Conjugate of dual agonist and carbohydrate antigen as well as preparation method and application of conjugate

Abstract

The invention relates to a conjugate of a dual agonist and a carbohydrate antigen, the conjugate contains the dual agonist and the carbohydrate antigen, the dual agonist is an iNKT cell agonist and a mincle receptor agonist, the iNKT cell agonist is an alpha-galactosylceramide analogue, and the mincle receptor agonist is a trehalose derivative; the structural general formula of the conjugate is as shown in formula (I): # imgabs0 # X is a carbohydrate antigen, and Y is a linker. The vaccine prepared from the conjugate can activate iNKT cells and mincle receptors at the same time, induce generation of high-titer IgG antibodies and effectively improve the immunogenicity of carbohydrate antigens, and has a good anti-tumor application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of carbohydrate chemistry and pharmaceutical technology, and specifically relates to a conjugate of a dual agonist and a carbohydrate antigen, and a preparation method and application thereof. Background Art

[0002] Tumor-associated carbohydrate antigens (TACAs) are effective targets for tumor immunity and have become one of the research hotspots in the field of anti-tumor. However, TACAs are T-cell-independent antigens with poor immunogenicity. They cannot induce a sufficiently effective immune response alone to treat cancer. At the same time, they are prone to immune tolerance during long-term use. At present, the fully synthetic tumor sugar vaccine obtained by conjugating TACAs with immunostimulatory adjuvants can covalently couple low-immunogenic TACAs with immune agonists as a whole. The immune agonist as a carrier can not only present TACAs to APCs, activate T cells by binding to MHC, promote B cells to transform into plasma cells and produce IgG antibodies, and stimulate B cells and T cells to mature into memory cells; and the immune agonist also acts as a built-in adjuvant, triggering innate immunity by activating specific immune signaling pathways and enhancing the adaptive response to TACAs. Therefore, the fully synthetic tumor sugar vaccine has the advantages of clear chemical structure, controllable quality, and no need for carrier proteins or external adjuvants.

[0003] Invariant natural killer T (iNKT) cells are a unique subtype of T lymphocytes that have phenotypic characteristics and immune effector properties of T cells and natural killer cells. Studies have shown that iNKT cells can initiate "T cell-dependent (TD) type II responses", which provides a basis for the study of iNKT cell agonists as vaccine carriers. α-Galactosylceramide analog (KRN7000), a natural α-GalGSL analog isolated from a sponge, is the first lipid that effectively activates iNKT cells. Anti-tumor vaccines using KRN7000 as a carrier, including STn-KRN7000, Tn-KRN7000, GM3-KRN7000 and MUC1-KRN7000, have been reported one after another; these vaccines can quickly induce high titer IgG antibodies and effectively mediate tumor cell lysis, indicating that KRN7000 can be used as an effective carrier for the construction of fully synthetic anti-tumor vaccines.

[0004] Mincle is a transmembrane C-type lectin receptor (CLR) expressed on activated macrophages and dendritic cells (DCs). Activation of Mincle can initiate the FcRg-Syk-Card9-Bcl10-Malt1 signaling pathway, leading to the activation of nuclear factor κB (NF-κB) protein and the production of proinflammatory cytokines, chemokines, and growth factors, thereby causing immune responses through helper T cells 1 and 17 (Th1 / Th17). Studies have shown that when trehalose-6,6-dimycolate derivatives (vizantin and TDB) bind to mincle, they can promote the maturation of antigen-presenting cells, activate Th1 and Th17 responses, and have adjuvant properties, indicating that vizantin and TDB can be used as effective carriers for tumor vaccines.

[0005] However, the low immunogenicity and effectiveness of the fully synthetic sugar vaccine hinder its further clinical application. Based on this, in order to further improve the immunogenicity and therapeutic effect of the TACAs vaccine, the present invention provides a fully synthetic sugar vaccine and its preparation method and application. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the deficiencies of the prior art and provide a conjugate of a dual agonist and a sugar antigen.

[0007] The second object of the present invention is to provide a method for preparing a conjugate of a dual agonist and a carbohydrate antigen.

[0008] The third object of the present invention is to provide a use of a conjugate of a dual agonist and a sugar antigen in the preparation of a drug for treating or preventing cancer.

[0009] The fourth object of the present invention is to provide an anti-tumor vaccine.

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

[0011] A conjugate of a dual agonist and a sugar antigen, the conjugate contains a dual agonist and a sugar antigen, the dual agonist is an iNKT cell agonist and a mincle receptor agonist, the iNKT cell agonist is an α-galactosylceramide analogue, and the mincle receptor agonist is a trehalose-6,6-dimycolate derivative; the general structural formula of the conjugate is shown in formula (I):

[0012]

[0013] Wherein, X represents a sugar antigen, selected from any one of the following sugar antigens, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

[0014]

[0015] L represents a linker, selected from the following CH2-NHC(O)-, (CH2CH2O) a -CH2CH2-NHC(O)-, (CH2) r -NHC(O)-, (CH2CH2O) r -、-NHC(O)-(CH2CH2O) r -CH2CH2-NHC(O)-, -(OCH2CH2) r -CH2CH2-NHC(O)-, -NHC(O)-(CH2) r -NHC(O)- or -NHC(O)-(CH2) a Any of the following, r is any integer from 0 to 20;

[0016] a is an integer from 1 to 9, b is an integer from 1 to 9, and c is an integer from 1 to 3;

[0017] n is an integer from 9 to 25;

[0018] R1 is -CH3 or

[0019] R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 Any of 2; R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

[0020] Preferably, the conjugate of the dual agonist and the carbohydrate antigen is a compound of structural formula (II) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of structural formula (II);

[0021]

[0022] Wherein, a is an integer from 1 to 9, b is an integer from 1 to 9, and c is an integer from 1 to 3;

[0023] n is an integer from 9 to 25;

[0024] R1 is -CH3 or

[0025] R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 Any of 2, R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

[0026] More preferably, the conjugate of the dual agonist and the carbohydrate antigen is a compound of structural formula (III) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of structural formula (III);

[0027]

[0028] Wherein, a is an integer of 1-9, b is an integer of 1-9, and c is an integer of 1-3.

[0029] Further preferably, the conjugate of the dual agonist and the carbohydrate antigen is a compound of structural formula (IV) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of the compound of structural formula (IV);

[0030]

[0031] The α-galactosylceramide analogue of the present invention is a compound of the general formula (V) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of the general formula (V);

[0032]

[0033] n is any integer from 9 to 25;

[0034] R1 is -H or

[0035] The trehalose derivative of the present invention is a compound of the general formula (VI) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of the general formula (VI);

[0036]

[0037] in:

[0038] R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR3 2. R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

[0039] The method for preparing the conjugate of the dual agonist and the sugar antigen, when X is L is When CH2-NHC(O)-, the preparation process includes:

[0040] S1. Compound 1 is dissolved in an organic solvent and reacted with propargylamine to form compound 2;

[0041] S2. Compound 2 is dissolved in an organic solvent, and the boc group is removed under the catalysis of trifluoroacetic acid to obtain compound 3;

[0042] S3. Compound 3 is dissolved in an organic solvent and reacted with a corresponding acid anhydride under the action of a basic catalyst to obtain compound 4;

[0043] S4. Compound 5 and compound 4 are dissolved in an organic solvent, a condensation agent is added, and an esterification reaction is performed to obtain compound 6;

[0044] S5. Compound 6 is dissolved in an organic solvent, and the Fmoc protecting group is removed under the catalytic action of piperidine to obtain compound 7;

[0045] S6. Compound 8 and compound 7 undergo an amide reaction under the catalytic action of a condensing agent to obtain compound 9;

[0046] S7. Compound 9 is catalyzed by boron trifluoride ether complex to remove the TBS group to obtain compound 10;

[0047] S9. Compound 10 and azide-containing TF sugar antigen 11 are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 12;

[0048] S10. Compound 12 is dissolved in an organic solvent, a catalyst is added, and a debenzylation reaction is performed to obtain the conjugate 13 of the dual agonist and the sugar antigen;

[0049] The structural formulas of compounds 1 to 13 are as follows:

[0050]

[0051]

[0052]

[0053] a is an integer from 1 to 9, b is an integer from 1 to 9, and cc is an integer from 1 to 3;

[0054] n is an integer from 9 to 25;

[0055] R1 is -CH3 or

[0056] R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 2. R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

[0057] The reaction formula of the preparation method of the present invention is as follows:

[0058]

[0059] Wherein, a is an integer from 1 to 9, b is an integer from 1 to 9, and c is an integer from 1 to 3;

[0060] n is an integer from 9 to 25;

[0061] R1 is -CH3 or

[0062] R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 2. R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

[0063] Preferably, in step S3, the alkaline catalyst is triethylamine or N,N-diisopropylethylamine; preferably N,N-diisopropylethylamine.

[0064] Preferably, in step S4, the organic solvent is dichloromethane, and the condensation agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA).

[0065] Preferably, in step S6, the organic solvent is dichloromethane, and the condensation agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine.

[0066] Preferably, in step S8, the organic solvent is dichloromethane, a mixture of dichloromethane and methanol, or a mixture of tetrahydrofuran and methanol, preferably a mixture of dichloromethane and methanol.

[0067] Preferably, in step S9, the organic solvent is dichloromethane, methanol, a mixture of dichloromethane and methanol, water, or a mixture of dichloromethane and methanol, preferably a mixture of dichloromethane and methanol, water; the catalyst is a mixture of hydrogen and palladium on carbon, palladium hydroxide, a mixture of hydrogen and palladium on carbon, or a mixture of hydrogen and palladium hydroxide, preferably a mixture of hydrogen and palladium on carbon, palladium hydroxide.

[0068] The present invention also protects the use of the conjugate of the dual agonist and the sugar antigen in preparing a drug for treating or preventing cancer.

[0069] Preferably, the cancer is selected from breast cancer, prostate cancer, colon cancer, melanoma, liver cancer, lung cancer, renal cell carcinoma, uterine cancer, ovarian cancer, lymphoma, brain cancer, gastric cancer, pancreatic cancer, thyroid cancer or leukemia.

[0070] The present invention also protects an anti-tumor vaccine, which comprises a conjugate of the dual agonist and a sugar antigen.

[0071] Preferably, the anti-tumor vaccine is in the form of an injection.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The present invention selects iNKT cell agonist α-galactosylceramide analog (KRN7000) and mincle receptor agonist trehalose-6,6-dimycolate derivative (vizantin) as embedded adjuvants by covalent bond connection, and couples with sugar antigen to construct a conjugate (KRN7000-TF-vizantin) containing iNKT cell agonist KRN7000, mincle receptor agonist vizantin and sugar antigen. The vaccine prepared by the conjugate has dual immune signaling pathway agonist activity, which can not only activate macrophages through vizantin, promote the uptake and presentation of TF antigen by antigen presenting cells, and enhance the immunogenicity of TF; but also can activate iNKT cells through KRN7000 to enhance the tumor killing effect of ADCC effect; induce stronger immune response, generate higher titer and memory T cells to regulate immune response, achieve the purpose of specifically killing tumor cells, and has good anti-tumor application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is a graph evaluating the IgG antibody immune activity of the KRN7000-TF-vizantin three-component sugar vaccine. DETAILED DESCRIPTION

[0075] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0076] Example 1

[0077] This embodiment is a conjugate provided by the present invention, wherein the conjugate is a compound of structural formula (IV) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (IV);

[0078]

[0079] The preparation method of the above conjugate comprises the following steps:

[0080] 1) Synthesis of compound 2:

[0081]

[0082] At room temperature, propargylamine (2.73 mL, 2.35 g, 42.71 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 12.3 g, 64.16 mmol) and 1-hydroxybenzotriazole (HOBt, 576.5 mg, 4.27 mmol) were added to a dichloromethane solution of N-α-fluorenylmethoxycarbonyl-N-ε-tert-butyloxycarbonyl-L-lysine (20.0 g, 42.73 mmol). The mixture was stirred at room temperature. The reaction was continued for 3 hours, during which a white solid precipitated. After the reaction was completed as monitored by TLC chromatography, dichloromethane was added to dilute the mixture, and the organic phase was washed with water twice and brine once. The organic layer was collected and dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed from the filtrate to obtain 20.0 g of a pale yellow powder crude product. The crude product was recrystallized from dichloromethane, and the filter cake was rinsed with water and then with a mixed solvent of dichloromethane and petroleum ether (1:1, v / v). After drying, a white solid compound 2 (17.0 g, yield 78.81%) was obtained. 1H NMR (400MHz, DMSO-d6) δ8.35 (t, J=5.5Hz, 1H, NHCO), 7.90 (d, J=7.5Hz, 2H, Ar-H), 7.78–7.70(m,2H,Ar-H),7.64–7.25(m,5H,Ar-H,NHCO),6.77(t,J=5.7Hz,1H,NHCO ),4.51–4.05(m,3H,COOCH2),4.02–3.79(m,3H,CONHCH,CONHCH2),3.10(t,J=2.6 Hz,1H,C≡CH),2.97–2.83(m,2H,CONHCH2),1.66–1.46(m,2H,CH2),1.37(s,13H,2x CH2,3x CH3).

[0083] 2) Synthesis of compound 3:

[0084]

[0085] At room temperature, compound 2 (10.0 g, 19.80 mmol) was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid (38.0 mL, 1:1, v / v). After the mixture was reacted at room temperature for 2 hours, the reaction solution was distilled under reduced pressure to remove the solvent. After dichloromethane was added to dissolve it, the solvent was distilled under reduced pressure again to obtain a pure white powder compound 3 (7.01 g, yield 87.50%). 1 H NMR(400MHz,DMSO-d6)δ8.38(t,J=5.5Hz,1H,NHCO),7.90(d,J=7.5Hz,2H,Ar-H),7.73(t ,J=7.4Hz,4H,NH2,Ar-H),7.50(d,J=8.3Hz,1H,NHCO),7.46–7.40(m,2H,Ar-H),7.38–7. 30(m,2H,Ar-H),4.34–4.17(m,3H,COOCH2),3.99–3.93(m,1H,CONHCH),3.89–3.83(m,2H ,CONHCH2),3.10(t,J=2.5Hz,1H,C≡CH),2.88–2.68(m,2H,CONHCH2),1.69–1.46(m,4H,2x CH2),1.38–1.30(m,2H,CH2).

[0086] 3) Synthesis of compound 4:

[0087]

[0088] After dissolving compound 3 (8.0 g, 19.75 mmol) and adipic anhydride (7.58 g, 59.21 mmol) in dichloromethane (80.0 mL), N,N-diisopropylethylamine (2.0 mL) was added to the mixture, and the reaction solution was reacted at room temperature for 6 hours; diluted with dichloromethane, washed with water twice, the organic layer was dried with anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate to obtain 7.8 g of a pale yellow powder crude product; the crude product was separated and purified by silica gel column (eluent: methanol: dichloromethane, 1:50) to obtain a white solid compound 4 (5.5 g, yield 52.28%). HR-ESI-MS (m / z): calcd for C 30 H 26 N3O6,[M+H] + ,534.2599;found 534.2589;calcd for C 30 H 25 N3NaO6,[M+Na] + ,556.2418; found 556.2410.

[0089] 5) Synthesis of compound 6:

[0090]

[0091] After dissolving compound 5 (100.0 mg, 73.80 μmol), compound 4 (57.0 mg, 106.94 μmol) and HATU (84 mg, 221.05 μmol) in dichloromethane (4.0 mL), DIEA (80 μL) was added to adjust the pH value of the reaction solution to 8, and the reaction was stirred at room temperature for 8 hours; after TLC monitoring, dichloromethane was added to dilute, and the solution was transferred to a separatory funnel, washed twice with saturated sodium bicarbonate aqueous solution, once with water, and once with brine, 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 187.0 mg of a crude product; purification by silica gel column (eluent: methanol: dichloromethane, 1:50) gave a colorless oily liquid compound 6 (98.0 mg, yield: 68.84%). 11H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 7.6 Hz, 2H, Ar-H), 7.54 (d, J = 7.4 Hz, 2H, Ar-H), 7.40–7.18 (m, 19H, Ar-H), 7.15 (t, J = 5.3 Hz, 1H, NHCO), 6.55 (s, 1H, NHCO), 6.24 (t, J = 6.5 Hz, 1H, NHCO), 6.07 (d, J = 8.1 Hz, 1H, NHCO), 5.80 (d, J = 8.0 Hz, 1H, NHCO), 4.91 (d, J = 10.9 Hz, 1H, PhCH2), 4.81 (d, J = 3.7 Hz, 1H, H-1), 4.79–4.51 (m, 5H, PhCH2), 4.33 (d, J = 7.2 Hz, 2H, COOCH2), 4.18–4.10 (m, 3H, 2x CONHCH), 4.03–3.88 (m, 4H, H-2, CONHCH2, OCH2), 3.84–3.71 (m, 4H, H-3, H-4, OCH, OCH2), 3.67–3.55 (m, 3H, H-6a, H-5, OCH), 3.32–3.04 (m, 3H, OCH2, H-6b), 2.21–2.11 (m, 3H, COCH2, C≡CH), 2.09–1.97 (m, 4H, 2xCOCH2), 1.86–1.72 (m, 1H, CH2), 1.68–1.40 (m, 10H, 5x CH2), 1.38–1.16 (m, 71H, 35x CH2), 0.85 (t, J = 6.8, 3.6 Hz, 24H, 8x CH3), 0.06– -0.03 (m, 12H, 4x SiCH3)..HR-ESI-MS (m / z): calcd for C 113 H 180 N5O 13 Si2, [M + H] + , 1871.3111;found 1871.3060;calcd forC 113 H 179 N5NaO 13 Si2, [M + Na] + , 1893.2930;found 1893.2886.

[0092] 6) Synthesis of Compound 7:

[0093]

[0094] After dissolving compound 6 (32.0 mg, 17.11 μmol) in N,N-dimethylformamide solution (1.0 mL), piperidine (5.0 μL) was added and reacted at room temperature for 2 hours; dichloromethane was added to dilute, the organic phase was washed with water once, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed from the filtrate to obtain a crude product of 101.0 mg; purification by silica gel column (eluent: methanol: dichloromethane, 1:10) gave a colorless oily liquid compound 7 (20.0 mg, yield: 70.93%). 1 H NMR (400MHz, CDCl3) δ7.82–7.70(m,1H,NHCO),7.37–7.24(m,15H,Ar-H),6.47(t,J=3.3Hz,1H,NHCO),6.14–6.07(m,1H,NHCO ),6.05(d,J=7.9Hz,1H,NHCO),4.93(d,J=11.1Hz,1H,PhCH2),4.83(d,J=3.7Hz,1H,H-1),4.79–4.62(m,5H,PhCH2),4.13(t, J=3.9Hz,1H,CONHCH),3.99–3.94(m,4H,H-2,CONHCH2,OCH2),3.84–3.71(m,4H,OCH2,H-3,H-4,OCH),3.67–3.59(m,2H,H-5, OCH),3.52–3.48(m,2H,H-6,CONHCH),3.19–3.10(m,3H,OCH2,H-6b),2.19–2.13(m,3H,COCH2,C≡CH),2.03(t,J=7.5Hz,4H,2x COCH2),1.62–1.38(m,10H,5x CH2),1.21(d,J=4.7Hz,72H,36x CH2),0.89–0.84(m,24H,8x CH3),0.08–-0.04(m,12H,4x SiCH3).HR-ESI-MS(m / z):calcd for C 98 H 169 N5NaO 11 Si2,[M+Na] + ,1671.2249; found 1671.2262.

[0095] 7) Synthesis of compound 9:

[0096]

[0097] After dissolving compound 7 (36.0 mg, 21.83 μmol), compound 8 (38.0 mg, 23.80 μmol) and HATU (25.0 mg, 65.79 μmol) in dichloromethane (10.0 mL), DIEA (200.0 μL) was added to adjust the pH value of the reaction solution to 8, and the reaction was stirred at room temperature for 8 hours; after TLC monitoring, dichloromethane was added to dilute, and the solution was transferred to a separatory funnel, washed twice with saturated sodium bicarbonate aqueous solution, once with water, and once with brine, 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; purification by silica gel column (eluent: methanol: dichloromethane, 1:40) gave a colorless oily liquid compound 9 (43.0 mg, yield: 60.99%). 1 H NMR (400MHz, CDCl3) δ7.51(t,J=5.4Hz,1H,NHCO),7.36–7.22(m,40H,Ar-H),6.76(d,J=8.0Hz,1H,NHCO),6.52(s,2H,2x NHCO),6.20(s,1H,NHCO),6.00(d,J=7.9Hz,1H,NHCO),5.13(d,J=3.6Hz,2H,2x H-1),5.02–4.55(m,17H,H-1,PhCH2),4.47(d,J=10.5Hz,1H,PhCH2),4.39–3.18(m,33H),3 .16–3.05(m,2H),2.63–2.53(m,1H,COCH2),2.46–2.26(m,3H,COCH2),2.21–2.00(m,10H,5x COCH2),1.76(s,2H,2xCH),1.56–1.32(m,10H,5x CH2),1.30–1.15(m,136H,78x CH2),0.89–0.78(m,36H,18x CH2),0.06–-0.05(m,12H,4x CH3).HR-ESI-MS(m / z):calcd for C 195 H 314 N6O 27 Si2,[M+2H] 2+ ,1614.1455;found 1614.1018;calcd for C 195 H 314 N6Na2O 27 Si2,[M+2Na] 2+ ,1636.1274;found1636.1154.

[0098] 8) Synthesis of compound 10:

[0099]

[0100] After dissolving compound 9 (43.0 mg, 13.32 μmol) in a mixed solution of acetonitrile and dichloromethane (2.0 mL, 1:1, v / v), boron trifluoride ether complex (3.0 μL, 24.3 μmol) was added. After reacting for 2 hours, the mixture was diluted with dichloromethane and washed twice with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was distilled under reduced pressure to remove the organic solvent to obtain a crude product, which was separated and purified by silica gel column (eluent: methanol: dichloromethane, 1:40) to obtain white solid compound 10 (32.0 mg, yield: 80.20%). 1 H NMR (400MHz, CDCl3) δ7.56 (s, 1H, NHCO), 7.35–7.18 (m, 40H, Ar-H), 6.94 (s, 1H, NHCO), 6.78 (d, J = 8.2Hz, 1H, NHCO), 6.59–6.53 (m, 2H, 2x NHCO),6.12(s,1H,NHCO),5.10(d,J=3.6Hz,2H,2x H-1),4.97–4.37(m,18H,H-1,PhCH2),4.37–3.60(m,19H),3.56–3.20(m,1 4H),3.10–2.98(m,2H),2.63–2.24(m,4H,2xCOCH2),2.22–1.92(m,10H,5x COCH2),1.81–1.69(m,2H,2x CH),1.64–1.39(m,10H,5x CH2),1.28–1.09(m,136H,78x CH2),0.80(t,J=6.2Hz,38H,6x CH3).HR-ESI-MS(m / z):calcd for C 183 H 284 N6O 27 ,[M+H] + ,2999.1107;found2999.0802;calcd for C 183 H 284 N6NaO 27 ,[M+Na] + ,3021.0927; found 3021.0825.

[0101] 9) Synthesis of compound 12:

[0102]

[0103] Compound 10 (10.0 mg, 3.3 μmol) and compound 11 (3.0 mg, 6.3 μmol) were dissolved in a mixed solution of methanol and dichloromethane (5.0 mL, 1:1.5, v / v), and then cuprous iodide (6.4 mg, 33.6 μmol) and N,N-diisopropylethylamine (100.0 μL, 575.2 μmol) were added. 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 on a silica gel plate (developing solvent was methanol: dichloromethane, 1:10) to obtain a white solid compound 12 (5.0 mg, yield was 43.47%). 1 H NMR (600MHz, CDCl3) δ7.80 (s, 1H, triazole-H), 7.43–7.16 (m, 40H, Ar-H), 6.68 (s, 2H), 5.17 (d, J=3.1Hz, 2H, 2x H-1),5.08(s,1H),5.03–4.64(m,16H),4.59–4.18(m,12H),4.13–3.21(m,42H),2.41–1.94(m,17H,7x COCH2,COCH3),1.79(d,J=6.5Hz,2H,2xCH),1.68–1.45(m,10H,5x CH2),1.36–1.05(m,136H,78xCH2),0.95–0.76(m,18H,6x CH3).HR-ESI-MS(m / z):calcd for C 199 H 314 N 10 O 38 ,[M+2H] 2+ ,1726.1467;found 1726.1353;calcdfor C 199 H 313 N 10 NaO 38 ,[M+H+Na] 2+ ,1737.1377;found 1737.1267;calcd for C 199 H 312 N 10 Na2O 38 ,[M+2Na] 2+ ,1748.1287; found 1748.1156.

[0104] 10) Synthesis of Compound 13

[0105]

[0106] Compound 12 (5.0 mg, 1.4 μmol) was dissolved in a mixed solvent (10.0 mL) of dichloromethane / methanol / water (3:1:0.1), palladium hydroxide (4.8 mg) and palladium carbon (5.3 mg) were added, hydrogen was introduced, sealed and stirred for 20 hours, filtered with diatomaceous earth, and the filter cake was rinsed three times with dichloromethane / methanol / water (3:1:0.1). The filtrate was distilled under reduced pressure to remove the solvent to obtain white solid compound 13 (compound of formula (IV)) (3.6 mg, 91.14%), i.e., three-component KRN7000-TF-vizantin. 1 H NMR (400MHz, CD3OD / CDCl3)δ7.83(s,1H,triazole-H),5.01(t,J=4.0Hz,2H,2x H-1),4.96(s,1H),4.76(m,3H),4.45–3.33(m,35H),3.15–3.03(m,4H),2.61(s,3H,COCH3),2.58–1.86(m,14H,7xCOCH2),1.78(d,J=13.6Hz,2H,2x CH),1.49(d,J=35.3Hz,10H,5x CH2),1.37–0.98(m,136H,78x CH2),0.79(t,J=5.8Hz,18H,6x CH3).HR-ESI-MS(m / z):calcd forC 143 H 265 N 10 NaO 38 ,[M+H+Na] 2+ ,1376.9499;found 1376.9414;calcd for C 143 H 264 N 10 Na2O 38 ,[M+2Na] 2+ ,1387.9409; found 1387.9424.

[0107] Example 2: ELISA immunoassay

[0108] 1) Mouse immunization: 6 Balb / c mice aged 6-8 weeks were taken. The conjugate (KRN7000-TF-vizantin) synthesized in Example 1 was prepared into liposomes, and the prepared vaccine was subcutaneously injected into the mice on days 0, 14, 21, and 28, respectively; blood was collected from the eye sockets on days 21, 27, and 38, and the whole blood was placed on ice for 1 hour, centrifuged at 4000 rpm for 15 minutes, and the upper clear serum was taken for ELISA detection and analysis.

[0109] 2) ELISA immunoassay: 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 plate, and incubated in a 4°C refrigerator overnight; incubated in a 37°C incubator for 1 hour the next day; and washed 3 times with PBST. After washing, 2% skim milk was added; 250 μL was added to each well; incubated at room temperature for 1 hour, and washed 3 times. Mouse serum samples were diluted with PBS to 300, 900, 2700, 8100, 24300, 72900, 218700, and 65400 times according to the half-and-half method; the diluted serum was added to a sealed 96-well plate, 100.0 μL per well, and three duplicate wells were made in parallel for each dilution gradient; incubated in a 37°C incubator for 1.5 hours; and washed 3 times. Dilute HRP-labeled Goat anti mouse IgG with PBS (dilution factor 1:2000) and add to the corresponding wells, add 100.0 μL to each well, incubate in a 37°C incubator for 1 hour; wash the plate 3 times. Add TMB solution, add 100.0 μL to each well, and color at room temperature in the dark for 15 minutes. Add 0.5MH2SO4 solution, add 100.0 μL to each well. After stopping color development, immediately detect the absorbance with an enzyme reader, the detection wavelength is 450nm, and the background wavelength is 570nm.

[0110] 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.

[0111] Experimental results:

[0112] from Figure 1 It can be seen that the KRN7000-TF-vizantin three-component conjugate synthesized in Example 1 of the present invention can produce a specific immune response more quickly in mice without the need for external adjuvants, and produce high-titer IgG antibodies, indicating that the dual adjuvant design of the KRN7000-TF-vizantin three-component vaccine can effectively improve the immunogenicity of the TF saccharide antigen. As the number of administrations increases, the antibody titer increases, indicating that multiple inductions can quickly and efficiently produce higher titer IgG antibodies, indicating that the saccharide vaccine can produce immune memory under the action of dual adjuvants.

[0113] Obviously, 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 of a dual agonist and a sugar antigen, characterized in that the conjugate contains a dual agonist and a sugar antigen, the dual agonist is an iNKT cell agonist and a mincle receptor agonist, the iNKT cell agonist is an α-galactosylceramide analogue, and the mincle receptor agonist is a trehalose derivative; the general structural formula of the conjugate is shown in formula (I): in, X represents a sugar antigen, selected from any one of the following sugar antigens, or a pharmaceutically acceptable salt, hydrate or solvate thereof: L represents a linker, selected from the following CH2-NHC(O)-, CH2CH2O) a -CH2CH2-NHC(O)-, (CH2) r -NHC(O)-, (CH2CH2O) r -、-NHC(O)-(CH2CH2O) r -CH2CH2-NHC(O)-, -(OCH2CH2) r -CH2CH2-NHC(O)-, -NHC(O)-(CH2) r -NHC(O)- or -NHC(O)-(CH2) a Any of the following, r is any integer from 0 to 20; a is an integer from 1 to 9, b is an integer from 1 to 9, and c is an integer from 1 to 3; n is an integer from 9 to 25; R1 is -CH3 or R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 Any of 2; R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

2. The conjugate of a dual agonist and a carbohydrate antigen according to claim 1, characterized in that the conjugate is a compound of structural formula (II) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (II); in, a is an integer from 1 to 9, b is an integer from 1 to 9, and c is an integer from 1 to 3; n is an integer from 9 to 25; R1 is -CH3 or R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 Any of 2, R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

3. The conjugate of a dual agonist and a carbohydrate antigen according to claim 1, characterized in that the conjugate is a compound of structural formula (III) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (III); in, a is an integer of 1-9, b is an integer of 1-9, and c is an integer of 1-3.

4. The conjugate of a dual agonist and a carbohydrate antigen according to claim 1, characterized in that the conjugate is a compound of structural formula (IV) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (IV); 5. A method for preparing a conjugate of a dual agonist and a sugar antigen according to any one of claims 1 to 4, characterized in that: When X is L is When CH2-NHC(O)-, the preparation process includes: S1. Compound 1 is dissolved in an organic solvent and reacted with propargylamine to form compound 2; S2. Compound 2 is dissolved in an organic solvent, and the boc group is removed under the catalysis of trifluoroacetic acid to obtain compound 3; S3. Compound 3 is dissolved in an organic solvent and reacted with a corresponding acid anhydride under the action of a basic catalyst to obtain compound 4; S4. Compound 5 and compound 4 are dissolved in an organic solvent, a condensation agent is added, and an esterification reaction is performed to obtain compound 6; S5. Compound 6 is dissolved in an organic solvent, and the Fmoc protecting group is removed under the catalytic action of piperidine to obtain compound 7; S6. Compound 8 and compound 7 undergo an amide reaction under the catalytic action of a condensing agent to obtain compound 9; S7. Compound 9 is catalyzed by boron trifluoride ether complex to remove the TBS group to obtain compound 10; S9. Compound 10 and azide-containing TF sugar antigen 11 are dissolved in an organic solvent, and a catalyst is added to react to obtain compound 12; S10. Compound 12 is dissolved in an organic solvent, a catalyst is added, and a debenzylation reaction is performed to obtain the conjugate 13 of the dual agonist and the sugar antigen; The structural formulas of compounds 1 to 13 are as follows: a is an integer from 1 to 9, b is an integer from 1 to 9, and cc is an integer from 1 to 3; n is an integer from 9 to 25; R1 is -CH3 or R2 is selected from -CH2-CH(OR 3 )-(CH2) m -CH3, -(CH2) m CH3 or -(CH2) m CHR 3 2. R 3 -(CH2) m -CH3 or -C(O)-(CH2) m -CH3, m is an integer selected from 8-26.

6. The method for preparing the conjugate of the dual agonist and sugar antigen according to claim 5, characterized in that: In step S3, the alkaline catalyst is triethylamine or N,N-diisopropylethylamine; In step S4, the organic solvent is dichloromethane, and the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine; In step S6, the organic solvent is dichloromethane, and the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine; In step S8, the organic solvent is dichloromethane, a mixture of dichloromethane and methanol, or a mixture of tetrahydrofuran and methanol; In step S9, the organic solvent is dichloromethane, methanol, a mixture of dichloromethane and methanol, water, or a mixture of dichloromethane and methanol; the catalyst is a mixture of hydrogen and palladium carbon, palladium hydroxide, a mixture of hydrogen and palladium carbon, or a mixture of hydrogen and palladium hydroxide.

7. Use of the conjugate of the dual agonist according to any one of claims 1 to 4 and a sugar antigen in the preparation of a drug for treating or preventing cancer.

8. The use according to claim 7, characterized in that: The cancer is selected from breast cancer, prostate cancer, colon cancer, melanoma, liver cancer, lung cancer, renal cell carcinoma, uterine cancer, ovarian cancer, lymphoma, brain cancer, stomach cancer, pancreatic cancer, thyroid cancer or leukemia.

9. An anti-tumor vaccine, characterized in that: The anti-tumor vaccine comprises a conjugate of the dual agonist according to any one of claims 1 to 4 and a sugar antigen.

10. The anti-tumor vaccine according to claim 9, characterized in that The anti-tumor vaccine is in the form of an injection.

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