Complex DACHPt-P-B-CD-IDOi and application thereof

Abstract

The invention discloses a complex DACHPt-P-B-CD-IDOi, which is characterized in that a compound PEG-BLG-CD and a compound DACHPt are uniformly dispersed in a solvent to obtain a mixed solution, and the mixed solution and a compound IDOi as shown in a formula C are subjected to post-treatment H to obtain the complex DACHPt-P-B-CD-IDOi, in a supramolecular nano-drug system, DACHPt is effectively loaded into nano-particles with the help of metal coordination, chemotherapy is carried out, and an anti-tumor immune response is activated. Meanwhile, catabolism of tryptophan and kynurenine is inhibited through subject-object complexed entrapped IDOi, and cold tumors are converted into hot tumors in a synergistic manner, so that chemical immunotherapy is enhanced. The supramolecular combination of IDOi and DACHPt performs powerful remodeling on an immunosuppressive tumor microenvironment, and achieves a remarkable anti-tumor effect in vitro and in vivo, which promotes the maturation of dendritic cells and the recruitment of T lymphocytes at tumor sites.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis route design and raw material drug and intermediate preparation, and particularly relates to an anticancer chemotherapy drug DACHPt-PB-CD-IDOi, its intermediates, and their preparation methods and applications in tumor treatment. Background Art

[0002] Malignant tumors are major diseases that threaten human life and health, and the incidence and mortality of malignant tumors are increasing year by year. Therefore, the development of efficient treatment methods for malignant tumors has very important social significance.

[0003] At present, the main treatment methods commonly used in clinical practice include surgical resection, chemotherapy, radiotherapy and immunotherapy. Among them, immunotherapy is a revolutionary cancer treatment method after traditional surgery, chemotherapy and radiotherapy. It changes people's way of thinking from directly destroying cancer cells to identifying and attacking cancer cells by activating the host's anti-tumor immune response. However, there are various immunosuppressive cells, cytokines and immune checkpoints that induce abnormal regulatory pathways in the tumor's immunosuppressive microenvironment. These factors can protect tumor cells from being recognized and eliminated by the host immune system.

[0004] Indoleamine 2,3-dioxygenase 1 (IDO-1) is one of the most important molecules in the formation of immunosuppressive tumor microenvironment (ITM). Inhibiting the activity and expression of IDO-1 can effectively activate the immune system to kill tumors. At present, a variety of chemotherapy IDO-1 immunotherapy nanomedicines have been designed and some progress has been made, but these combined therapy nanomedicines usually use encapsulation or chemical covalent connection to carry chemotherapy drugs and IDO-1 inhibitors, which can easily cause problems such as low drug loading, premature drug leakage and complex chemical synthesis, which seriously hinders the clinical transformation of these drugs.

[0005] By using supramolecular strategies, the above challenges faced by combination therapy nanomedicines can be effectively overcome. Cyclodextrin (CD), especially β-cyclodextrin (β-CD), has become the most attractive and suitable host for supramolecular nanomedicines in aqueous solution because of its strong binding affinity and good biocompatibility for a variety of different guests and anticancer drugs. Therefore, we can use supramolecular strategies to construct supramolecular nanomedicines that can not only efficiently carry chemotherapeutic drugs but also achieve selective drug release in the tumor microenvironment, so as to enhance the efficiency of cancer chemotherapy IDO-1 immunotherapy. Summary of the invention

[0006] In order to solve the defects of the prior art in cancer treatment, such as strong toxicity of chemotherapy system, easy tumor recurrence, lack of systemic immunity, etc., the present invention provides a novel method for preparing and synthesizing the anticancer chemotherapy drug DACHPt-PB-CD-IDOi and its intermediates.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The present invention discloses a complex DACHPt-PB-CD-IDOi as shown in formula D:

[0009]

[0010] The present invention also provides a method for preparing the complex DACHPt-PB-CD-IDOi, which comprises:

[0011] The compound PEG-BLG-CD of formula A and the compound DACHPt of formula B are uniformly dispersed in a solvent to obtain a mixed solution, and the mixed solution and the compound IDOi of formula C are post-treated to obtain the complex DACHPt-PB-CD-IDOi, wherein the molar ratio of the compound PEG-BLG-CD of formula A, the compound DACHPt of formula B, and the compound IDOi of formula C is 1:2-4:2-4 (preferably 1:2:2);

[0012]

[0013] In formula A, m is 100-10000 (preferably 2000), n is 1-14 (preferably 7), and o is 1-14 (preferably 7).

[0014] Furthermore, the solvent is dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide or water.

[0015] Furthermore, the volume of the solvent is 10-25 mL / g based on the total mass of the compound of formula A PEG-BLG-CD, the compound of formula B DACHPt and the compound of formula C IDOi.

[0016] Furthermore, the post-treatment H is: slowly adding the compound represented by the formula C to the mixed solution, and freeze-drying to obtain the complex DACHPt-PB-CD-IDOi.

[0017] The present invention also provides an intermediate for preparing the complex DACHPt-PB-CD-IDOi, such as a compound PEG-BLG-CD shown in formula A:

[0018]

[0019] Further, the compound represented by formula A is synthesized as follows:

[0020] (1) Preparation of OTs-CD: β-cyclodextrin and water are mixed to obtain a cyclodextrin solution, and an aqueous NaOH solution and an acetonitrile solution of toluenesulfonyl chloride are sequentially added dropwise to the cyclodextrin solution to obtain a mixed solution, the mixed solution is stirred at room temperature for 2-5 hours, the filtrate is collected by filtration, the pH of the filtrate is adjusted to 7-8 (with a saturated ammonium chloride solution), and the precipitate is collected by centrifugation. The obtained precipitate is subjected to post-treatment A to obtain the compound OTs-CD represented by formula 1; the molar ratio of NaOH contained in the β-cyclodextrin and the aqueous NaOH solution to the toluenesulfonyl chloride contained in the acetonitrile solution of toluenesulfonyl chloride is 1:1-10:1-5 (preferably 1:3.4:1.1);

[0021]

[0022] (2) NH 2 Preparation of OTs-CD: The compound OTs-CD described in step (1) is reacted with ethylenediamine at 50-90°C for 2-8h (preferably at 75°C for 4h), and the resulting reaction solution B is subjected to post-treatment B to obtain the compound NH 2 -CD; the volume of the ethylenediamine is 2-10mL / g (preferably 5mL / g) based on the mass of the compound OTs-CD;

[0023]

[0024] (3) Preparation of PEG-BLG: Compound BLG represented by Formula 3 and compound PEG-NH represented by Formula 4 2 The mixture is dissolved in anhydrous tetrahydrofuran, and then bis(trimethylsilyl) lithium amide (LiHMDS) is slowly added thereto, stirred at room temperature for 10-60 min (preferably 30 min), and then formic acid (FA) is added dropwise to quench, and the resulting solution is washed with cold acetone C, and dried to obtain a compound PEG-BLG shown in formula 5, wherein the compound BLG and the compound PEG-NH 2 The amount of substance ratio is 30-10:1 (preferably 15:1).

[0025]

[0026] (4) Preparation of PEG-BLG-COOH: The compound PEG-BLG described in step (3) is dissolved in dichloroacetic acid, a mixture of hydrobromic acid and acetic acid is added thereto, and the mixture is slowly stirred at 25-35° C. for 50-70 min (preferably 60 min) to obtain a product, and the product is washed with cold ether A, and dried to obtain the compound PEG-BLG-COOH represented by formula 6.

[0027]

[0028] (5) Preparation of PEG-BLG-CD: The PEG-BLG-COOH obtained in step (4) was dissolved in N,N-dimethylformamide A, and N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and DMAP were added thereto in sequence, and stirred at room temperature for 12-24 h (preferably 18 h) to obtain a product solution, and the product solution was dropped into diethyl ether B for washing, and vacuum dried to obtain a carboxyl-activated polymer; the carboxyl-activated polymer and the compound CD-NH obtained in step 2 were mixed. 2 The mixture is dissolved in N,N-dimethylformamide B, triethylamine is added thereto, and the mixture is reacted at room temperature. After the reaction is completed, a product solution is obtained, and the product solution is dropped into acetone D for precipitation. The precipitate is washed with ether C and then dried in vacuo to obtain a compound PEG-BLG-CD represented by formula A; the compound PEG-BLG-COOH, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and the compound NH 2 -The molar ratio of CD to triethylamine is 1:1-5:1-5:1-3:5-10:0.5-1 (preferably 1:2:3:1.6:6.25:0.6)

[0029]

[0030] Furthermore, the post-treatment A in step (1) is: washing the precipitate with water and acetone in sequence and then drying it in vacuum to obtain the compound shown in formula 1;

[0031] The post-treatment B in step (2) is: cooling the reaction solution B to room temperature, adding acetone A (the volume of acetone A is 10-100 mL / g, preferably 20 mL / g, based on the mass of the compound 1) for precipitation, centrifuging, dissolving the resulting precipitate in water, and precipitating again with acetone B (the volume of acetone B is 20-100 mL / g, preferably 20 mL / g, based on the mass of the compound 1), centrifuging, and vacuum drying the resulting precipitate to obtain compound NH 2 -CD. (The acetone A and acetone B both refer to acetone, and A and B distinguish the acetone used in different reaction steps).

[0032] Further, the volume of the water in step (1) is 5-10 mL / mmol (preferably 8.6 mL / mmol) based on the amount of the β-cyclodextrin;

[0033] The NaOH aqueous solution in step (1) is obtained by dissolving NaOH in water A, wherein the volume of water A is 0.1-0.2 mL / mmol (preferably 0.18 mL / mmol) based on the amount of NaOH substance;

[0034] The acetonitrile solution of toluenesulfonyl chloride in step (1) is obtained by dissolving toluenesulfonyl chloride in acetonitrile, wherein the volume of acetonitrile is 0.1-0.3 mL / mmol (preferably 0.284 mL / mmol) based on the amount of toluenesulfonyl chloride;

[0035] The volume of tetrahydrofuran in step (3) is 50-200 mL / g (preferably 100 mL / g) based on the mass of compound BLG;

[0036] The amount of lithium bis(trimethylsilyl)amide in step (3) is 2.5-10 mL / g (preferably 10 mL / g) based on the mass of compound BLG.

[0037] The temperature of the cold acetone C in step (3) is 5°C-20°C, and the amount used for each washing is 500-2000mL / g (preferably 1000mL / g) based on the mass of compound BLG.

[0038] The volume of dichloroacetic acid in step (4) is 40-160 mL / g (preferably 100 mL / g) based on the mass of the compound PEG-BLG.

[0039] The mass ratio of the mixed solution of hydrobromic acid and acetic acid in step (4) is 3:7, and the volume of the aqueous hydrobromic acid solution or the aqueous acetic acid solution is 5-50 mL / g (preferably 25 mL / g) based on the mass of the compound PEG-BLG.

[0040] The temperature of the cold ether A in step (4) is 5-20°C. The amount of the cold ether A used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG.

[0041] The volume of N,N-dimethylformamide A in step (5) is 50-200 mL / g (preferably 100 mL / g) based on the mass of the compound PEG-BLG-COOH.

[0042] The volume of N,N-dimethylformamide B in step (5) is 100-1000 mL / g (preferably 125 mL / g) based on the mass of the compound PEG-BLG-COOH.

[0043] The temperature of the ether B in step (5) is 5-20° C., and the amount used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG-COOH.

[0044] The temperature of acetone D in step (5) is 5-20° C., and the amount used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG-COOH.

[0045] The temperature of the ether C in step (5) is 5-20° C. The amount of the ether C used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG-COOH.

[0046] The present invention also provides an application of the complex DACHPt-PB-CD-IDOi in preparing anti-tumor supramolecular nanomedicine.

[0047] Preferably, the tumor is a CT26 tumor.

[0048] Acetone A, B, C, D and ether A, B, C are only for the purpose of distinguishing acetone and ether added at different stages, for the convenience of description, and have no other special meanings. N,N-dimethylformamide A and B are only for the purpose of distinguishing N,N-dimethylformamide added at different stages, for the convenience of description, and have no other special meanings. Water A is also only for the convenience of description, and has no special meaning.

[0049] Compared with the prior art, the beneficial effect of the present invention is that the present invention designs and synthesizes a supramolecular nanodrug DACHPt-PB-CD-IDOi that can regulate the tumor microenvironment and enhance tumor chemoimmunotherapy. In the supramolecular nanodrug system, DACHPt is effectively loaded into nanoparticles with the help of metal coordination, implements chemotherapy and activates anti-tumor immune response. At the same time, IDOi encapsulated by host-guest complexation inhibits the catabolism of tryptophan and kynurenine, synergistically transforms "cold" tumors into "hot" tumors, thereby enhancing chemoimmunotherapy. This supramolecular combination of IDOi and DACHPt has a powerful remodeling of the immunosuppressive tumor microenvironment and has achieved significant anti-tumor effects in vitro and in vivo, which promotes dendritic cell maturation and the recruitment of T lymphocytes in the tumor site. In vitro cytological experiments and in vivo mouse experiments have proved that the constructed supramolecular nanodrug not only has low systemic toxicity and good biocompatibility, but also can effectively inhibit tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Example 1 The intermediate chemical compound OTs-CD prepared 1 H NMR spectra.

[0051] Figure 2 The intermediate compound NH obtained in Example 2 2 -CD's 1 H NMR spectra.

[0052] Figure 3 (a) is the intermediate compound PEG-BLG prepared in Example 3 1 H NMR; FIG3( b ) is a graph showing the molecular weight of PEG-BLG measured by GPC at different ratios of BLG in Example 4.

[0053] Figure 4 The intermediate compound PEG-BLG-COOH obtained in Example 5 1 H NMR spectra.

[0054] Figure 5 (a) is a graph showing the PEG-BLG-CD prepared in Example 6. 1 H NMR diagram; Figure 5(b) is the PEG-BLG-CD prepared in Example 7 at different ratios of CD-NH 2 The molecular weight graph measured by GPC is shown below.

[0055] Figure 6 Example 8 (a) is the DLS image of PEG-BLG-CD:IDOi at a ratio of 1:2; (b) is the DLS image of PEG-BLG-CD:IDOi at a ratio of 1:4; (c) is the TEM image of PEG-BLG-CD:IDOi at a ratio of 1:2; (d) is the TEM image of PEG-BLG-CD:IDOi at a ratio of 1:4.

[0056] Figure 7 is the β-CD, IDOi and NMR spectrum of .

[0057] Figure 8 This is a graph of cell viability of CT26 cells incubated with different concentrations of IDOi, DACHPt, β-CD+IDOi+DACHPt, IDOi+DACHP, and DACHPt-PB-CD-IDOi in Example 10 for 24 hours.

[0058] Figure 9 This is a graph showing the changes in the ratio of tryptophan to kynurenine in cells of different treatment groups in Example 11.

[0059] Figure 10 These are (a) tumor photos of different treatment groups in Example 12; (b) tumor volume change diagram of mice in different treatment groups; and (c) weight change trend diagram of mice in different treatment groups.

[0060] Figure 11This is a flow cytometry study of immune cells in mice in different treatment groups in Example 13. DETAILED DESCRIPTION

[0061] For ease of understanding, the present invention will be described in detail below through specific embodiments. It should be particularly noted that the specific embodiments are only for illustration, and it is obvious that a person skilled in the art can make various modifications to the present invention within the scope of the present invention based on the description herein.

[0062] Example 1: Preparation of intermediate OTs-CD

[0063] First, 40g (32.6mmol) of β-CD (β-cyclodextrin) was added to 280mL of water to obtain a turbid cyclodextrin solution. Then, 4.32g (109.4mmol) of NaOH was dissolved in 20mL of water to obtain an aqueous NaOH solution, which was slowly added dropwise to the cyclodextrin solution. After the addition, the solution was light yellow and clear. Then, 6.72g (35.2mmol) of p-toluenesulfonyl chloride was dissolved in 10mL of acetonitrile to obtain an acetonitrile solution of toluenesulfonyl chloride, which was slowly added to the above mixed solution. The toluenesulfonyl chloride solution was added dropwise to produce a precipitate immediately. After the addition was completed, the mixed solution was stirred at room temperature for 3h. The filtrate was collected and a saturated ammonium chloride solution was continuously added thereto. A precipitate was precipitated and the solution was adjusted to pH = 7. The precipitate was collected by centrifugation. The precipitate was washed once with water and twice with acetone and then vacuum dried to obtain a solid product OTs-CD4.2 g with a yield of 10%.

[0064] Figure 1 This is the 1H NMR spectrum of the intermediate OTs-CD, which proves that the compound was successfully prepared.

[0065] Example 2: Intermediate NH 2 -CD preparation

[0066] 2.0 g OTs-CD and excess EDA (10 mL) were reacted at 75 ° C for 4 h. After the reaction, the obtained mixture (reaction solution B) was cooled to room temperature and 40 mL acetone was added for precipitation. The precipitate was centrifuged (8000 r, 3 min) and dissolved in 5 mL water. The precipitate was precipitated again with 40 mL acetone, centrifuged (8000 r 3 min) and dried in vacuum at 50 ° C to obtain the product NH 2 -CD 1.8 g, yield 90%.

[0067] Figure 2 It is the intermediate NH 2 The 1H NMR pattern of -CD proved that the compound was successfully prepared.

[0068] Example 3: Preparation of PEG-BLG

[0069] BLG (0.39 g, 1.5 mmol) and compound PEG-NH 2 (0.20 g, 0.1 mmol) was dissolved in 39 mL of anhydrous tetrahydrofuran (THF), and then 0.1 M lithium bis(trimethylsilyl)amide (LiHMDS) (3.9 mL) was slowly added, stirred at room temperature for 30 min, and then quenched by adding 0.2 mL of formic acid (FA) dropwise. The resulting solution was poured into 400 mL of cold acetone and washed twice, and the precipitate was collected by centrifugation to obtain the product PEG-BLG, with a yield of 95%.

[0070] Figure 3a This is the 1H NMR graph of PEG-BLG, which proves that the compound was successfully prepared.

[0071] Example 4: Preparation of PEG-BLG

[0072] BLG (0.26 g, 1.0 mmol) and compound PEG-NH 2 (0.20 g, 0.1 mmol) was dissolved in 26 mL of anhydrous tetrahydrofuran (THF), and then 0.1 M lithium bis(trimethylsilyl)amide (LiHMDS) (2.6 mL) was slowly added, stirred at room temperature for 30 min, and then quenched by adding 0.13 mL of formic acid (FA) dropwise. The resulting solution was poured into 270 mL of cold acetone and washed twice, and the precipitate was collected by centrifugation to obtain the product PEG-BLG with PEG:BLG=1:10.

[0073] BLG (0.78 g, 3.0 mmol) and compound PEG-NH 2 (0.20 g, 0.1 mmol) was dissolved in 78 mL of anhydrous tetrahydrofuran (THF), and then 0.1 M lithium bis(trimethylsilyl)amide (LiHMDS) (7.8 mL) was slowly added, stirred at room temperature for 30 min, and then quenched by adding 0.4 mL of formic acid (FA). The resulting solution was poured into 800 mL of cold acetone and washed twice, and the precipitate was collected by centrifugation to obtain the product PEG-BLG with PEG:BLG=1:30.

[0074] Figure 3b This is the molecular weight diagram of PEG-BLG measured by GPC at different BLG ratios. As the amount of BLG increases, the molecular weight of the polymer also increases accordingly, proving that the polymerization of the block polymer is feasible.

[0075] Example 5: Preparation of PEG-BLG-COOH

[0076] PEG-BLG (5 g) was dissolved in 500.0 mL of dichloroacetic acid, and 125.0 mL of a mixture of hydrobromic acid and acetic acid (the mass ratio of hydrobromic acid to acetic acid was 3:7) was added, and the mixture was slowly stirred at 30°C for 50-70 minutes to obtain the product. The product was poured into 5000 mL of cold ether and washed twice, and the precipitate was collected by centrifugation to obtain the final product PEG-BLG-COOH, with a yield of 89.8%.

[0077] Figure 4 This is the 1H NMR graph of PEG-BLG-COOH, which proves that the compound was successfully prepared.

[0078] Example 6: Preparation of PEG-BLG-CD

[0079] 50 mg (0.05 mmol) of PEG-BLG-COOH (PEG-BLG-COOH reference Triggered In Situ Dimorphic Transformation of BF 2 -Azadipyrromethene Nanoaggregates for Enhanced Solid Tumor Penetration.ACS Nano.2020Mar 24;14(3):3640-3650) was dissolved in 6.25mL DMF (N,N-dimethylformamide A), followed by the addition of 11.5mg NHS (N-hydroxysuccinimide), 29.6mg EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 10mg DMAP (4-dimethylaminopyridine). The reaction was stirred at room temperature for 24h to obtain a product solution, which was then dropped into 50mL of ether for washing. The washed solid was vacuum dried to obtain a carboxyl-activated polymer. The carboxyl-activated polymer and 385mg (0.34mmol) CD-NH 2 Dissolve in 10 mL N,N-dimethylformamide (DMF), add 1.5 μL triethylamine, and react at room temperature. After the reaction is completed, drop the product solution into 50 mL acetone for precipitation and centrifuge. The precipitate is washed three times with 50 mL of ether and vacuum dried to obtain PEG-BLG-CD with a yield of 89.8%.

[0080] FIG5 is a 1H NMR graph of PEG-BLG-CD, which proves that the compound was successfully prepared.

[0081] Example 7: Preparation of PEG-BLG-CD

[0082] 50 mg (0.34 mmol) of PEG-BLG-COOH was dissolved in 6.25 mL of DMF (N,N-dimethylformamide A), followed by the addition of 11.5 mg of NHS (N-hydroxysuccinimide), 29.6 mg of EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg of DMAP (4-dimethylaminopyridine). The reaction was stirred at room temperature for 24 h to obtain a product solution, which was then dropped into 50 mL of ether for washing. The washed solid was vacuum dried to obtain a carboxyl-activated polymer. The carboxyl-activated polymer and 308 mg (0.27 mmol) of CD-NH 2 Dissolve in 10 mL N,N-dimethylformamide (DMF), add 1.5 μL triethylamine, and react at room temperature. After the reaction is completed, drop the product solution into 50 mL acetone for precipitation and centrifuge. Wash the precipitate three times with 50 mL of ether and vacuum dry to obtain the product PEG-BLG:CD=1:5.

[0083] 50 mg (0.34 mmol) of PEG-BLG-COOH was dissolved in 6.25 mL of DMF (N,N-dimethylformamide A), followed by the addition of 11.5 mg of NHS (N-hydroxysuccinimide), 29.6 mg of EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 10 mg of DMAP (4-dimethylaminopyridine). The reaction was stirred at room temperature for 24 h to obtain a product solution, which was then dropped into 50 mL of ether for washing. The washed solid was vacuum dried to obtain a carboxyl-activated polymer. The carboxyl-activated polymer and 3.08 g (2.72 mmol) of CD-NH 2 Dissolve in 10 mL N,N-dimethylformamide (DMF), add 1.5 μL triethylamine, and react at room temperature. After the reaction is completed, drop the product solution into 50 mL acetone for precipitation and centrifuge. Wash the precipitate three times with 50 mL of ether and vacuum dry to obtain PEG-BLG:CD-NH 2 =1:10 product PEG-BLG-CD.

[0084] Figure 5b PEG-BLG-CD at different ratios of CD-NH 2 The molecular weight diagram measured by GPC is shown below. 2 As the amount of 2 Can be grafted into polymers.

[0085] Example 8: Preparation and size characterization of DACHPt-PB-CD-IDOi nanodrug

[0086] 40 mg PEG-PLG-CD (0.01 mmol) was dissolved in 0.4 mL N,N-dimethylformamide DMF and ultrasonically treated for 60 min to obtain a PEG-BLG-CD solution. 12 mg (0.02 mmol) dichloro(1,2-diaminocyclohexane) platinum (II) (DACHPt.) was dissolved in 0.2 mL DMF, and the resulting solution was added to the PEG-BLG-CD solution to obtain a mixed solution. 7 mg (0.02 mmol) IDO-1 inhibitor (IDOi) was dissolved in 0.1 mL DMF and slowly added to the above mixed solution, and then 5 mL deionized water was slowly added to the solution, and the mixture was ultrasonically treated for another 60 min, and then stirred at 50 ° C for 48 h. After freeze-drying, the supramolecular nanodrug DACHPt-PB-CD-IDOi (A) was obtained.

[0087] 40 mg PEG-PLG-CD (0.01 mmol) was dissolved in 0.4 mL N,N-dimethylformamide DMF and ultrasonically treated for 60 min to obtain a PEG-BLG-CD solution. 12 mg (0.02 mmol) dichloro(1,2-diaminocyclohexane) platinum (II) (DACHPt.) was dissolved in 0.0.2 mL DMF, and the resulting solution was added to the PEG-BLG-CD solution to obtain a mixed solution. 14 mg (0.04 mmol) IDO-1 inhibitor (IDOi) was dissolved in 0.2 mL DMF and slowly added to the above mixed solution, and then 5 mL of deionized water was slowly added to the solution, and the mixture was ultrasonically treated for another 60 min, and then stirred at 50 ° C for 48 h. After freeze-drying, the supramolecular nanodrug DACHPt-PB-CD-IDOi (B) was obtained.

[0088] 5 mg of DACHPt-PB-CD-IDOi drug powder A and B were weighed and dissolved in 10 mL of deionized water. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were then performed to characterize the size of the material. Figure 6 As shown, Figure 6 a, 6b are the DLS images of PEG-BLG-CD:IDOi of 1:2 and 1:4, Figure 6c, 6d are TEM images of PEG-BLG-CD:IDOi of 1:2 and 1:4, respectively. It can be seen from the figure that DACHPt-PB-CD-IDOi can self-assemble into nanoparticles with a particle size of about 100nm in aqueous solution. And as the content of IDOi increases, the particle size will increase slightly, but it is still around 100nm, which is consistent with the TEM structure. Therefore, it can be proved that DACHPt-PB-CD-IDOi forms nanoparticles of about 100nm, and nanoparticles of this size can be gathered at the tumor site through the EPR effect (hypertonic retention effect), thereby improving the therapeutic efficiency of nanomedicines.

[0089] Example 9: Study on the complexation of β-CD and IDOi

[0090] In order to study the host-guest interaction between β-CD and IDOi, β-CD, IDOi and Conducted 1 H NMR spectroscopy analysis, such as Figure 7 As shown, when 10 mg (0.01 mmol) of β-CD and 4 mg (0.01 mmol) of IDO in a 1:1 molar ratio were complexed in 1 mL of deuterated DMSO, the proton signals on IDOi and β-CD both underwent obvious chemical shift changes due to the formation of the inclusion complex, providing strong evidence for the host-guest complexation between β-CD and IDOi.

[0091] Example 10: Cell viability assay

[0092] CT26 (mouse breast cancer cells) were inoculated in 96-well culture plates under 5% CO 2 After culturing in a 37°C incubator for 24 h, 100 μL of IDOi, DACHPt, β-CD+IDOi+DACHPt, IDOi+DACHP, and DACHPt-PB-CD-IDOi (0, 1.125, 2.25, 4.5, 9, 18, 36 μM) were added to each well, and after culturing for 24 h, 50 μL of MTT was added to each well. The cells were then incubated in 5% CO. 2 , continue to culture in a 37°C incubator for 4 h, discard the culture medium, add 150 μL of DMSO, shake well on a plate shaker, read the plate at 495 nm with an enzyme reader, and calculate the cell inhibition rate based on the measured absorbance value. Figure 8 As shown in Figure 2, DACHPt-PB-CD-IDOi exhibited concentration-dependent cytotoxicity, with the maximum inhibitory concentration (IC 50) was 3.49μg / mL, which was much lower than DACHPt (6.68μg / mL), IDOi (21.0μg / mL) and IDOi@DACHPt (4.69μg / mL). This indicates that DACHPt-PB-CD-IDOi has a good effect in inhibiting the growth of tumor cells.

[0093] Example 11: Changes in the ratio of tryptophan to kynurenine in cells of different treatment groups.

[0094] CT26 (mouse breast cancer cells) were inoculated in 96-well culture plates under 5% CO 2 , and cultured in a 37°C incubator for 24 hours. 100 μL of each of IDOi, DACHPt, β-CD+IDOi+DACHPt, IDOi+DACHP, and DACHPt-PB-CD-IDOi (0, 1.125, 2.25, 4.5, 9, 18, 36 μM) was added to each well, and recombinant mouse IFN-γ (100 ng / mL) was added to each well and incubated with the cells for 24 hours. The supernatant of each well was transferred to a new 96-well plate, and acetonitrile / water (v / v=8 / 92, pH=3.6) was used as the mobile phase. The tryptophan content was determined by high performance liquid chromatography, and the tryptophan content was determined by a microplate reader (490 nm). The inhibitory effect of SNPs (DACHPt-PB-CD-IDOi) on the IDO-1 pathway was detected on CT26 cells by high performance liquid chromatography and colorimetry. The results are shown in Figure 9 As shown, SNPs significantly reduced the ratio of Kyn to Trp compared with other groups, indicating that the inhibitory ability of IDOi was not weakened after loading with SNPs.

[0095] Example 12: In vivo anti-tumor evaluation of DACHPt-PB-CD-IDOi

[0096] After CT26 tumors were implanted in the abdomen of Balb / c mice (the tumor size grew to 100 mm 3 The mice were randomly divided into 5 groups: PBS group, IDOi group, DACHPt group, IDOi@DACHP group, and SNPs (DACHPt-PB-CD-IDOi) for anti-tumor treatment study (the drug concentration was 200 μM, and the mice were injected with drugs through the tail vein, once every 3 days, for 3 times. Figure 10 As shown in c, during the treatment period, there was no significant change in the body weight of mice, indicating that DACHPt-PB-CD-IDOi had little systemic toxicity. Figure 10As shown in a and b, the tumor volume of mice in the DACHPt-PB-CD-IDOi group was significantly reduced, the tumor in the DACHPt-PB-CD-IDOi group did not increase significantly, and the tumor in the PBS group increased significantly, indicating that the photothermal-chemotherapy synergistic effect of DACHPt-PB-CD-IDOi can effectively inhibit tumor growth. After the treatment, the mice were dissected and their hearts, livers, spleens, lungs, kidneys and tumors were weighed. Figure 10 As shown in c, the tumor in the treatment group was significantly smaller than that in the control group. It is worth noting that the spleen in the treatment group was also significantly smaller than that in the control group. It is speculated that the reason may be that the immune system of the untreated control group mice was disordered, resulting in spleen enlargement.

[0097] Example 13: Study on immune cells of mice in different treatment groups

[0098] In order to evaluate the effect of DACHPt-PB-CD-IDOi on the immune system of mice. In implementation case 12, after the mice were killed, their tumors were removed and lymph node cells were studied. The tumor was homogenized in PBS to obtain a single cell suspension, and the cell suspension was stained with anti-CD3ε-PE, anti-CD4-FITC, anti-CD8a-PE-Cy7, anti-CD25-APC and anti-Foxp3-PE, and analyzed by flow cytometry. The mouse lymph nodes were collected, homogenized with PBS, filtered to obtain a single cell suspension, and the lymph node cell suspension was stained with anti-CD11c-FITC, anti-CD80-PE, and anti-CD86-APC antibodies, and analyzed by flow cytometry. Figure 11 As shown in the flow cytometry, the CD3+, CD4+, and CD3+, CD8+ infiltration rates of the SNPs group were much higher than those of the other groups, indicating that the SNPs group activated T cells in mice. In tumor-draining lymph nodes (TDLNs). The high expression of CD80+ and CD86+ on DC cells after SNPs treatment indicated that this treatment promoted the maturation of DC cells, thereby enhancing the immunogenicity of tumors. In addition, CD4+ helper T cells can differentiate not only into effective T cells (CD3+CD4+Foxp3-), but also into immunosuppressive Treg cells (CD3+CD4+Foxp3+). Compared with the PBS group, the number of Tregs in the SNPs group decreased by 65%, which was also much lower than that in the IDOi, DACHPt, and IDOi@DACHPt groups, indicating that SNPs effectively reprogrammed the immunosuppressive tumor microenvironment (ITM) and improved the response rate to immunotherapy.

Claims

1. A complex DACHPt-PB-CD-IDOi as shown in formula D:

2. The method for preparing the complex DACHPt-PB-CD-IDOi according to claim 1, characterized in that The method is: The compound PEG-BLG-CD of formula A and the compound DACHPt of formula B are uniformly dispersed in a solvent to obtain a mixed solution, and the mixed solution and the compound IDOi of formula C are post-treated to obtain the complex DACHPt-PB-CD-IDOi, wherein the molar ratio of the compound PEG-BLG-CD of formula A, the compound DACHPt of formula B, and the compound IDOi of formula C is 1:2-4:2-4; In formula A, m is 100-10000, n is 1-14, and o is 1-14.

3. The method for preparing the complex DACHPt-PB-CD-IDOi according to claim 2, characterized in that: The solvent is dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide or water.

4. The method for preparing the complex DACHPt-PB-CD-IDOi according to claim 2, characterized in that: The volume of the solvent is 10-25 mL / g based on the total mass of the compound of formula A PEG-BLG-CD, the compound of formula B DACHPt and the compound of formula C IDOi.

5. The method for preparing the complex DACHPt-PB-CD-IDOi according to claim 2, characterized in that The post-treatment H is: slowly adding the compound represented by the formula C to the mixed solution, and freeze-drying to obtain the complex DACHPt-PB-CD-IDOi.

6. An intermediate for preparing the complex DACHPt-PB-CD-IDOi, such as the compound PEG-BLG-CD shown in formula A:

7. The compound of formula A as claimed in claim 6, characterized in that The compound represented by formula A is synthesized as follows: (1) Preparation of OTs-CD: β-cyclodextrin and water are mixed to obtain a cyclodextrin solution, and a NaOH aqueous solution and a methylbenzenesulfonyl chloride acetonitrile solution are sequentially added dropwise to the cyclodextrin solution to obtain a mixed solution, the mixed solution is stirred at room temperature for 2-5 hours, the filtrate is collected by filtration, the pH of the filtrate is adjusted to 7-8, and the precipitate is collected by centrifugation. The obtained precipitate is subjected to post-treatment A to obtain the compound OTs-CD represented by formula 1; the molar ratio of NaOH contained in the β-cyclodextrin and NaOH aqueous solution to the methylbenzenesulfonyl chloride contained in the acetonitrile solution is 1:1-10:1-5; (2) Preparation of NH2-CD: The compound OTs-CD described in step (1) is reacted with ethylenediamine at 50-90° C. for 2-8 hours, and the resulting reaction solution B is subjected to post-treatment B to obtain the compound NH2-CD as shown in Formula 2; the volume of the ethylenediamine is 2-10 mL / g based on the mass of the compound OTs-CD; (3) Preparation of PEG-BLG: The compound BLG represented by Formula 3 and the compound PEG-NH2 represented by Formula 4 are dissolved in anhydrous tetrahydrofuran, and then lithium bis(trimethylsilyl)amide is slowly added thereto, and stirred at room temperature for 10-60 minutes, followed by quenching by adding formic acid dropwise. The resulting solution is washed with cold acetone C, and dried to obtain the compound PEG-BLG represented by Formula 5, wherein the molar ratio of the compound BLG to the compound PEG-NH2 is 30-10:

1. (4) Preparation of PEG-BLG-COOH: The compound PEG-BLG described in step (3) is dissolved in dichloroacetic acid, a mixture of hydrobromic acid and acetic acid is added thereto, and the mixture is slowly stirred at 25-35° C. for 50-70 min to obtain a product. The product is washed with cold ether A, and dried to obtain the compound PEG-BLG-COOH represented by formula 6. (5) Preparation of PEG-BLG-CD: The PEG-BLG-COOH obtained in step (4) was dissolved in N,N-dimethylformamide A, and N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added thereto in sequence, and the mixture was stirred at room temperature for 12-24 hours to obtain a product solution, and the product solution was dropped into diethyl ether B for washing, and vacuum dried to obtain a carboxyl-activated polymer; the carboxyl-activated polymer and the compound CD-NH2 obtained in step 2 were dissolved in N,N-dimethylformamide A. triethylamine is added to amide B, and the mixture is reacted at room temperature. After the reaction is completed, a product solution is obtained, and the product solution is dropped into acetone D for precipitation. The precipitate is washed with ether C and then dried in vacuo to obtain a compound PEG-BLG-CD represented by formula A; the molar ratio of the compound PEG-BLG-COOH, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, the compound NH2-CD and triethylamine is 1:1-5:1-5:1-3:5-10:0.5-1.

8. The compound of formula A according to claim 7, characterized in that: The post-treatment A in step (1) is: washing the precipitate with water and acetone in sequence and then drying it in vacuum to obtain the compound shown in formula 1; The post-treatment B in step (2) is: cooling the reaction solution B to room temperature, adding acetone A for precipitation, centrifuging, dissolving the obtained precipitate in water, precipitating again with acetone B, centrifuging, and vacuum drying the obtained precipitate to obtain compound NH2-CD.

9. The compound as shown in formula A according to claim 7, characterized in that The volume of the water in step (1) is 5-10 mL / mmol based on the amount of the β-cyclodextrin; The NaOH aqueous solution in step (1) is obtained by dissolving NaOH in water A, wherein the volume of water A is 0.1-0.2 mL / mmol based on the amount of NaOH substance; The acetonitrile solution of toluenesulfonyl chloride in step (1) is obtained by dissolving toluenesulfonyl chloride in acetonitrile, wherein the volume of the acetonitrile is 0.1-0.3 mL / mmol based on the amount of toluenesulfonyl chloride; The volume of tetrahydrofuran in step (3) is 50-200 mL / g based on the mass of compound BLG; The amount of lithium bis(trimethylsilyl)amide in step (3) is 2.5-10 mL / g based on the mass of compound BLG. The temperature of the cold acetone C in step (3) is 5°C-20°C, and the amount used for each washing is 500-2000mL / g based on the mass of compound BLG. The volume of dichloroacetic acid in step (4) is 40-160 mL / g based on the mass of the compound PEG-BLG. The mass ratio of the hydrobromic acid and acetic acid mixed solution in step (4) is 3:7, and the volume of the hydrobromic acid aqueous solution or the acetic acid aqueous solution is 5-50 mL / g based on the mass of the compound PEG-BLG. The temperature of the cold ether A in step (4) is 5-20°C. The amount of the cold ether A used for each washing is 500-2000 mL / g based on the mass of the compound PEG-BLG. The volume of dimethyl N,N-formamide A in step (5) is 50-200 mL / g based on the mass of the compound PEG-BLG-COOH. The volume of dimethyl N,N-formamide B in step (5) is 100-1000 mL / g based on the mass of the compound PEG-BLG-COOH. The temperature of the ether B in step (5) is 5-20° C., and the amount used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG-COOH. The temperature of acetone D in step (5) is 5-20° C., and the amount used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG-COOH. The temperature of the ether C in step (5) is 5-20° C. The amount of the ether C used for each washing is 500-2000 mL / g (preferably 1000 mL / g) based on the mass of the compound PEG-BLG-COOH.

10. Use of the complex DACHPt-PB-CD-IDOi according to claim 1 in the preparation of anti-tumor supramolecular nanomedicine.

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