Compound for preparing artificial tumor target as well as preparation method and application of compound

Abstract

The invention discloses a compound for preparing an artificial tumor target as well as a preparation method and application of the compound. The compound AzoManAc3Az disclosed by the invention is specifically reduced under an anoxic condition to obtain ManAc3AzOH, and an azido group can be expressed to the surface of a tumor cell membrane through a glycometabolism way to form an artificial tumor target. Meanwhile, the bionic nano drug delivery system DBCO-mPD-1NV (at) AuNC # R848 of the gold nano cage and the R848, which are encapsulated by the dibenzocyclooctyne group labeled macrophage membrane vesicles for overexpressing PD-1, can be used for specifically targeting an artificial tumor target spot. The compound AzoManAc3Az provided by the invention can realize azide labeling at a tumor site, and can effectively inhibit tumor growth, prolong the survival time of mice, activate natural immunity and improve infiltration of T cells when being combined with a bionic nano drug delivery system for use, thereby showing a good anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a compound for preparing artificial tumor targets, a preparation method and an application thereof. Background Art

[0002] At present, immunotherapy is commonly used in clinical treatment of tumors. Immunotherapy that blocks the PD-1 / PD-L1 pathway has achieved significant clinical efficacy. However, due to various drug resistance mechanisms, many patients fail to respond. The key to improving efficacy lies in taking corresponding combined therapies for different immune environments in the tumor tissue stroma. In tumor immunotherapy, there is an "on-target off-tumor" effect due to the lack of selective targeting, which not only limits the efficacy of anti-tumor immunity, but also induces serious immune-related adverse events, such as cytokine storm, neurotoxicity, etc., and cannot achieve targeting for non-targetable diseases such as triple-negative breast cancer. Therefore, it is urgent to develop an artificial targeting strategy. In "Selective in vivometabolic cell-labeling-mediated cancer targeting", a composition tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) is disclosed, and the acetyl group at the C1 position of the molecule is converted into an ether bond, and a protective group is connected to form a DCL-AAM molecule. The protective group on DCL-AAM will only be removed by the specifically overexpressed histone deacetylase (HDAC) and cathepsin L (CTSL) in cancer cells, forming a free hydroxyl group at the C1 position, thereby generating the Ac3ManAzOH molecule. Ac3ManAzOH can bind to the cell membrane, thereby labeling the cell with an azide group, becoming a specific target for dibenzocyclooctyne (DBCO), and at the same time, anticancer drugs can be linked to DBCO to kill cancer cells in a targeted manner. However, the labeling efficiency of Ac4ManAz and DCL-AAM for tumor cells needs to be improved. Summary of the invention

[0003] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a compound AzoManAc3Az.

[0004] The second object of the present invention is to provide a method for preparing the above compound AzoManAc3Az.

[0005] The third object of the present invention is to provide the use of the above compounds as artificial tumor targets in the preparation of drugs for inhibiting tumor growth.

[0006] The fourth object of the present invention is to provide an anti-tumor pharmaceutical composition.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a compound AzoManAc3Az, wherein the compound is AzoManAc3Az, and the structural formula of the compound AzoManAc3Az is shown in the following formula (I):

[0009]

[0010] The present invention couples a hypoxia-responsive group ((4-((4-(dimethylamino)phenyl)azo)phenyl)methanol) and azidoacetylmannose ((2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate) to obtain a sugar metabolism-labeled precursor compound AzoManAc3Az. The compound AzoManAc3Az is a hypoxia-responsive sugar derivative that can be specifically reduced under tumor hypoxia conditions to obtain ManAc3AzOH. The azido group is expressed on the surface of the tumor cell membrane through the sugar metabolism pathway to form an artificial tumor target. In normal tissues, under normoxic conditions, the precursor compound AzoManAc3Az maintains its original form and cannot be labeled on the cell membrane, thereby achieving the effect of tumor-specific labeling.

[0011] The present invention provides a method for preparing the above-mentioned compound AzoManAc3Az, comprising the following steps:

[0012] S1. 4-aminobenzyl alcohol (compound 1) is subjected to a diazo coupling reaction with N,N-dimethylaniline to obtain (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol (compound 2);

[0013]

[0014] S2. 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride (Compound 4) is subjected to a dehydration condensation reaction with azidoacetic acid to form an amide bond to obtain (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate (Compound 5);

[0015]

[0016] S3. reacting (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate (Compound 5) with acetic acid hydrazine to obtain (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 6);

[0017]

[0018] S4. (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 6) is reacted with 4-nitrobenzene chloroformate to obtain (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 7);

[0019]

[0020] S5. (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 7) is reacted with (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol (Compound 2) to obtain (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-((((4-((4-(dimethylamino)phenyl)azo)benzyl)oxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, i.e., AzoManAc3Az.

[0021]

[0022] Furthermore, in step S1, the molar ratio of 4-aminobenzyl alcohol (compound 1) to N,N-dimethylaniline is 16-17:24-25.

[0023] Preferably, in step S1, the molar ratio of 4-aminobenzyl alcohol (compound 1) to N,N-dimethylaniline is 16.24:24.36.

[0024] Preferably, the diazo coupling reaction in step S1 is to use sodium nitrite to react with 4-aminobenzyl alcohol under acidic conditions to convert the amino group of 4-aminobenzyl alcohol into a diazonium salt. The diazonium salt is a very active intermediate that can further react with other compounds. The generated diazonium salt then undergoes a coupling reaction with N,N-dimethylaniline. The reaction conditions of sodium nitrite and 4-aminobenzyl alcohol are to react in an ice bath for 0.4 to 0.6 hours; the reaction conditions of diazonium salt and N,N-dimethylaniline are to react in an ice bath for 2 to 4 hours.

[0025] Furthermore, in step S2, the molar ratio of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride (compound 4) to azidoacetic acid is 5-5.5:5.5-6.

[0026] Preferably, in step S2, the molar ratio of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride (Compound 4) to azidoacetic acid is 5.21:5.73.

[0027] Preferably, the dehydration condensation reaction in step S2 is to react 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride with azidoacetic acid under the mixed conditions of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide), HOBt (1-hydroxybenzotriazole) and triethylamine overnight to form an amide bond.

[0028] Furthermore, in step S3, the molar ratio of (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate (Compound 5) to hydrazine acetate is 4-4.5:5-5.5.

[0029] Preferably, in step S3, the molar ratio of (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate (Compound 5) to hydrazine acetate is 4.18:5.23.

[0030] Preferably, in step S3, (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate (Compound 5) and acetic acid hydrazine are dissolved in anhydrous dimethylformamide and reacted at 45° C. to obtain (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 6).

[0031] Furthermore, in step S4, the molar ratio of (2R, 3S, 4R, 5S, 6R)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 6) to 4-nitrobenzene chloroformate is 1 to 1.5:1 to 1.5.

[0032] Preferably, in step S4, the molar ratio of (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 6) to 4-nitrobenzene chloroformate is 1.29:1.35.

[0033] Preferably, in step S4, (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (compound 6) is reacted with 4-nitrobenzene chloroformate and N,N-diisopropylethylamine (DIPEA) to obtain (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (compound 7).

[0034] Furthermore, in step S5, the molar ratio of (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 7) to (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol (Compound 2) is 0.5-0.6:1.3-1.4.

[0035] Preferably, in step S5, the molar ratio of (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (Compound 7) to (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol (Compound 2) is 0.542:1.36.

[0036] Preferably, in step S5, (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (compound 7) is reacted with (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol (compound 2), 4-dimethylaminopyridine (DMAP), and N,N-diisopropylethylamine (DIPEA) overnight to obtain (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-((((4-((4-(dimethylamino)phenyl)azo)benzyl)oxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (AzoManAc3Az).

[0037] As described above, the compound AzoManAc3Az of the present invention is specifically reduced under hypoxic conditions to obtain ManAc3AzOH, and the azide group can be expressed on the surface of the tumor cell membrane through the sugar metabolism pathway to form an artificial tumor target. At the same time, the bionic nano-drug delivery system DBCO-mPD-1NV@AuNC#R848 of the macrophage membrane vesicles overexpressing PD-1 labeled with dibenzocyclooctyne groups and loaded with gold nanocages and R848 is used to specifically target the above-mentioned artificial tumor targets. The compound AzoManAc3Az of the present invention can achieve metabolic labeling of azide at the tumor site, and the combination with the bionic nano-drug delivery system can effectively inhibit tumor growth, prolong the survival time of mice, activate natural immunity, and increase the infiltration of T cells, showing good anti-tumor effects.

[0038] The R484 is a TLR agonist that can convert "cold tumors" into "hot tumors", making the combination of TLR and immune checkpoint inhibitors a potential therapy in cancer treatment.

[0039] The gold nanocages can be used for photothermal therapy to directly ablate tumors and induce immunogenic cell death (ICD) in tumors, thereby enhancing the immunogenicity of tumors.

[0040] The macrophage-derived biomimetic nanovesicle (NV) is a nanovesicle with a phospholipid bilayer. As a natural biomaterial, it has low immunogenicity and good biocompatibility, and also has the advantages of long circulation and small toxic side effects, which can improve the safety of treatment. At the same time, the nanovesicle can be modified by means of genetic engineering, lipid insertion, etc., and the drug can be encapsulated by means of electroporation, etc., so it has great application potential in the field of nano delivery. It is obtained by post-treatment of macrophages infected with PD-1 lentivirus, and the combination of PD-1 and PD-L1 on the tumor relieves the inhibition of tumors on T cells, significantly improving the infiltration of T cells.

[0041] Therefore, the present invention also provides the use of the above compound AzoManAc3Az as an artificial tumor target in the preparation of a drug for inhibiting tumor growth.

[0042] The present invention also provides a pharmaceutical composition, comprising the compound AzoManAc3Az and a DBCO (dibenzocyclooctyne group) labeled anti-tumor drug.

[0043] Furthermore, the DBCO (dibenzocyclooctyne group) labeled anti-tumor drug is a DBCO-labeled bionic nano drug delivery system DBCO-mPD-1NV@AuNC#R848 of macrophage membrane vesicles overexpressing PD-1 encapsulating gold nanocages and R848.

[0044] Furthermore, the preparation method of the bionic nano drug delivery system DBCO-mPD-1NV@AuNC#R848 comprises the following steps:

[0045] S1. Mixing R848, lauric acid and gold nanocages to obtain gold nanocage-encapsulated R848 nanoparticles AuNC@R848;

[0046] S2. The macrophage membrane vesicles overexpressing PD-1 are mixed with dibenzocyclooctyne-active ester to obtain the macrophage membrane vesicles overexpressing PD-1 labeled with dibenzocyclooctyne group DBCO-mPD-1NV;

[0047] S3. Mix the AuNC@R848 in step S1 and the DBCO-mPD-1NV in step S2 to obtain the dibenzocyclooctyne group-labeled macrophage membrane vesicles overexpressing PD-1 encapsulating gold nanocages and R848 biomimetic nanodrug delivery system DBCO-mPD-1NV@AuNC#R848.

[0048] Furthermore, in step S1, the mass ratio of R848, lauric acid and gold nanocage is 1:8-12:23-27.

[0049] Furthermore, in step S1, the mass ratio of R848, lauric acid and gold nanocages is 1:10:25.

[0050] Furthermore, in step S2, the macrophage membrane nanovesicles are secreted by cells and obtained by mechanical grinding, gradient centrifugation and extrusion.

[0051] Preferably, the cells are Raw 264.7 cells.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The present invention discloses a compound for preparing an artificial tumor target, a preparation method and an application thereof. The compound AzoManAc3Az of the present invention can be specifically reduced to obtain ManAc3AzOH under the condition of tumor hypoxia, and can express the azide group to the surface of the tumor cell membrane through the sugar metabolism pathway to form an artificial tumor target. At the same time, the bionic nano-drug delivery system DBCO-mPD-1NV@AuNC#R848 of the macrophage membrane vesicles overexpressing PD-1 labeled with dibenzocyclooctyne groups and encapsulating gold nanocages and R848 can specifically target the artificial tumor target. The compound AzoManAc3Az of the present invention can achieve metabolic labeling of azide at the tumor site, and the labeling effect is significantly better than Ac4ManAz. The combined use with the bionic nano-drug delivery system can effectively inhibit tumor growth, prolong the survival time of mice, activate natural immunity, and increase the infiltration of T cells, showing a good anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The figure is the result of the nuclear magnetic resonance hydrogen spectrum detection of Ac4ManAz. Figure 1 A in the figure is the H-NMR spectrum of Ac4ManAz; B is the C-NMR spectrum of Ac4ManAz; and C is the high-resolution mass spectrum of Ac4ManAz.

[0055] Figure 2 The figure is the result of the nuclear magnetic resonance hydrogen spectrum detection of AzoManAc3Az. Figure 2 A in the figure is the H-NMR spectrum of AzoManAc3Az; B is the C-NMR spectrum of AzoManAc3Az; and C is the high-resolution mass spectrum of AzoManAc3Az.

[0056] Figure 3 It is the result detection of tumor artificial targeting of AzoManAc3Az. Figure 3 A in the figure is the result of AzoManAc3Az being labeled with DBCO-Cy5.5; B is the comparison of the metabolic labeling effect of azide in the tumor site between the AzoManAc3Az group and the Ac4ManAz group.

[0057] Figure 4 Characterization of EVs overexpressing PD-1. Figure 4 A in the figure is a stable cell line overexpressing Mcherry-mPD-1 on the cell membrane surface; BC are the size and Zeta potential of cell vesicles.

[0058] Figure 5 The effects of different treatments on tumor size and survival of breast cancer mice. Figure 5AB in the figure represents the therapeutic effect of DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az on tumor cells; C represents the weight of mice; and D represents the survival curve of mice.

[0059] Figure 6 The effects of different treatments on adaptive immunity. Figure 6 A and B are the effects of DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az on T cells; C is the results of CD3 immunohistochemistry and Ki67 immunohistochemistry. DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] Example 1 Preparation method and test results of compound AzoManAc3Az

[0063]

[0064] Synthesis of (4-((4-(dimethylamino)phenyl)diazenyl)phenyl)methanol)(2): 4-aminobenzyl alcohol (compound 1) (2.0 g, 16.24 mmol, 1.0 eq) was dissolved in 75% concentrated HCl at room temperature, stirred in an ice bath, and slowly added with sodium nitrite aqueous solution (1.18 g, 17.05 mmoll, 1.05 eq) and placed in an ice bath for 0.5 h. Then, a hydrochloric acid solution of N,N-dimethylaniline (2.95 g, 24.36 mmol, 1.5 eq) was added and continued to be placed in an ice bath for 3 h. At this time, the reaction slowly turned reddish brown and a large amount of flocs were generated. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution, dissolved with ethyl acetate, and filtered with diatomaceous earth. Silica gel was added and dried through a column to obtain a yellow product.

[0065] Synthesis of (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (Ac4ManAz) (5): 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride ((2S,3R,4R,5S,6R)-6-(Acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate) was added. hydrochloride, beta-Glucosamine, tetraacetate, hydrochloride, BiDe Pharmaceuticals, Product No.: BD223740) (4) (2.0 g, 5.21 mmol, 1.0 eq), azidoacetic acid (0.579 g, 5.73 mmol, 1.1 eq), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (1.5 g, 7.82 mmol, 1.5 eq), 1-hydroxybenzotriazole (HOBt) (1.06 g, 7.82 mmol, 1.5 eq), triethylamine (1.58 g, 15.63 mmol, 3.0 eq), add 100 mL of anhydrous tetrahydrofuran to dissolve and react overnight at room temperature. TLC monitors the reaction, Hanessian staining solution is used for color development, and the plate is baked. After the reaction is completed, filter with diatomaceous earth, wash the filter cake with ethyl acetate, collect the organic phase, dry the solvent under reduced pressure, wash with ethyl acetate solvent, add water twice, and wash once with saturated brine. Add silica gel, dry the solvent under reduced pressure, and add the sample (petroleum ether: ethyl acetate = 1:1) to the column to obtain the product in the form of white powder.

[0066] Synthesis of (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate (6): Compound (5) (1.80 g, 4.18 mmol, 1.0 eq) and acetic acid hydrazine (0.482 g, 5.23 mmol, 1.25 eq) were added separately, and then 30 mL of anhydrous dimethylformamide (DMF) was added to dissolve, and the mixture was placed in an oil bath at 45°C to react until complete. The reaction was monitored by TLC, and the color was developed with Hanessian staining solution, and the plate was baked. TLC showed that the raw materials were reacted completely. Remove part of the solvent under reduced pressure, add ethyl acetate, wash with a large amount of water, and extract with ethyl acetate three times. Combine the organic phases, and wash the organic phase once with saturated brine. Add silica gel, decompress and spin dry the solvent, load the sample, (petroleum ether: ethyl acetate = 1:1) and pass through the column to obtain the product in the form of white powder.

[0067] (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate Synthesis of diacetate)(7): Compound (6) (0.50 g, 1.29 mmol, 1.0 eq), 4-nitrobenzene chloroformate (0.272 g, 1.35 mmol, 1.05 eq), N,N-diisopropylethylamine (DIPEA) (0.39 g, 3.86 mmol, 3 eq) were added under ice bath, and 100 mL of anhydrous tetrahydrofuran was added to dissolve the mixture and the temperature was naturally raised to room temperature for reaction. The reaction was monitored by TLC, and the color was developed by Hanessian staining solution. The plate was baked. After the reaction was completed, the mixture was washed with ethyl acetate solvent, water twice, and saturated brine once. Silica gel was added to mix the sample, and the solvent was dried under reduced pressure and then loaded onto the sample (petroleum ether: ethyl acetate = 2:1) to obtain the product as a white powder.

[0068] (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-((((4-((4-(dimethylamino)phenyl)azo)benzyl)oxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate Synthesis of (mino)phenyl)diazenyl)benzyl)oxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate)(AzoManAc3Az): Compound (7) (0.300 g, 0.542 mmol, 1.0 eq), compound (2) (0.355 g, 1.36 mmol, 2.0 eq), 4-dimethylaminopyridine (DMAP) (6.62 mg, 0.054 mmol, 0.1 eq), N,N-diisopropylethylamine (DIPEA) (0.715 g, 1.08 mmol, 2.5 eq) were added respectively, and 5 mL of anhydrous tetrahydrofuran was added to dissolve and reacted overnight at room temperature. TLC was used to monitor the reaction. After the reaction was completed, silica gel was directly added, the solvent was dried under reduced pressure and loaded, and the product was passed through a column (petroleum ether: ethyl acetate = 1:1) to obtain the product in the form of a reddish brown powder. The product obtained after passing through the column was then added to HCl / EA and stirred for 6 hours and filtered to obtain the product.

[0069] H NMR, C NMR and high resolution mass spectrometry Figure 1 AC shows that Ac4ManAz was successfully synthesized. 1HNMR (400MHz, Chloroform-d) δ6.66 (d, J = 9.2Hz, 1H), 5.27 (dd, J = 10.7, 9.5Hz, 1H), 5.08 (t, J = 9.5Hz, 1H), 4.39 (d, J = 4.0Hz, 1H), 4.16 (d, J = 11.3Hz, 2H), 4.12–4.05 (m, 1H), 3.87 (s, 2H), 2.04 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H).13C NMR (101MHz, CDCl3) δ171.07,170.68,169.39,169.35,167.18,92.16,72.83,72 .23,67.91,61.69,53.05,52.60,20.87,20.71,20.60,20.57.HRMS(ESI):calcd for C16H22N4O10([M + H] +):431.1336, found 431.1406.

[0070] H NMR, C NMR and high resolution mass spectrometry Figure 2 As shown in AC, it proves that AzoManAc3Az was successfully synthesized. NMR(400MHz,Chloroform-d)δ7.80(t,J=8.5Hz,4H),7.44(d,J=8.4Hz,2H),6.69(d ,J=9.2Hz,2H),6.47(d,J=8.8Hz,1H),6.03(d,J=3.7Hz,1H),5.23–5.19(m,2H),5.1 6(d,J=9.6Hz,1H),4.39(ddd,J=10.8,8.8,3.7Hz,1H),4.20(dd,J=13.0,4.4Hz,1H ), 4.03–3.98 (m, 2H), 3.83 (s, 2H), 3.02 (s, 6H), 2.00 (s, 3H), 1.97 (d, J = 1.9Hz, 6H). HRMS(ESI):calcd for C30H35N7O11([M + H] + ):670.2395, found 670.2466.

[0071] Example 2 Verification of artificial targeting function of AzoManAc3Az

[0072] (1) The 4T1 cell line (Pnosai, CL-0007) was placed in a three-gas incubator under hypoxia for 24 h, and then the Ac4ManAz and AzoManAc3Az synthesized in Example 1 were administered. After 48 h, the DBCO-Cy5.5 probe (MCE, HY-D1583) was added to detect the expression of azide groups on the cell membrane.

[0073] The results are as follows Figure 3 As shown in A, the cell membrane of the AzoManAc3Az group can be labeled with DBCO-Cy5.5, indicating that artificial targeting function can be achieved at the cellular level.

[0074] (2) To construct an orthotopic breast cancer model in mice, 6-8 week-old SPF BALB / c mice were prepared. After anesthesia, the mice were depilated and injected with 2×10 7 100 μL of 4T1 cells were injected into the right mammary gland of BALB / c mice. 7100 μL of 4T1 cells were injected into BALB / c mice to establish the model, and the model was successfully established after 10-14 days. Different doses of Ac4ManAz and AzoManAc3Az were injected through the tail vein, and DBCO-Cy5.5 (5 mg / kg) fluorescent probe was injected through the tail vein 24 hours after administration to detect the expression of azide groups in mice.

[0075] The results are as follows Figure 3 As shown in B, fluorescence was found in the tumor site after 24 hours, and it had a concentration-dependent characteristic, indicating that AzoManAc3Az could metabolically label azide in the tumor site, and the fluorescence intensity of the AzoManAc3Az group was higher than that of the Ac4ManAz group, indicating that AzoManAc3Az had a better effect in metabolically labeling azide in the tumor site.

[0076] Example 3 Extraction and characterization of cell membrane vesicles (EVs) overexpressing PD-1

[0077] When the macrophage Raw 264.7 culture reaches a density of 40%-50%, the culture medium is removed, the PD-1 lentiviral solution is slowly dripped into it, and polybrene is added to make its final concentration 8μg / mL. After 24h, the virus suspension is discarded and 10% FBSDMEM culture medium is added to continue culturing. Screening of stable cell lines overexpressing Mcherry-mPD-1 on the cell membrane surface. Add HM buffer solution to the cell line and resuspend it, transfer it to a grinder and grind it for 3min, then centrifuge it at 5000rpm, 4℃, 10min, collect the supernatant and centrifuge it again at 15000rpm, 4℃, 20min, resuspend the precipitate with 2mL PBS, and pass it through a 0.45μm filter membrane and a 0.22μm filter membrane in turn to obtain cell membrane vesicles (mPD-1NV) overexpressing mPD-1.

[0078] The results are as follows Figure 4 As shown in A, a stable cell line overexpressing Mcherry-mPD-1 on the cell membrane surface was obtained; the results are shown in Figure 4 As shown in BC, the vesicle sizes are between 100-180 nm, and the Zeta potential is between 10-20 mV.

[0079] Example 4 Anti-tumor effect of bionic nano drug delivery system DBCO-mPD-1NV@AuNC#R848 Preparation method of DBCO-mPD-1NV@AuNC#R848:

[0080] (1) 0.2 mg / mL of dibenzocyclooctyne-active ester (DBCO-NHS) was added to the cell membrane vesicles (mPD-1NV) overexpressing mPD-1 prepared in Example 3, and the mixture was shaken at 4°C for 30 min. After the reaction was completed, the mixture was centrifuged at 15000 rpm, 4°C, 20 min, and resuspended in PBS. The mixture was centrifuged at 15000 rpm, 4°C, 20 min, and the supernatant was removed to obtain DBCO-mPD-1NV.

[0081] (2) Weigh 20 mg of R848 and 200 mg of lauric acid, place them in a 2 mL EP tube, then add 1 mL of methanol (MeOH), and fully dissolve them in a water bath by ultrasonication until transparent. Add the above solution to 0.5 g of gold nanocage (AuNC), disperse them by ultrasonication, and stir them at room temperature for 4-6 hours. Centrifuge at 12000 rpm, 4 °C, 20 min, discard the supernatant, and wash twice with 1 mL of MeOH. Add 1 mL of PBS and ultrasonically resuspend to prepare AuNC@R848;

[0082] (3) Add 0.2 mL of AuNC@R848 suspension to DBCO-mPD-1NV, mix thoroughly by low-temperature rapid sonication in a water bath for 10-30 min, and then filter through a 0.22 μm filter membrane 2-3 times to prepare DBCO-mPD-1NV@AuNC#R848.

[0083] Example 5 Effects of different treatments on tumor size and survival of breast cancer mice.

[0084] To establish a bilateral orthotopic tumor-bearing model of mouse breast cancer 4T1, 6-8 week-old SPF-grade BALB / c mice were prepared, and hair removal was performed after anesthesia. 2×10 7 100 μL of 4T1 cells were injected into the right mammary gland of BALB / c mice. 7 100 μL of 4T1 cells were injected into BALB / c mice to establish the model, and the model was successfully established after 10-14 days. AzoManAc3Az was injected into the tail vein on days 0, 1, and 2 after the model was established, and DBCO-mPD-1NV@AuNC#R848 was administered into the tail vein on the third day. 24 hours after the administration, 808nm laser 0.1W / cm2 was irradiated for 10 minutes for photothermal treatment, which was repeated 3 times.

[0085] The results are as follows Figure 5 As shown in AB, the DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az+Laser treatment group had a significant ability to inhibit tumor growth after the first photothermal treatment. The data showed that the tumor did not grow during the 28-day observation period, but the tumor volume and weight decreased significantly. Figure 5As shown in C, there was no significant statistical difference in the weight of mice, indicating that the photothermal therapy mediated by DBCO-mPD-1NV@AuNC#R848 had no obvious toxicity. The mouse survival curve results are shown in Figure 5 As shown in D, the DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az+Laser treatment group significantly prolonged the survival time of mice.

[0086] Example 6 Effects of different treatments on adaptive immunity

[0087] (1) Prepare 6-8 week old SPF grade C57BL / 6 mice and inject 2×10 7 100 μL of 4T1 cells were injected into C57BL / 6 mice, and the model was successfully established after 10-14 days. On the 18th day, spleen tissues of tumor-bearing mice were extracted and memory T cells were analyzed by flow cytometry.

[0088] T cell extraction method:

[0089] Mouse spleen tissue was collected and soaked in PBS, then ground through a 100-mesh filter to obtain a tissue single-cell suspension, centrifuged at 2000 rpm for 10 min, and the supernatant was discarded; 3 mL of red blood cell lysis buffer was added, lysed on ice for 2 min, and centrifuged at 1500 rpm at 37°C for 5 min to remove the supernatant, and lysed twice in total.

[0090] The results are as follows Figure 6 As shown in A and B, the central memory T cell population and effector memory T cell population in the DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az+Laser group reached 8.45% and 17.0%, respectively, indicating the activation of immune memory T cells and long-term anti-tumor immunity.

[0091] (2) CD3 immunohistochemistry and Ki67 immunohistochemistry in in situ tumors

[0092] Paraffin section preparation method:

[0093] The tumor tissue was completely immersed in 4% paraformaldehyde and rinsed with running water for 30 minutes after fixation. Then, it was fully immersed in 70% EtOH, 80% EtOH, 90% EtOH, 95% EtOH, anhydrous EtOH, and anhydrous EtOH for 20 minutes;

[0094] Then soak in 50% EtOH and 50% xylene solution for 20 min, and then transfer to xylene I, xylene II, and xylene III solution in sequence, soaking for 40 min each time;

[0095] Melt the paraffin, put the tissue into the wax melting box, repeat the wax dipping 3 times, cool and solidify, and embed. Use a microtome to cut into 5 μm slices, seal the slices, and dry them in a 45℃ thermostat.

[0096] Immunohistochemistry method:

[0097] a. Place the paraffin sections in xylene for 10 min, anhydrous EtOH for 5 min, 95% EtOH for 5 min, 80% EtOH for 5 min, 70% EtOH for 5 min, and rinse carefully with distilled water twice;

[0098] b. Immerse the slices in 3% H2O2 solution for 10 minutes and carefully rinse with distilled water twice;

[0099] c. Transfer the slices to 0.01 mol / L citric acid repair solution, boil in a microwave oven on medium heat for about 3 minutes, cool to room temperature, and repeat once;

[0100] d. Wash the sections with PBS for 5 min, repeat 3 times, incubate at 37°C, and block with serum for 30 min;

[0101] e. Add 50 μL of primary antibody, incubate overnight at 4°C, wash with PBS for 5 minutes, and repeat 3 times;

[0102] f. Add 50 μL of secondary antibody, incubate at 37°C for 60 min, wash with PBS for 5 min, repeat 3 times;

[0103] g. Add DAB color developer, observe the staining under a microscope, and terminate the staining with distilled water;

[0104] h. Soak in 95% EtOH for 5 min, anhydrous EtOH for 5 min, and xylene for 5 min, and dehydrate until transparent.

[0105] The results are as follows Figure 6 As shown in C, the CD3 immunohistochemistry results showed that the DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az+Laser group had more brown plaques, indicating that T cells infiltrated more in the tumor area. The Ki67 immunohistochemistry results showed that the DBCO-mPD-1NV@AuNC#R848+AzoManAc3Az+Laser treatment group had fewer brown plaques, and tumor proliferation was significantly inhibited.

Claims

1. A compound AzoManAc3Az, characterized in that The structural formula of the compound AzoManAc3Az is shown in the following formula (I):

2. The method for preparing the compound AzoManAc3Az according to claim 1, characterized in that: The following steps are involved: S1. diazo coupling reaction of 4-aminobenzyl alcohol and N,N-dimethylaniline to obtain (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol; S2. 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride is subjected to a dehydration condensation reaction with azidoacetic acid to form an amide bond to obtain (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate; S3. reacting (2S,3S,4R,5S,6R)-6-(acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triacetate with acetic acid hydrazine to obtain (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate; S4. reacting (2R,3S,4R,5S,6R)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-hydroxy-tetrahydro-2H-pyran-3,4-diyl diacetate with 4-nitrobenzene chloroformate to obtain (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetylamino)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; S5. (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate is reacted with (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol to obtain (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-((((4-((4-(dimethylamino)phenyl)azo)benzyl)oxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, i.e., AzoManAc3Az.

3. The preparation method according to claim 2, characterized in that: In step S5, the molar ratio of (2R,3S,4R,5S,6S)-2-(acetoxymethyl)-5-(2-azidoacetamido)-6-(((4-nitrophenoxy)carbonyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to (4-((4-(dimethylamino)phenyl)azo)phenyl)methanol is 0.5-0.6:1.3-1.

4.

4. The preparation method according to claim 2, characterized in that: In the step S1, the molar ratio of 4-aminobenzyl alcohol to N,N-dimethylaniline is 16-17:24-25.

5. The preparation method according to claim 2, characterized in that: In step S2, the molar ratio of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucose hydrochloride to azidoacetic acid is 5-5.5:5.5-6.

6. Use of the compound AzoManAc3Az according to claim 1 as an artificial tumor target in the preparation of a drug for inhibiting tumor growth.

7. A pharmaceutical composition, characterized in that The invention comprises the compound AzoManAc3Az as claimed in claim 1, and an anti-tumor drug labeled with a dibenzocyclooctyne group.

8. The pharmaceutical composition according to claim 7, characterized in that: The dibenzocyclooctyne group-labeled anti-tumor drug is a dibenzocyclooctyne group-labeled macrophage membrane vesicle overexpressing PD-1 encapsulating gold nanocages and R848 bionic nano drug delivery system DBCO-mPD-1NV@AuNC#R848.

9. The pharmaceutical composition according to claim 7, characterized in that: The preparation method of the bionic nano drug delivery system DBCO-mPD-1NV@AuNC#R848 comprises the following steps: S1. Mixing R848, lauric acid and gold nanocages to obtain gold nanocage-encapsulated R848 nanoparticles AuNC@R848; S2. The macrophage membrane vesicles overexpressing PD-1 are mixed with dibenzocyclooctyne-active ester to obtain the macrophage membrane vesicles overexpressing PD-1 labeled with dibenzocyclooctyne group DBCO-mPD-1NV; S3. Mix the AuNC@R848 in step S1 and the DBCO-mPD-1NV in step S2 to obtain the dibenzocyclooctyne group-labeled macrophage membrane vesicles overexpressing PD-1 encapsulating gold nanocages and R848 biomimetic nanodrug delivery system DBCO-mPD-1NV@AuNC#R848.

10. The pharmaceutical composition according to claim 9, characterized in that: In the step S1, the mass ratio of R848, lauric acid and gold nanocage is 1:8-12:23-27.

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