen;

[0032] The trehalose compound is a compound of general formula (IV) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of general formula (IV);

[0033]

[0034] wherein X is selected from O or N;

[0035] R1 and R2 are independently selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH 3、 -(CH2) x CHR 3 2 or -(CH2) m CHR 3 2. R 3 Selected from -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26; x is an integer selected from 1-5;

[0036] The sugar antigen is the sugar antigen TF, and its structural formula is:

[0037]

[0038] A method for preparing a conjugate comprises the following steps:

[0039] (1) reacting compound 1 and benium bromide under the catalytic action of sodium hydride to obtain compound 2;

[0040] (2) reacting the compound 2 and the compound 11 under the catalysis of sodium hydride to obtain the compound 3;

[0041] (3) removing the PMB group from the compound 3 under the action of trifluoroacetic acid to obtain compound 4;

[0042] (4) subjecting the compound 4 to an esterification condensation reaction with a fatty acid chain monomer to obtain a compound 5;

[0043] (5) reacting the compound 5 under the catalysis of zinc powder and acetic acid to obtain compound 6;

[0044] (6) reacting the compound 6 with an acid anhydride in the presence of a basic catalyst to obtain a compound 7;

[0045] (7) subjecting the compound 7 to an amide condensation reaction with propargylamine to obtain compound 8;

[0046] (8) reacting the compound 8 with the compound 13 to obtain the compound 9;

[0047] (9) reacting the compound 9 under the catalysis of hydrogen and palladium hydroxide and / or palladium on carbon to obtain compound 10;

[0048] Its synthetic route is:

[0049]

[0050] Wherein, n is an integer from 1 to 7;

[0051] R is each independently selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH 3、 -(CH2) x CHR 3 2 and -(CH2) m CHR 3 2; R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26; x is an integer selected from 1-5;

[0052] Among them, the structural formulas of compound 11 and compound 13 are:

[0053]

[0054] In some embodiments, in step (4), the fatty acid chain monomer is HOOC-CH2-CH(OR 3 )-(CH2) m -CH3, HOOC-(CH2) m CH3 or HOOC-(CH2) m CHR 3 2. R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

[0055] In some embodiments, in step (6), the alkaline catalyst is triethylamine or N,N-diisopropylethylamine.

[0056] Preferably, the acid anhydride is succinic anhydride.

[0057] In some embodiments, in step (8), the catalyst used in the reaction is cuprous iodide / N,N-diisopropylethylamine or cuprous iodide / N,N-diisopropylethylamine / glacial acetic acid, etc.; preferably cuprous iodide / N,N-diisopropylethylamine; the solvent of the reaction is dichloromethane, dichloromethane / methanol or tetrahydrofuran / methanol, etc.; preferably dichloromethane / methanol;

[0058] In some embodiments, in step (9), the catalyst for the debenzylation reaction is hydrogen / palladium carbon / palladium hydroxide, hydrogen / palladium carbon or hydrogen / palladium hydroxide, preferably hydrogen / palladium carbon; the solvent used in the reaction is dichloromethane / methanol / water, dichloromethane, methanol or dichloromethane / methanol, etc.; preferably dichloromethane / methanol.

[0059] The present invention protects an anti-tumor sugar vaccine, including the conjugate or its isomer, pharmaceutically acceptable salt, hydrate or solvent compound.

[0060] The present invention protects the use of a conjugate or its isomer, pharmaceutically acceptable salt, hydrate or solvate in preparing a drug for preventing and / or treating cancer.

[0061] In some embodiments, the cancer is at least one of breast cancer, lung cancer, uterine cancer, ovarian cancer, liver cancer, prostate cancer, melanoma, colon cancer, renal cell carcinoma, cellular lymphoma, brain cancer, pancreatic cancer, thyroid cancer, gastric cancer or leukemia.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The present invention provides a conjugate containing a trehalose derivative and a sugar antigen TF (vizantin-TF sugar vaccine), which is obtained by conjugating the sugar antigen TF with a trehalose derivative (vizantin) which is an immunostimulant of the mincle receptor as an embedded adjuvant. Vizantin can not only improve the immunogenicity of the TF sugar antigen, present the TF sugar antigen to corresponding immune cells, and generate a higher titer of immune response specific to the tumor sugar antigen TF; but also can target and bind to the mincle receptor, activate the innate immune system and generate an immune response.

[0064] The method for preparing the conjugate containing trehalose derivative and sugar antigen TF provided by the present invention has a short synthesis route, mild reaction conditions, high yield, convenient operation, and can be used for industrial preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a graph showing the antibody immunoactivity evaluation of the conjugate (III) prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0067] Example 1

[0068] This example is a conjugate containing a trehalose derivative and a sugar antigen provided by the present invention, and its structural formula is shown in the following formula (III):

[0069]

[0070] The method for preparing the conjugate containing the trehalose derivative and the sugar antigen comprises the following steps:

[0071] Synthesis of compound 2:

[0072]

[0073] Compound 1 (12.3 g, 13.04 mmol) was dissolved in N,N-dimethylformamide solution (50.0 mL), and the reaction solution was cooled to 0°C in an ice bath. 60% sodium hydride (1.35 g, 33.67 mmol) and benzyl bromide solution (0.77 mL, 1.12 g, 10.43 mmol) were added in an ice bath, and the reaction solution was gradually restored to room temperature. The reaction was stirred at room temperature for 1 hour; methanol (10.0 mL) was added in an ice bath to quench the reaction, and the solution was diluted with ethyl acetate, washed twice with a saturated sodium bicarbonate aqueous solution, once with water, and once with brine, dried over anhydrous sodium sulfate, filtered, and ethyl acetate was removed from the filtrate to obtain a pale yellow oily crude product. The crude product was separated and purified by silica gel column (eluent: ethyl acetate: petroleum ether, 1:5) to obtain a colorless oily compound 2 (5.1 g, yield: 37.86%).

[0074] Synthesis of compound 3:

[0075]

[0076] A mixture of compound 2 (1.37 g, 1.33 mmol) and compound 11 (0.45 g, 1.59 mmol) in N,N-dimethylformamide (5.0 mL) was cooled to 0°C, 60% sodium hydride (0.16 g, 39.79 mmol) was added at 0°C, and the reaction solution was gradually restored to room temperature. The reaction was stirred at room temperature for 2 hours; methanol (2.0 mL) was added under an ice bath to quench the reaction, and the mixture was diluted with ethyl acetate, washed twice with a saturated sodium bicarbonate aqueous solution, once with water, and once with brine, dried over anhydrous sodium sulfate, filtered, and ethyl acetate was removed from the filtrate to obtain a pale yellow oily crude product. The crude product was separated and purified by silica gel column (eluent: ethyl acetate: petroleum ether, 1:5) to obtain a colorless oily compound 3 (1.36 g, yield: 89.47%).

[0077] Synthesis of compound 4:

[0078]

[0079] To a mixture of compound 3 (1.36 g, 1.19 μmol) and dichloromethane (5.0 mL) was added trifluoroacetic acid (400.0 μL) dropwise and reacted for 10 minutes; the mixture was diluted with dichloromethane and the solvent was directly distilled off under reduced pressure to obtain a pale yellow crude product; the crude product was separated and purified by silica gel column (eluent: ethyl acetate: petroleum ether, 1:5) to obtain a colorless oily compound 4 (0.61 g, yield: 56.74%).

[0080] Synthesis of compound 5:

[0081]

[0082] At room temperature, compound 4 (480.0 mg, 529.80 μmol), compound 12 (691.0 mg, 2.12 mmol), EDCI (400.0 mg, 2.11 mmol) and 4-dimethylaminopyridine (DMAP, 70.0 mg, 530.30 μmol) were dissolved in dichloromethane, and the reaction solution was refluxed at 50°C for 8 hours; after the reaction was completed as monitored by TLC chromatography, dichloromethane was added to dilute, and the mixture was washed twice with saturated sodium bicarbonate solution. The organic layer was collected and dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed from the filtrate to obtain a pale yellow oily crude product; the crude product was separated and purified by silica gel column (eluent: ethyl acetate: petroleum ether, 1:5) to obtain a colorless oily product 5 (700.0 mg, yield: 86.84%).

[0083] Synthesis of compound 6:

[0084]

[0085] At room temperature, activated zinc powder (250.0 mg, 3.85 mmol) and acetic acid (200 μL, 3.50 mmol) were added to a dichloromethane solution of compound 5 (230.0 mg, 151.12 μmol), and the mixture was reacted at room temperature for 7 hours. After the reaction was completed by TLC chromatography, the mixture was filtered with filter paper, the filter cake was rinsed with dichloromethane, and the filtrate was washed twice with a saturated aqueous solution of sodium bicarbonate and once with water. The organic layer was collected and dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed from the filtrate. After vacuum pump drying, a colorless oily compound 6 (202.0 mg, yield 89.34%) was obtained.

[0086] 1 H NMR (400MHz, CDCl3) δ7.29–7.14(m,25H,Ar-H),5.09(t,J=4.2Hz,2H,H-1,H-1'),4.99–4.72(m,5H,PhCH2),4. 66–4.40(m,7H),4.20–3.85(m,9H),3.49–3.45(m,8H),3.26(t,J=9.5Hz,1H),2.79(s,1H),2.18–2.04(m,4H,2x COCH2), 1.73 (s, 2H, 2x CH), 1.15 (d, J = 13.5Hz, 64H, 32x CH2), 0.79 (t, J = 6.7Hz, 12H, 4x CH3).

[0087] 13 C NMR (100MHz, CDCl3) δ172.32,172.16,137.67,136.91,136.81,136.78,127.45,127.43,127.40,127.36,127.33,127.06,126.98 ,126.90,126.86,126.82,126.76,126.73,126.59,126.57,126.40,93.10,80.57,80.21,78.35,78.28,77.49,76.63,74.70,74.4 8,74.25,72.06,71.88,71.42,70.62,69.54,68.18,68.11,61.51,61.35,39.97,38.08,34.19,33.90,32.92,32.79,32.74,32.68,30.92,30.90,29.07,28.97,28.94,28.91,28.71,28.66,28.63,28.59,28.36,28.33,25.58,25.52,25.49,21.68,13.12,0.00.

[0088] HR-ESI-MS(m / z):calcd for C 93 H 142 NO 14 ,[M+H] + ,1497.0425;found1497.0397.

[0089] Synthesis of compound 7:

[0090]

[0091] After dissolving compound 6 (340.0 mg, 227.12 μmol) and succinic anhydride (114.0 mg, 1.14 mmol) in dichloromethane (8.0 mL), N,N-diisopropylethylamine (562.0 μL, 439.5 mg, 3.41 mmol) was added to the mixture to adjust the pH value to 8. The reaction solution was reacted at room temperature for 6 hours; it was diluted with dichloromethane, washed once with a saturated sodium bicarbonate solution and once with water, the organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate to obtain 460.0 mg of a pale yellow oily crude product; the crude product was separated and purified by silica gel column (eluent: methanol: dichloromethane, 1:50) to obtain a colorless oily compound 7 (330.0 mg, yield: 90.98%).

[0092] 1 H NMR (400MHz, CDCl3) δ7.39–7.23 (m, 25H, Ar-H), 6.85 (t, J = 5.3Hz, 1H, NHCO), 5.18 ( t,J=2.9Hz,2H,H-1,H-1'),5.03–4.80(m,5H,PhCH2),4.73–4.67(m,4H,PhCH2),4.5 2(d,J=10.5Hz,1H,PhCH2),4.39–4.34(m,1H),4.22(d,J=9.7,2H),4.17–3.97(m,6H ),3.62–3.50(m,5H),3.50–3.41(m,4H),3.35(t,J=9.4Hz,2H),2.65–2.43(m,4H,2x COCH2), 2.26–2.16 (m, 4H, 2x CH), 1.80 (d, J = 5.4Hz, 2H, 2x COCH2), 1.31–1.14 (m, 64H, 32x CH2), 0.87 (t, J = 6.8Hz, 12H, 4x CH3).

[0093] 13C NMR (100MHz, CDCl3) δ174.83,174.21,173.25,173.04,138.58,137.85,137.80,137.72,128.50,128.49,128.45,128.12, 127.99,127.92,127.90,127.87,127.85,127.73,127.53,127.47,94.25,81.61,81.20,79.39,79.25,78.40,77.67,75.74 ,75.54,75.34,73.07,72.96,72.75,70.53,69.82,69.20,69.15,62.85,62.33,39.72,39.17,39.12,35.01,34.94,33.82,33.73,33.71,33.66,31.92,30.37,30.28,30.02,29.97,29.95,29.67,29.63,29.37,29.36,26.55,26.52,22.71,14.15.

[0094] HR-ESI-MS(m / z):calcd for C 97 H 146 NO 17 ,[M+H] + ,1597.0585; found1597.0613; calcd for C 97 H 145 NNaO 17 ,[M+Na] + ,1619.0405; found 1619.0459.

[0095] Synthesis of compound 8:

[0096]

[0097] At room temperature, compound 7 (50.0 mg, 31.31 μmol), EDCI (12.0 mg, 62.62 μmol) and HOBt (0.4 mg, 3.13 μmol) were dissolved in dichloromethane, propargylamine (2.4 μL, 2.0 mg, 37.57 mmol) was added, and the reaction solution was reacted at room temperature for 4 hours; after the reaction was completed as monitored by TLC chromatography, dichloromethane was added to dilute, washed twice with water, the organic layer was collected and dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed from the filtrate to obtain a pale yellow oily crude product; the crude product was separated and purified by silica gel column (eluent: ethyl acetate: petroleum ether, 1:1) to obtain colorless oil 8 (35.0 mg, yield 68.43%).

[0098] 1 H NMR (400MHz, CDCl3) δ7.34–7.13(m,25H,Ar-H),6.76(s,1H,NHCO),6.54(s,1H,NHCO),5.10(d,J=3.6Hz,2H,2x H-1),5.00–4.37(m,10H,5x PhCH2),4.29–3.83(m,11H),3.57–3.19(m,11H),2.39(s,4H,2xCOCH2),2.23–2.03(m,5H,2x COCH2,C≡CH),1.73(s,2H,2x CH),1.34–1.03(m,64H,32x CH2),0.80(t,J=6.7Hz,12H,4x CH3).

[0099] 13 C NMR (100MHz, CDCl3) δ173.89,173.24,172.41,171.98,138.67,138.64,137.89,137.85,137.77,128.49,128.47,128.45,128.11,127. 96,127.92,127.89,127.86,127.84,127.81,127.69,127.56,127.46,94.24,81.63,81.25,79.75,79.37,79.26,78.47,75.74,75.52,7 5.32,73.06,72.93,72.70,71.30,70.57,70.03,69.18,62.74,62.36,39.55,39.18,39.12,34.99,34.93,33.81,33.73,33.71,33.66,31.93,31.56,31.17,30.02,29.98,29.95,29.72,29.68,29.66,29.65,29.63,29.53,29.38,29.35,29.14,26.55,26.52,22.71,14.15.

[0100] HR-ESI-MS(m / z):calcd for C 100 H 148 N2NaO 16 ,[M+Na] + ,1656.0721; found1656.0731.

[0101] Synthesis of compound 9:

[0102]

[0103] After dissolving compound 8 (21.0 mg, 12.85 μmol) and compound TF (11.6 mg, 25.70 μmol) in a mixed solution of methanol and dichloromethane (5 mL, 1:1.5, v / v), cuprous iodide (25.0 mg, 131.57 μmol) and N,N-diisopropylethylamine (100.0 μL, 575.20 μmol) were added, and the reaction was stirred at room temperature for 24 hours; filtered through diatomaceous earth, and the filtrate was distilled under reduced pressure to remove the solvent to obtain a crude product; separated and purified by silica gel column (eluent: methanol: dichloromethane, 1:10) to obtain white solid compound 9 (14.0 mg, yield 52.24%).

[0104] 1 H NMR (400MHz, CD3OD / CDCl3)δ7.73(s,1H,triazole-H),7.30–7.14(m,25H,Ar-H),5.11(d,J=3.7Hz,2H,2x H-1),4.95–4.57(m,10H,5xPhCH2),4.52–4.46(m,3H),4.37–4.23(m,4H),4.17–4.05(m,4H),3.9 9–3.89(m,5H),3.82–3.36(m,19H),3.32–3.21(m,4H),3.06(t,J=6.1Hz,1H),2.44–2.32(m,4H,2x COCH2),2.19–2.06(m,4H,2x COCH2),1.91(s,3H,COCH3),1.72(s,2H,2x CH),1.20–1.11(m,64H,32x CH2),0.84–0.71(m,12H,4x CH3).

[0105] 13C NMR(100MHz,CD3OD / CDCl3)δ173.87,173.60,173.03,172.85,172.70,138.47 ,137.79,137.75,137.65,128.42,128.39,128.35,127.97,127.89,127.84,12 7.76,127.65,127.55,127.37,123.29,104.51,97.44,93.90,93.85,81.53,81.02,79.29,75.65,75.45,75.26,75.18,73.19,73.08,72.91,72.41,71.16,7 0.43,70.40,69.62,69.09,69.04,68.18,65.46,62.60,62.39,61.72,61.13,50.01,49.42,49.21,49.07,49.00,48.86,48.78,48.57,48.35,48.30,48.14, 39.27,39.13,39.08,34.97,34.93,34.69,33.71,33.62,33.57,31.84,30.80,30.72,29.88,29.84,29.66,29.57,29.55,29.28,26.44,26.40,22.60,13.94.

[0106] HR-ESI-MS(m / z):calcd for C 116 H 176 N6NaO 27 ,[M+Na] + ,2108.2476; found2108.2408; calcd for C 116 H 177 N6O 27 ,[M+H] + ,2086.2656; found 2086.2598.

[0107] Synthesis of compound 10:

[0108]

[0109] Compound 9 (10.0 mg, 4.79 μmol) was dissolved in a mixed solution of methanol and dichloromethane (9.0 mL, 2:1, v / v), palladium hydroxide (10.0 mg) and palladium carbon (10.0 mg) were added, hydrogen was introduced, sealed and stirred for 20 hours, filtered through celite, and the filter cake was rinsed three times with dichloromethane and methanol (2:1). The filtrate was distilled under reduced pressure to remove the solvent to obtain white solid compound 10 (6.0 mg, 76.53%).

[0110] 1 H NMR (400MHz, CD3OD / CDCl3) δ7.86(s,1H,triazole-H),4.98(t,J=3.5Hz,2H,2xH-1),4.75(d,J=3.7Hz,1H),4.53( t,J=4.2Hz,2H),4.40–4.09(m,8H),4.02–3.34(m,25H),3.33–3.23(m,3H),3.21–3.11(m,1H),2.51–2.39(m,4H,2x COCH2),2.20(t,J=7.3Hz,4H,2x COCH2),1.89(s,2H,2x CH),1.76(s,3H,COCH3),1.19(s,64H,32x CH2),0.86–0.73(m,12H,32x CH2).

[0111] 13 C NMR(100MHz,CD3OD / CDCl3)δ174.00,173.84,173.30,173.27,172.70,145.15,123.48,104.72,99.99,97.5 5,93.72,93.58,79.26,77.24,75.39,73.31,73.22,73.18,71.84,71.76,71.13,70.85,70.49,70.38,70.00 ,69.22,68.89,68.81,68.33,65.72,63.10,62.72,61.32,61.24,49.90,39.11,38.89,38.86,34.96,34.89,34.51,33.58,33.55,31.75,30.85,29.66,29.64,29.41,29.17,26.30,26.28,26.25,22.44,21.88,13.36.

[0112] HR-ESI-MS(m / z):calcd for C 81 H 148 N6NaO 27,[M+Na] + ,1658.0128; found1658.0247.

[0113] Example 2

[0114] In this example, the conjugate (fully synthetic sugar vaccine) prepared in Example 1 was used to immunize mice, and its immune effect was preliminarily evaluated by ELSA experiment.

[0115] 1.ELISA immunoassay

[0116] 1) Mouse immunization:

[0117] Six Balb / c mice aged 6-8 weeks were selected. After the sugar vaccine was prepared into liposomes, the immune test was performed by subcutaneous injection of mice. The prepared vaccine was injected on days 0, 14, 21, and 28, with an injection volume of 0.1 mL per mouse each time; on days 21, 27, and 38, the mice were bled from the eye sockets, placed at 0°C for 60 minutes, centrifuged at 4000 rpm for 15 minutes, and the upper clear serum was taken for ELISA detection and analysis.

[0118] 2) ELISA immunoassay:

[0119] Coating: TF-HSA was prepared into a 2.0 μg / mL solution with 0.1 M carbonate buffer (pH 9.6), and 100.0 μL was added to each well of a 96-well ELISA plate. The plate was sealed and wrapped with tin foil, and incubated overnight in a 4°C refrigerator. The next day, the plate was incubated in a 37°C incubator for 1 hour. The plate was washed three times with PBST (PBS + 0.1% Tween 20), and 300.0 μL of washing solution was added to each well, and the plate was washed using a plate washer.

[0120] Blocking: After washing, add 2% skim milk; add 250 μL to each well; incubate at room temperature for 1 hour, and wash the plate 3 times using a plate washer.

[0121] Add primary antibody: mouse serum samples were diluted with PBS to 300, 900, 2700, 8100, 24300, 72900, 218700 and 65400 times according to the split-half method; the proportionally diluted serum was added to a blocked 96-well plate, 100.0 μL per well, and three replicate wells were made in parallel for each dilution gradient; the plate was sealed with film, wrapped with tin foil, and incubated in a 37°C incubator for 1.5 hours; the plate was washed 3 times.

[0122] Add secondary antibody: dilute HRP-labeled Goat anti mouse IgG with PBS (dilution factor 1:2000); add the diluted secondary antibody to the corresponding wells, add 100.0 μL to each well, and incubate in a 37°C incubator for 1 hour; wash the plate 3 times with a plate washer.

[0123] Color development: Add TMB solution, 100.0 μL per well, and develop color at room temperature in the dark for 15 minutes.

[0124] Stop color development: add 0.5M H2SO4 solution, add 100.0μL to each well.

[0125] On-machine detection: After stopping the color development, immediately use an enzyme-labeled instrument to detect the absorbance, the detection wavelength is 450nm, and the background wavelength is 570nm.

[0126] The absorbance (OD) values ​​were plotted against the antiserum dilution values ​​and a best fit line was obtained. The equation of this line was used to calculate the dilution value when the OD value reached 0.2, and the antibody titer was calculated based on the reciprocal of the dilution value. Figure 1 shown.

[0127] 3) Experimental results:

[0128] from Figure 1 It can be seen that the vizantin-TF sugar vaccine (conjugate III) synthesized in Example 1 of the present invention can produce a specific immune response more quickly in mice without the need for an external adjuvant, and produce high-titer IgG antibodies; as the number of administrations increases, the antibody titer increases, indicating that multiple inductions can quickly and efficiently produce higher titer IgG antibodies.

[0129] The present invention provides a conjugate containing a trehalose derivative and a sugar antigen TF (vizantin-TF sugar vaccine), which is obtained by conjugating the sugar antigen TF with a trehalose derivative (vizantin), an immunostimulant of the mincle receptor, as an embedded adjuvant. Vizantin can not only improve the immunogenicity of the TF sugar antigen, present the TF sugar antigen to the corresponding immune cells, and generate a higher titer of immune response specific to the tumor sugar antigen TF; it can target and bind to the mincle receptor, activate the innate immune system and generate an immune response; and under the action of the embedded adjuvant vizantin, the vaccine can generate immune memory.

[0130] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A conjugate, characterized in that The conjugate is a compound of general formula (I) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of general formula (I); Wherein, L represents a linker, and L is selected from R'-C(O)NH-R”-C(O)NH-R”’-, R"'-R"-C(O)NH-, R*-C(O)NH-, R”'-, -NHC(O)-R”’-R”-C(O)NH-, -R”’-R”-C(O)NH-, -NHC(O)-R*-C(O)NH- or -NHC(O)-R* R' is selected from C1-C4 alkyl; R" is selected from C1-C4 alkyl; R"' is selected from (CH2CH2O) a , a is selected from any integer between 0 and 20; R* is selected from C0-C 20 alkyl; X is selected from O or N; R is selected from -R**-CH(OR 3 )-R 4 , -R 4 、-(CH2) x CHR 3 2 or -R 4 R 3 2; x is an integer selected from 1-5; R** is selected from C1-C4 alkyl, R 4 For C9-C 27 Alkyl, R 3 Selected from -(CH2) m -CH3 or -C(O)-R 4 ; m is selected from an integer of 8-26.

2. The conjugate according to claim 1, characterized in that L is selected from CH2-C(O)NH-CH2CH2-C(O)NH-(CH2CH2O) a - (CH2CH2O) a -CH2CH2-C(O)NH-、 (CH2) a -C(O)NH-, (CH2CH2O) a -、-NHC(O)-(CH2CH2O) a -CH2CH2-C(O)NH-, -(OCH2CH2) a -CH2CH2-C(O)NH-, -NHC(O)-(CH2) a -C(O)NH- or -NHC(O)-(CH2) a a is selected from any integer between 0 and 20.

3. The conjugate according to claim 1, characterized in that The conjugate is a compound of structural formula (II) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of structural formula (II); n is an integer from 1 to 7; R is selected from -R**-CH(OR 3 )-R 4 , -R 4 、-(CH2) x CHR 3 2 or -R 4 R 3 2; x is an integer selected from 1-5; R** is selected from C1-C4 alkyl, R 4 For C9-C 27 Alkyl, R 3 Selected from -(CH2) m -CH3 or -C(O)-R 4 ; m is selected from an integer of 8-26.

4. The conjugate according to claim 3, characterized in that R is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH 3、 -(CH2) x CHR 3 2 or -(CH2) m CHR 3 2; R 3 Selected from -(CH2) m -CH3 and -C(O)-(CH2) m -CH3, m is selected from an integer of 8-26, and x is selected from an integer of 1-5.

5. The conjugate according to claim 3, characterized in that The conjugate is a compound of structural formula (III) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of structural formula (III); 6. A conjugate, characterized in that It is formed by the reaction of trehalose compounds and sugar antigens; The trehalose compound is a compound of general formula (IV) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of general formula (IV); wherein X is selected from O or N; R1 and R2 are independently selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH 3、 -(CH2) x CHR 3 2 or -(CH2) m CHR 3 2. R 3 Selected from -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26; x is an integer selected from 1-5; The sugar antigen is the sugar antigen TF, and its structural formula is:

7. A method for preparing a conjugate, characterized in that: The steps include: (1) reacting compound 1 and benium bromide under the catalytic action of sodium hydride to obtain compound 2; (2) reacting the compound 2 and the compound 11 under the catalysis of sodium hydride to obtain the compound 3; (3) removing the PMB group from the compound 3 under the action of trifluoroacetic acid to obtain compound 4; (4) subjecting the compound 4 to an esterification condensation reaction with a fatty acid chain monomer to obtain a compound 5; (5) reacting the compound 5 under the catalysis of zinc powder and acetic acid to obtain compound 6; (6) reacting the compound 6 with an acid anhydride in the presence of a basic catalyst to obtain a compound 7; (7) subjecting the compound 7 to an amide condensation reaction with propargylamine to obtain compound 8; (8) reacting the compound 8 with the compound 13 to obtain the compound 9; (9) reacting the compound 9 under the catalysis of hydrogen and palladium hydroxide and / or palladium on carbon to obtain compound 10; Its synthetic route is: Wherein, n is an integer from 1 to 7; R is each independently selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH 3、 -(CH2) x CHR 3 2 and -(CH2) m CHR 3 2; R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26; x is an integer selected from 1-5; Among them, the structural formulas of compound 11 and compound 13 are:

8. An anti-tumor sugar vaccine, characterized in that: The invention comprises the conjugate according to any one of claims 1 to 6 or its isomer, pharmaceutically acceptable salt, hydrate or solvate.

9. Use of the conjugate according to any one of claims 1 to 6 or its isomer, pharmaceutically acceptable salt, hydrate or solvate in the preparation of a medicament for preventing and / or treating cancer.

10. The use according to claim 9, characterized in that The cancer is at least one of breast cancer, lung cancer, uterine cancer, ovarian cancer, liver cancer, prostate cancer, melanoma, intestinal cancer, renal cell carcinoma, cellular lymphoma, brain cancer, pancreatic cancer, thyroid cancer, gastric cancer or leukemia.

Images