PD-L1 targeting small-molecule inhibitor, nano-particles prepared by combining PD-L1 targeting small-molecule inhibitor with photodynamic therapy, medicine and application of PD-L1 targeting small-molecule inhibitor and nano-particles

Abstract

The invention provides a PD-L1 targeting small-molecule inhibitor, nanoparticles prepared by combining the PD-L1 targeting small-molecule inhibitor with a photodynamic therapy, a medicine and application, and belongs to the technical field of biological medicine. The invention provides a PD-L1 targeting small molecule inhibitor, which is an alanine substituted isoindoline bitoluene derivative C2 as shown in a formula I which is described in the specification. Tests show that the alanine substituted isoindoline bitoluene derivative C2 can effectively inhibit PD-1 / PD-L1 binding and has certain anti-tumor activity, and nanoparticles of a nano delivery system prepared from the inhibitor and a photosensitizer have better anti-tumor (colon cancer) activity. The technical scheme of the invention provides a new thought for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small molecule inhibitor targeting PD-L1 and nanoparticles, drugs and applications prepared by combining the inhibitor with photodynamic therapy. Background Art

[0002] Cancer immunotherapy has been widely studied. Immunotherapy aims to restore the balance between the immune system and tumor cells by enhancing the immune system's recognition and elimination of cancer cells. One important approach is immune checkpoint inhibition. Immune checkpoint inhibitors competitively bind to checkpoint molecules to inhibit checkpoint-mediated immune system suppression. Therefore, immune checkpoint blockade therapy has become an emerging immunotherapy strategy for treating tumors. PD-1 / PD-L1 inhibition is the most successful form of immune checkpoint blockade therapy, achieving anti-tumor effects by blocking the interaction between PD-1 (programmed death receptor 1) and PD-L1 (programmed death ligand 1). Currently, the PD-L1 inhibitors used in clinical practice are mainly antibodies, which have been approved for the treatment of various hematological malignancies and solid tumors. However, despite the widespread use of therapeutic antibodies in clinical practice, antibodies still have drawbacks such as high production costs and poor tumor tissue penetration.

[0003] In addition, the use of immunotherapy alone has limitations. Although immune checkpoint blockade therapy has high efficiency and long-lasting tumor killing effects, many cancer patients are not sensitive to immune checkpoint inhibitors due to low tumor immunogenicity, and its efficacy is largely limited by low response rates. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a small molecule inhibitor targeting PD-L1, which has good anti-tumor activity by blocking the PD-1 / PD-L1 pathway.

[0005] The present invention also aims to provide a new type of nanoparticle, which is a nanoparticle prepared by combining an alanine-substituted isoindoline dimethylbenzene derivative C2 as a PD-L1 small molecule inhibitor with a photosensitizer Ce6, and exerts an anti-tumor effect based on photodynamic therapy and immunotherapy.

[0006] The present invention provides a small molecule inhibitor targeting PD-L1, which is an alanine-substituted isoindoline-bimethylol derivative C2 represented by Formula I:

[0007]

[0008] The present invention provides a method for preparing the small molecule inhibitor targeting PD-L1, comprising the following steps:

[0009] Compound 1 having a structure shown in Formula II is subjected to a dehydration condensation reaction with Boc-alanine in the presence of a condensing agent, carbodiimide and 1-hydroxybenzotriazole, and the organic phase is collected to obtain a reaction product;

[0010] The reaction product is subjected to a Boc-deprotection reaction with trifluoroacetic acid, and the product is extracted to obtain a small molecule inhibitor targeting PD-L1;

[0011]

[0012] Preferably, the molar ratio of the compound 1, Boc-alanine, carbodiimide and 1-hydroxybenzotriazole is 1:(1.2-2):(1.2-2):(1.2-2).

[0013] Preferably, the method for collecting the organic phase is to extract the organic phase with NaHCO3, NaCl and KHSO4 in sequence.

[0014] Preferably, the method for extracting the product is to extract the organic phase with dichloromethane at a pH value of 7.8 to 8.2.

[0015] The present invention provides a nanoparticle based on a small molecule inhibitor targeting PD-L1 combined with photodynamic therapy, comprising a photosensitizer Ce6 and the small molecule inhibitor targeting PD-L1 or the small molecule inhibitor targeting PD-L1;

[0016] The molar ratio of the small molecule inhibitor targeting PD-L1 and the photosensitizer Ce6 is (2.8-3.2):1.

[0017] The present invention provides an anti-tumor drug, the active ingredient of which includes the small molecule inhibitor targeting PD-L1, the small molecule inhibitor targeting PD-L1 or the nanoparticle.

[0018] Preferably, it also includes pharmaceutically acceptable excipients;

[0019] The pharmaceutically acceptable excipient includes 1% by volume of ethanol solution.

[0020] Preferably, the drug concentration of the small molecule inhibitor targeting PD-L1 is 0.045 to 0.055 mg / mL.

[0021] The present invention provides the small molecule inhibitor targeting PD-L1, and the use of the small molecule inhibitor targeting PD-L1 or the nanoparticle in the preparation of a drug for treating colon cancer.

[0022] The present invention provides a small molecule inhibitor targeting PD-L1, which is an alanine-substituted isoindoline-dimethylbenzene derivative C2 having a formula I. Molecular docking simulation results show that C2 binds to PD-L1 and achieves anti-tumor activity by inhibiting the binding between PD-1 and PD-L1. Animal experiments of the present invention show that compared with the control group, the administration of the alanine-substituted isoindoline-dimethylbenzene derivative C2 can effectively inhibit tumor growth (reduce tumor volume and total mass). By detecting the content of cytokines and immune cells, it can be seen that C2 increases the proportion and activity of CD8 in the tumor immune microenvironment. At the same time, C2 has low cytotoxicity and good safety.

[0023] The present invention provides nanoparticles for combined photodynamic therapy with a small molecule inhibitor targeting PD-L1, comprising a photosensitizer Ce6 and the small molecule inhibitor targeting PD-L1 or the small molecule inhibitor targeting PD-L1; the molar ratio of the small molecule inhibitor targeting PD-L1 to the photosensitizer Ce6 is (2.8-3.2):1. The present invention combines the photosensitizer Ce6 with the small molecule inhibitor targeting PD-L1 to prepare nanoparticles, which are co-delivered to the tumor site. This not only achieves the effects of combined photodynamic therapy and immunotherapy, but also improves the hydrophobic properties of Ce6 and the small molecule PD-L1 inhibitor, addressing the issues of tumor accumulation and low bioavailability. Experiments have shown that the nanoparticles have excellent particle stability and nanometer-scale particle size, meeting the requirements for drug delivery. Furthermore, under the action of Ce6, they enhance the accumulation of the small molecule PD-L1 inhibitor at the tumor site, improving drug utilization while enhancing the tumor immune response and effectively clearing the tumor. In the examples of the present invention, drug administration experiments in tumor mouse models demonstrated that, compared with the C2 and Ce6 groups alone, the nanoparticles prepared by the present invention had excellent PD-L1 targeting in mice, more effectively inhibited the growth of MC38 tumor tissue, and increased the proportion and activity of CD8 in the tumor immune microenvironment. This suggests that the present invention provides a new approach to tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The synthetic route of inhibitor C2 is shown below; wherein a. DCC, 1-hydroxybenzotriazole (HOBt), anhydrous tetrahydrofuran, and Boc-alanine; b. dichloromethane and 20% trifluoroacetic acid;

[0025] Figure 2The docking interaction analysis between inhibitor C2 and PD-L1 protein (PDB ID: 5J89) and the HTRF results of compound C2; A is the interaction result between compound C2 and PD-L1 (PDB ID: 5J89), B is the interaction result between compound BMS202 and PD-L1 (PDB ID: 5J89), and C is the affinity curve of compound C2 targeting PD-L1;

[0026] Figure 3 The nano-property characterization results of C2-Ce6 NPs; A is the Tyndall effect diagram of C2-Ce6 NPs; B is the hydrated particle size result; C is the PDI and Zeta potential results; D is the transmission electron microscopy result;

[0027] Figure 4 7-day stability test results of C2-Ce6 NPs;

[0028] Figure 5 Figure 3 is the cytotoxicity results of C2-Ce6 NPs, C2 NPs, and Ce6 under illumination or non-illumination conditions; A is the cytotoxicity results of C2-Ce6 NPs, Ce6 alone, and C2 alone on MC38 cells measured by CCK-8 without laser irradiation; B is the cytotoxicity results of C2-Ce6 NPs, Ce6 alone, and C2 alone on MC38 cells measured by CCK-8 with laser irradiation; C is the relationship between the concentration of C2-Ce6 NPs and the antiproliferative effect of cells;

[0029] Figure 6 The results of in vivo antitumor activity determination of C2-Ce6 NPs are shown in Figure 1, where A is the animal experiment flow chart; B is the tumor volume photos of each group; C is the daily tumor volume monitoring results; D is the statistical analysis results of tumor weight; E is the tumor tissue apoptosis and HE staining results;

[0030] Figure 7 These are the results of ELISA determination of cytokine levels in mouse serum. A is the result of IL-6 content in mouse serum; B is the result of TNF-α content in mouse serum; C is the result of IFN-γ content in mouse serum. DETAILED DESCRIPTION

[0031] The present invention provides a small molecule inhibitor targeting PD-L1, which is an alanine-substituted isoindoline-bimethylol derivative C2 represented by Formula I:

[0032]

[0033] In the present invention, small molecule inhibitors targeting PD-L1 can effectively inhibit the binding of PD-1 and PD-L1 and have certain anti-tumor activity. In an embodiment of the present invention, molecular docking technology was used to simulate the binding of C2 to PD-L1. The results showed that the binding site of the pseudo compound C2 to PD-L1 was consistent with that of the positive control BMS202. Specifically, C2 had hydrogen bond interactions with the glutamine of the PD-L1 protein and π-π interactions with tyrosine. The docking interaction energy (cdocker interaction energy) was -57.342 kJ / mol. This experiment demonstrated that C2 may have the potential to inhibit PD-L1. The affinity of compound C2 for targeting PD-L1 was 43.65 nmol / L obtained through HTRF experiments. In another embodiment of the present invention, a tumor mouse model was used as the experimental subject and treated with C2. The results showed that compared with the blank control group, C2 significantly suppressed tumor volume and weight and effectively inhibited tumor growth. Therefore, the small molecule inhibitor targeting PD-L1 can be used as an active ingredient in the preparation of anti-tumor drugs.

[0034] The present invention provides a method for preparing the small molecule inhibitor targeting PD-L1, comprising the following steps:

[0035] Compound 1 represented by Formula II is subjected to a dehydration condensation reaction with Boc-alanine under the action of condensing agents, carbodiimide and 1-hydroxybenzotriazole, and the organic phase is collected to obtain a reaction product;

[0036] The reaction product is subjected to a Boc-deprotection reaction with trifluoroacetic acid, and the product is extracted to obtain a small molecule inhibitor targeting PD-L1;

[0037]

[0038] The present invention conducts a dehydration condensation reaction between the compound 1 represented by formula II and Boc-alanine under the action of condensing agents carbodiimide and 1-hydroxybenzotriazole, and collects the organic phase to obtain a reaction product.

[0039] In the present invention, the molar ratio of Compound 1, Boc-alanine, carbodiimide, and 1-hydroxybenzotriazole is 1:(1.2-2):(1.2-2):(1.2-2), can be 1:(1.4-1.8):(1.4-1.8):(1.4-1.8), or can be 1:(1.5-1.7):(1.5-1.7):(1.5-1.7). The pH value of the solution of Compound 1 is preferably neutral, for example, 6.8-7.2, or 7.0. The solvent for Compound 1 is preferably dichloromethane. The solvent for Boc-alanine is preferably anhydrous tetrahydrofuran. The temperature of the dehydration condensation reaction is preferably 18-25°C, or 20-23°C. The dehydration condensation reaction is preferably accompanied by stirring. The stirring speed is preferably 500-1000, or 700. The carbodiimide and 1-hydroxybenzotriazole are preferably mixed with a solution of Boc-alanine.

[0040] In the present invention, the method for collecting the organic phase is preferably to extract the organic phase with NaHCO3, NaCl, and KHSO4 in sequence. During the extraction process, the working concentrations of NaHCO3, NaCl, and KHSO4 are preferably 4.8% to 5.2%, and can be 5%. After the organic phase is extracted, the organic solvent in the organic phase is preferably removed by rotary evaporation to obtain a dry powder of the reaction product.

[0041] After obtaining the reaction product, the present invention performs a Boc protection deprotection reaction on the reaction product with trifluoroacetic acid, extracts the product, and obtains a small molecule inhibitor targeting PD-L1.

[0042] In the present invention, the reaction product is preferably dissolved before the Boc deprotection reaction. The solvent for the reaction product is preferably dichloromethane. The molar amount of compound 1, the volume of dichloromethane, and the volume ratio of trifluoroacetic acid are preferably 0.76 mmol:4 mL:1 mL. The temperature of the Boc deprotection reaction is preferably 18-25°C, and can be 20-23°C. The time of the Boc deprotection reaction is preferably 2.5-3.5 hours, and can be 3 hours.

[0043] In the present invention, the method for extracting the product is preferably to extract the organic phase with dichloromethane under the condition of pH 7.8 to 8.2 (8). The organic phase is a dichloromethane layer. After the extraction, the product is dried and filtered, and the obtained filtrate is purified. The drying is preferably carried out by drying the dichloromethane layer with anhydrous Na2SO4 to remove water. The filtering method is preferably suction filtration. The purification method is preferably column chromatography purification. The total yield of the small molecule inhibitor targeting PD-L1 prepared by the preparation method of the present invention is 50.7%.

[0044] In the present invention, nanoparticle stability testing was performed on the prepared small molecule inhibitor targeting PD-L1. The solvent type, solvent volume fraction, and concentration of the small molecule inhibitor targeting PD-L1 were optimized. The results showed that 1% by volume of ethanol can maintain good nanoparticle stability of the small molecule inhibitor targeting PD-L1. At the same time, the nanoparticle properties of the small molecule inhibitor targeting PD-L1 in an ethanol aqueous solution (C2 NPs) with a concentration of 0.05 mg / mL were the best. In another embodiment of the present invention, the PDI of the small molecule inhibitor targeting PD-L1 was 0.12-0.13, the potential was positive, specifically 28-29 mV, and the particle size was in the range of 120.1-157.3 nm.

[0045] The present invention provides nanoparticles (C2-Ce6 NPs) based on a small molecule inhibitor targeting PD-L1 combined with photodynamic therapy, comprising a photosensitizer Ce6 and the small molecule inhibitor targeting PD-L1 or the small molecule inhibitor targeting PD-L1; the molar ratio of the small molecule inhibitor targeting PD-L1 to the photosensitizer Ce6 is (2.8-3.2):1.

[0046] In the present invention, the molar ratio of the small molecule inhibitor targeting PD-L1 to the photosensitizer Ce6 is 3:1. Different molar ratios of the small molecule inhibitor targeting PD-L1 and the photosensitizer Ce6 affect the particle size and PDI of the nanoparticles. The results show that both too high (4:1) and too low (1:1 / 2:1) molar ratios tend to increase the particle size and PDI of the nanoparticles, thereby reducing the stability of the nanoparticles.

[0047] In the present invention, the particle size of the nanoparticles is 111.1 nm to 142.9 nm. The potential of the nanoparticles is -22 to -23 mV. The PDI of the nanoparticles is lower than that of C2, which may be due to the binding of Ce6 to the hydrophobic site of the small molecule PD-L1 inhibitor, thereby improving the stability of the nanoparticles. In the present invention, the ethanol-water solution of the nanoparticles exhibits a Tyndall effect, indicating that the nanoparticles have good water solubility. The 7-day stability test of the nanoparticles showed that both C2 NPs and C2-Ce6 NPs have good stability in aqueous solution.

[0048] In the present invention, the cytotoxicity of C2 NPs and C2-Ce6 NPs was evaluated. The results showed that in the absence of laser irradiation, the cell viability of the C2-Ce6 NPs group was greater than that of the Ce6 group or the C2 group, indicating that C2-Ce6 NPs can reduce dark toxicity to a certain extent and improve safety. 2) When irradiated for 3 to 6 minutes, free Ce6 showed no obvious PDT effect, while the C2-Ce6 NPs group showed a significant concentration-dependent anti-proliferative effect, IC 50 It is 230.3nmol / L.

[0049] The present invention provides an anti-tumor drug, the active ingredient of which includes the small molecule inhibitor targeting PD-L1, the small molecule inhibitor targeting PD-L1 or the nanoparticle.

[0050] In the present invention, the anti-tumor drug preferably further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient comprises a 1% by volume ethanol solution. The drug concentration of the small molecule inhibitor targeting PD-L1 is preferably 0.045 to 0.055 mg / mL, and may be 0.05 mg / mL. The present invention does not particularly limit the preparation method of the anti-tumor drug, and the preparation method of anti-tumor drugs well known in the art can be used.

[0051] The present invention provides the small molecule inhibitor targeting PD-L1, and the use of the small molecule inhibitor targeting PD-L1 or the nanoparticle in the preparation of a drug for treating colon cancer.

[0052] In the present invention, small molecule inhibitors targeting PD-L1 can effectively inhibit PD-1 / PD-L1 binding and have certain anti-tumor activity, and the nanoparticles have even better anti-tumor activity. In an example of the present invention, using a tumor mouse model induced by injection of MC38 cells, the nanoparticles significantly inhibited tumor growth compared to the positive control drug BMS-202.

[0053] The following examples describe in detail the nanoparticles of the present invention for combining a small molecule inhibitor targeting PD-L1 with photodynamic therapy, the drugs prepared therefrom, and their applications. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] Chemical Synthesis Method of Alanine-Substituted Isoindoline-Bis(methylbenzene) Derivative C2

[0056] according to Figure 1 The reaction formula for synthesizing C2 is as follows:

[0057] To 216 mg (0.76 mmol) of compound 1, 2 mL of N-methylmorpholine was added to adjust the pH to 7, and the mixture was stirred for 20 minutes to obtain liquid A. 172 mg (0.91 mmol) of Boc-L-Ala was dissolved in 4 mL of anhydrous tetrahydrofuran, followed by the addition of 123 mg (0.91 mmol) of HOBt. After stirring in an ice bath for 10 minutes, 187 mg (0.91 mmol) of DCC was added to obtain liquid B. Liquid A was added to liquid B, and the mixture was stirred at room temperature overnight. Upon completion of the reaction, the starting materials were essentially eliminated, as monitored by TLC. The combined organic phases were extracted with various 5% saline solutions (NaHCO3, NaCl, and KHSO4), then spin-dried and dissolved in 4 mL of dichloromethane. 1 mL of trifluoroacetic acid was then added to the solution for Boc-protection removal. The reaction was allowed to proceed at room temperature for 3 hours, with the starting materials essentially eliminated, as monitored by TLC. The pH value of the reaction solution was adjusted to 8 with saturated NaHCO3 solution under ice bath, and extracted with dichloromethane solvent. The combined dichloromethane layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated and purified by column chromatography to obtain 136.9 mg of compound C2 as a white solid. The total reaction yield was 50.7%.

[0058] The prepared alanine-substituted isoindoline-bimethylbenzene derivative C2 was detected by nuclear magnetic resonance, mass spectrometry and high performance liquid chromatography. The test results are as follows: 1 H NMR (300MHz, DMSO-d6) δ7.50-7.14(m,11H),5.04(d,J=14.4Hz,1H),4.86(d,J=14.4Hz,1H),4.74(d,J=16.2Hz,1H),4.66 (d,J=16.2Hz,1H),3.69(3.68)(q,J=6.6Hz,1H),3.28(s,2H),2.06(2.05)(s,3H),1.17(d,J=6.6Hz,3H); MS(ESI):exact mass calculated for C 24 H 24 N2O[M+H] + 357.1961, found 357.1965. Analyt. HPLC purity: 98.9%.

[0059] Example 2

[0060] Molecular docking test of the binding of compound C2 to PD-L1 protein

[0061] The co-crystal of PD-L1 and BMS202 (PDB ID: 5J89) was downloaded from the PDB library and imported into DiscoveryStudio software. The protein was prepared by hydrogenation and dehydration. The planar structure of compound C2 was copied into DS software, and the three-dimensional structure of the compound was calculated using the energy minimization function of the software. The binding site of the simulated compound C2 to PD-L1 was consistent with that of BMS202. The interaction between compound C2 and PD-L1 was calculated using the CDocker module.

[0062] See the results Figure 2 . The results showed that compound C2 had hydrogen bond interactions with glutamine of PD-L1 protein and π-π interactions with tyrosine, and the docking interaction energy (cdocker interaction energy) was -57.342 kJ / mol. Compound C2 may have the potential to inhibit PD-L1. The affinity of compound C2 targeting PD-L1 was 43.65 nmol / L obtained by HTRF experiment. Compound C2 can significantly inhibit the mutual binding of PD-1 / PD-L1 and can be used to prepare PD-1 / PD-L1 inhibitors, including its stereoisomers, pharmaceutically acceptable salts, prodrugs and hydrates or solvates and medically acceptable excipients. The anti-tumor effect of this inhibitor is significant.

[0063] Example 3

[0064] Screening of solvents and concentrations of compound C2

[0065] When screening solvents, the fixed concentration of compound C2 was 2.0 mg / mL and it was dissolved in different organic solvents (Table 1). 1 mL was added dropwise to 5 mL of water under vigorous stirring. The mixture was stirred for 5 minutes, and the organic solvent was evaporated to remove the organic solvent. The volume was made up to 5 mL with water, and the particle size was measured.

[0066] The results showed that ethanol was the best organic phase for preparing nanostructured systems (Table 1). As shown in Table 1, ethanol had the smallest PDI, with a particle size of 155.9 nm. This indicates that ethanol is a good organic phase for preparing nanostructured systems.

[0067] Table 1 Effect of solvent type on particle size and PDI of nano drug delivery system

[0068] Solvent type Methanol acetone Tetrahydrofuran ethanol Particle size / nm 115.2 163.3 170.8 155.8 PDI 0.237 0.196 0.162 0.118

[0069] When ethanol was used as the solvent and the final concentration of the fixed compound C2 was 0.05 mg / mL, ethanol solutions with different volume fractions were prepared (see Table 2).

[0070] The results showed that the nanostructured particles had the best properties when the ethanol content was 1% (Table 2).

[0071] Table 2 Effect of ethanol volume fraction on particle size and PDI of nano drug delivery system

[0072] Ethanol volume fraction Particle size / nm PDI 0.50％ 299.5±7.312 0.197±0.043 1％ 194.7±0.9849 0.131±0.005 1.50％ 264.1±4.073 0.178±0.031 2％ 227.6±4.026 0.335±0.030

[0073] The volume fraction of ethanol was fixed at 0.5%, ethanol solutions of compound C2 with different concentrations were prepared, and the stability was investigated (the particle size and PDI of the nanoparticles were measured).

[0074] The results showed that the nanostability was the best when the final concentration was 0.05 mg / mL (Table 3).

[0075] Table 3 Effect of C2 final concentration on particle size and PDI of nano drug delivery system

[0076]

[0077]

[0078] Example 4

[0079] A preparation method of nanoparticles (C2-Ce6NPs) based on targeted PD-L1 small molecule inhibitors combined with photodynamic therapy

[0080] Prepare a 5 mg / mL ethanol solution of C2 (14 mM) and an 8.4 mg / mL DMSO solution of Ce6 (14 mM) using 100 μL of organic solvent and 10 mL of water. Mix the two and sonicate for 5 minutes. Adjust the volume ratio of the two to create nanoparticles with varying molar ratios (see Table 4).

[0081] The results showed that the nanostructured products had the best properties when the final molar ratio of compound C2 (75 μL) to photosensitizer Ce6 (25 μL) was 3:1 (Table 4).

[0082] Table 4 Effect of the molar ratio of C2 to Ce6 on the particle size and PDI of the nanodrug delivery system

[0083] C2 to Ce6 molar ratio Particle size / nm PDI 1:1 693.7±22.49 0.477±0.104 2:1 294.9±0.4041 0.297±0.044 3:1 191.4±0.9074 0.091±0.008 4:1 745.0±121.2 0.683±0.182

[0084] C2 was prepared into a 14mmol / L ethanol solution, 100μL of C2 ethanol solution was taken, and 10mL of water was added thereto in batches, and each time it was sonicated until clear; C2 was prepared into a 14mmol / L ethanol solution and Ce6 was prepared into a 14mmol / L DMSO solution, 75μL of C2 ethanol solution and 25μL of Ce6 DMSO solution were taken, mixed and stirred at room temperature overnight, and water was added to the mixed solution in batches, a total of 10mL, and each time it was sonicated until clear. The molar ratio of C2 to Ce6 in C2-Ce6 NPs is 3:1. The prepared nanosolution exhibits Tyndall effect ( Figure 3 Middle A).

[0085] The particle sizes of C2 NPs and C2-Ce6 NPs were determined to be 194.7±0.98nm and 191.4±0.91nm, respectively, and the PDIs were 0.131±0.005 and 0.091±0.008, respectively, using DLS. The Zeta potential of the nanoparticles was determined to be positive for C2 NPs at 28.6mV, while that of C2-Ce6 NPs was negative at -22.5mV. Figure 3 B and C).

[0086] The morphology of the nanoparticles was observed by TEM, and the average particle sizes of C2 NPs and C2-Ce6 NPs were 138.7±18.6nm and 127.0±15.9nm, respectively. Figure 3 Middle D).

[0087] Example 5

[0088] C2-Ce6 NPs stability test

[0089] DLS was used to measure the changes in particle size, PDI and Zeta potential of C2-Ce6 NPs during storage at room temperature for 7 days, and the stability of C2 NPs and C2-Ce6 NPs in aqueous solution was evaluated, respectively.

[0090] See the results Figure 4 The results showed that the particle size and PDI of the nanoparticles did not change much within 7 days, and were roughly a straight line, indicating that C2-Ce6 NPs had good storage stability.

[0091] Example 6

[0092] Cytotoxicity test of C2-Ce6 NPs

[0093] The cytotoxicity of C2-Ce6 NPs, Ce6 alone, and C2 alone on MC38 cells with and without laser irradiation was determined by CCK-8 assay. The specific steps are as follows. MC38 cells were seeded in 96-well plates and treated with C2-Ce6 NPs, Ce6, and C2 for 9 h after iron blocking. Then, the cells were irradiated with 660 nm laser (0.4 W / cm 2 ) for 3 minutes and then cultured for 15 hours. CCK-8 was added to each well, and after 2 hours of incubation, absorbance at 450 nm was measured using a microplate reader. Without laser irradiation, cell viability in the C2-Ce6 NPs group was greater than that in the Ce6 or C2 groups, indicating that C2-Ce6 NPs can reduce cytotoxicity to a certain extent and improve safety.

[0094] See the results Figure 5 . At 660nm laser (0.4W / cm 2) irradiated for 3 min, free Ce6 showed no obvious PDT effect, while the C2-Ce6 NPs group showed a significant concentration-dependent anti-proliferative effect, IC 50 It is 230.3nmol / L (expressed as Ce6 concentration).

[0095] Example 7

[0096] Verification of anti-tumor efficacy at the animal level

[0097] The efficacy study was conducted by establishing a tumor mouse model and dividing the mice into 5 groups, with 6 mice in each group. 4 of the groups were intraperitoneally injected with BMS202 (10 mg / kg), C2 (10 mg / kg), Ce6 (+) (2.5 mg / kg) and C2-Ce6 NPs (+) (2.5 mg / kg, Ce6 concentration), and the remaining group was used as a blank control without any treatment. The drug was administered once every two days. The light group was treated with a laser (0.4 W / cm 2 ) irradiated the tumor site of mice for 6 minutes. The mice in the dark group did not receive any treatment. The tumor volume and body weight of the mice were monitored every two days (see Figure 6 Middle A). Mice were sacrificed on day 12.

[0098] Figure 6 The results in Figures B to D show that C2-Ce6 NPs have relatively ideal in vivo antitumor activity, with a TGI of 90%.

[0099] Blood samples were collected for ELISA testing, and tumor tissues were stained with H&E and Tunnel staining (see Figure 6 (E) Flow cytometry of tumor tissues: Tumor tissues from each group of mice were cut into small pieces, placed in a 2 cm culture dish, and ground with the rubber end of a syringe. The cells were then filtered through a nylon mesh and rinsed with PBS.

[0100] ELISA was used to detect two cytokines in the serum of mice in different groups. ELISA was used to detect two cytokines in the serum of mice in different groups. Commercially available kits were used to detect three cytokines in the serum of mice in different groups according to the manufacturer's instructions. Figure 7 The results showed that C2-Ce6NPs promoted the production of IFN-γ, TNF-α and IL-6, which also confirmed that nanoparticles can more effectively restore immune function.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A small molecule inhibitor targeting PD-L1, characterized in that: It is an alanine-substituted isoindoline-bimethylbenzene derivative C2 having formula I:

2. A method for preparing the small molecule inhibitor targeting PD-L1 according to claim 1, characterized in that: The following steps are involved: Compound 1 having a structure shown in Formula II is subjected to a dehydration condensation reaction with Boc-alanine in the presence of a condensing agent, carbodiimide and 1-hydroxybenzotriazole, and the organic phase is collected to obtain a reaction product; The reaction product is subjected to a Boc-deprotection reaction with trifluoroacetic acid, and the product is extracted to obtain a small molecule inhibitor targeting PD-L1; 3. The preparation method according to claim 2, characterized in that: The molar ratio of the compound 1, Boc-alanine, carbodiimide and 1-hydroxybenzotriazole is 1:(1.2-2):(1.2-2):(1.2-2).

4. The preparation method according to claim 2, characterized in that The method for collecting the organic phase is to extract the organic phase with NaHCO3, NaCl and KHSO4 in sequence.

5. The preparation method according to claim 2, characterized in that: The method for extracting the product is to extract the organic phase with dichloromethane under the condition of pH value of 7.8-8.

2.

6. A nanoparticle based on a small molecule inhibitor targeting PD-L1 combined with photodynamic therapy, characterized in that: Comprising the photosensitizer Ce6 and the small molecule inhibitor targeting PD-L1 according to claim 1 or the small molecule inhibitor targeting PD-L1 according to any one of claims 2 to 5; The molar ratio of the small molecule inhibitor targeting PD-L1 and the photosensitizer Ce6 is (2.8-3.2):

1.

7. An anti-tumor drug, characterized in that: The active ingredient includes the small molecule inhibitor targeting PD-L1 according to claim 1, the small molecule inhibitor targeting PD-L1 according to any one of claims 2 to 5, or the nanoparticle according to claim 6.

8. The anti-tumor drug according to claim 7, characterized in that: Also includes pharmaceutically acceptable excipients; The pharmaceutically acceptable excipient includes 1% by volume of ethanol solution.

9. The antitumor drug according to claim 7 or 8, characterized in that: The drug concentration of the small molecule inhibitor targeting PD-L1 is 0.045 to 0.055 mg / mL.

10. Use of the small molecule inhibitor targeting PD-L1 according to claim 1, the small molecule inhibitor targeting PD-L1 according to any one of claims 2 to 5, or the nanoparticle according to claim 6 in the preparation of a drug for treating colon cancer.

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