Antitumor small molecule compounds, methods of making and using the same

Abstract

The present application relates to a kind of antitumor small molecule compounds and its preparation method and application, the small molecule compound has the structure shown in the following formula I.This new antitumor small molecule compound can inhibit LLC cell proliferation in vitro, and has good drugability, at animal level, no obvious toxic character to mouse, has better tumor inhibiting effect, and can enhance the inhibiting effect of PD-L1 antibody to non-small cell lung cancer, the combined administration of both can synergistically improve the treatment effect, safe and effective, can be used to prepare the drug for treating non-small cell lung cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to an antitumor drug, in particular to an antitumor small-molecule compound and a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is the highest incidence of cancer worldwide and is the main cause of cancer death worldwide. About 85% of lung cancer patients are non-small cell line cancer (NSCLC), of which lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the most common subtypes. With the introduction of corresponding tyrosine kinase inhibitors in patients with EGFR, ALK, ROS1 and NTRK mutations, the treatment of non-small cell lung cancer has been developed. In recent years, the application of immune checkpoint inhibitors (ICIs) mainly based on anti-PD-1 / PD-L1 has greatly changed the pattern of NSCLC treatment. Unfortunately, many factors in the tumor microenvironment (TME) will inhibit the therapeutic effect of immune checkpoints such as anti-PD-1 / PD-L1. Such as lack of antigenic protein (low mutation load), inefficient antigen presentation (down-regulation of major histocompatibility complex MHC-I or transporter associated with antigen processing TAP), insensitivity to T cells (PD-L1 expression, mutations in the interferon gamma pathway), etc. The effective rate of conventional chemotherapy combined with immunotherapy is only 50%. In addition, a number of clinical trials have shown that the incidence of ICI treatment-related ILD (such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis) is about 3.5-14.5%. This will lead to the interruption of ICI treatment and tumor progression. Therefore, it is urgent to find a combined drug that can overcome ICI resistance and alleviate lung complications caused by ICI treatment to improve the overall efficiency and safety of anti-PD-1 / PD-L1 in the treatment of NSCLC. SUMMARY

[0003] Based on this, the purpose of the present application is to provide a new antitumor small-molecule drug which has good antitumor effect and can improve the efficiency and safety of anti-PD-1 / PD-L1 in the treatment of NSCLC.

[0004] The technical solutions for achieving the above-mentioned purposes include the following.

[0005] In a first aspect, the present application provides a small-molecule compound having the structure shown in formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

[0006]

[0007] In a second aspect, the present application provides a use of a small molecule compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of an antitumor drug.

[0008] In a third aspect, the present application provides a use of a small molecule compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in combination with a PD-L1 antibody in the preparation of an antitumor drug.

[0009] Preferably, the tumor is non-small cell lung cancer.

[0010] In a fourth aspect, the present application provides an antitumor drug prepared from an active ingredient and pharmaceutically acceptable adjuvants, wherein the active ingredient comprises a small molecule compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0011] In a fifth aspect, the present application provides another antitumor drug, wherein the active ingredient comprises a pharmaceutical component 1 and a PD-L1 antibody, the pharmaceutical component 1 being a small molecule compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; the pharmaceutical component 1 and the PD-L1 antibody are respectively independent administration units, or the pharmaceutical component 1 and the PD-L1 antibody jointly form a combined administration unit.

[0012] In a sixth aspect, the present application provides a preparation method of the small molecule compound, comprising the following steps:

[0013] (1) reacting 2-hydroxybenzaldehyde with diethyl malonate to obtain coumarin-3-carboxylic acid ethyl ester;

[0014] (2) performing a hydrolysis reaction on the coumarin-3-carboxylic acid ethyl ester to obtain coumarin-3-carboxylic acid;

[0015] (3) reacting the coumarin-3-carboxylic acid with thionyl chloride to obtain coumarin-3-chloroformate;

[0016] (4) reacting the coumarin-3-chloroformate with N-hydroxy-4-methoxybenzamide to obtain a small molecule compound having a structure as shown in Formula I;

[0017] The reaction formula is as follows:

[0018]

[0019] The application synthesizes a novel antitumor small molecule compound by structural design and modification of coumarin in a simple and efficient method, which can inhibit LLC cell proliferation in vitro; at the animal level, it has no obvious toxicity to mice, has good tumor inhibition effect, and can enhance the tumor inhibition effect of PD-L1 antibody on LLC tumor-bearing C57BL / 6 mice. The novel antitumor small molecule compound can improve the tumor immune infiltration of LLC tumor-bearing C57BL / 6 mice, increase CD8 positive T cell infiltration, reduce Tre cells, and promote the polarization of M2 macrophages to M1 macrophages, which can effectively inhibit the tumor growth of tumor-bearing mice without affecting the animal body weight, clinical symptoms and blood indexes, and has good safety. When the antitumor small molecule compound is combined with PD-L1 antibody, the tumor growth inhibition effect is more obvious, which proves that the novel antitumor small molecule compound has a significant therapeutic effect on tumor diseases such as non-small cell lung cancer, and can enhance the therapeutic effect of PD-L1 antibody on LLC non-small cell lung cancer, and the combination of the two has a significant synergistic effect, which can significantly improve the therapeutic effect, is safe and effective, and can be used for preparing a drug for treating non-small cell lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the antitumor small molecule compound ZYZ338 is shown in the figure;

[0021] Figure 2 The nuclear magnetic resonance carbon spectrum of the antitumor small molecule compound ZYZ338 is shown in the figure;

[0022] Figure 3 The mass spectrum of the molecular weight characterization of the antitumor small molecule compound is shown in the figure;

[0023] Figure 4 The detection results of the LLC non-small cell lung cancer in vitro proliferation inhibited by the compound ZYZ338 are shown in the figure;

[0024] Figure 5 The effects of the compound ZYZ338 and the combination of the compound ZYZ338 and PD-L1 antibody on the tumor volume and weight of LLC tumor-bearing C57BL / 6 mice are shown in the figure;

[0025] Figure 6 The inhibition effects of the compound ZYZ338 and the combination of the compound ZYZ338 and PD-L1 antibody on the in vivo tumor and ex vivo tumor of LLC tumor-bearing C57BL / 6 mice are shown in the figure;

[0026] Figure 7 The effects of the compound ZYZ338 and the combination of the compound ZYZ338 and PD-L1 antibody on the body weight of LLC tumor-bearing C57BL / 6 mice are shown in the figure;

[0027] Figure 8 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0028] Figure 9 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0029] Figure 10 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0030] Figure 11 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0031] Figure 12 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0032] Figure 13 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0033] Figure 14 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0034] Figure 15 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0035] Figure 16 The detection results of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on liver function and kidney function of LLC tumor-bearing C57BL / 6 mice;

[0036] Figure 17 The results of histopathological examination of the main organs of LLC tumor-bearing C57BL / 6 mice for compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody;

[0037] In each figure, control is the control group. DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0039] The experimental methods in the following examples not specified in the specific conditions, generally in accordance with conventional conditions or in accordance with the manufacturer's recommended conditions. The various common chemical reagents used in the examples, are commercially available products.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0041] In addition, as used in this application, the term "or" is the inclusive "or" and is equivalent to the term "and / or", unless the context clearly indicates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly indicates otherwise. Also, throughout the specification, "one", "a", and "the" are inclusive of plural references unless the context clearly dictates otherwise. The meaning of "in" includes "in" and "on".

[0042] In some embodiments of the present application, a small molecule compound having the structure shown in formula I, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

[0043]

[0044] In some embodiments of the present application, the preparation method of the small molecule compound comprises the following steps:

[0045] (1) 2-hydroxybenzaldehyde is reacted with diethyl malonate to obtain coumarin-3-carboxylic acid ethyl ester;

[0046] (2) Coumarin-3-carboxylic acid ethyl ester is subjected to hydrolysis reaction to obtain coumarin-3-carboxylic acid;

[0047] (3) reacting coumarin-3-carboxylic acid with thionyl chloride to obtain coumarin-3-acyl chloride;

[0048] (4) reacting coumarin-3-acyl chloride with N-hydroxy-4-methoxybenzamide to obtain a small molecule compound having the structure shown in formula I as follows:

[0049] The reaction formula is as follows:

[0050]

[0051] In some preferred embodiments, the reaction in step (1) is carried out in the presence of a base, which is preferably piperidine.

[0052] In some preferred embodiments, the solvent for the reaction in step (1) is anhydrous ethanol.

[0053] In some preferred embodiments, the molar ratio of 2-hydroxybenzaldehyde to diethyl malonate in step (1) is 1:1.2-1.8.

[0054] In some preferred embodiments, the temperature for the reaction in step (1) is 15-30°C, and the reaction time is 0.5-3h, preferably 1-2h.

[0055] In some preferred embodiments, the hydrolysis reaction in step (2) is carried out in a basic solution.

[0056] In some preferred embodiments, the base in the basic solution is NaOH.

[0057] In some preferred embodiments, the basic solution is composed of anhydrous ethanol and an aqueous NaOH solution in a volume ratio of 1:1.5-2.5, and the concentration of NaOH in the aqueous NaOH solution is 3-5mol / mL.

[0058] In some preferred embodiments, the temperature for the hydrolysis reaction in step (2) is 60-80°C, and the reaction time is 0.5-3h, preferably 1-2h.

[0059] In some preferred embodiments, the hydrolysis reaction in step (2) is carried out in an atmosphere of an inert gas.

[0060] In some preferred embodiments, after the hydrolysis reaction in step (2) is completed, the following post-treatment step is further included: pouring the reaction mixture into an aqueous hydrochloric acid solution, filtering and drying the precipitated crystals, and recrystallizing to obtain coumarin-3-carboxylic acid.

[0061] In some preferred embodiments, the concentration of hydrochloric acid in the aqueous hydrochloric acid solution is 0.8 mol / mL to 1.5 mol / mL.

[0062] In some preferred embodiments, the molar ratio of coumarin-3-carboxylic acid to thionyl chloride in step (3) is 1:1.2 to 1.8.

[0063] In some preferred embodiments, the temperature of the reaction in step (3) is 60°C to 80°C, and the reaction time is 0.5 h to 3 h, preferably 1 h to 2 h.

[0064] In some preferred embodiments, the reaction in step (3) is carried out under an atmosphere of an inert gas.

[0065] In some preferred embodiments, the reaction in step (4) is carried out in the presence of a base, which is preferably triethylamine.

[0066] In some preferred embodiments, the solvent of the reaction in step (4) is dichloromethane.

[0067] In some preferred embodiments, the molar ratio of coumarin-3-carbonyl chloride to N-hydroxy-4-methoxybenzamide in step (4) is 1:1 to 1.5.

[0068] In some preferred embodiments, the temperature of the reaction in step (4) is 15°C to 30°C, and the reaction time is 0.5 h to 3 h, preferably 1 h to 2 h.

[0069] In some preferred embodiments, the reaction in step (4) is carried out under an atmosphere of an inert gas.

[0070] In some embodiments of the present application, the use of the small-molecule compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of the structure shown in formula I in the preparation of an antitumor drug is disclosed.

[0071] In some embodiments of the present application, the use of the small-molecule compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of the structure shown in formula I in combination with a PD-L1 antibody in the preparation of an antitumor drug is disclosed.

[0072] Preferably, the tumor is non-small cell lung cancer.

[0073] In some embodiments of the present application, an antitumor drug is disclosed, which is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the small-molecule compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of the present application.

[0074] In some embodiments of the present application, an anti-tumor drug is involved, the active ingredient of which comprises a drug component 1 and a PD-L1 antibody, the drug component 1 being the small molecule compound or the pharmaceutically acceptable salt or the stereoisomer thereof according to the present application; the drug component 1 and the PD-L1 antibody are respectively independent administration units, or the drug component 1 and the PD-L1 antibody together form a combined administration unit.

[0075] The small molecule compound or the pharmaceutically acceptable salt or the stereoisomer thereof according to the present application and the PD-L1 antibody can be mixed together in a combined form for administration; can also be administered separately as independent administration units; can be administered simultaneously, or can be administered separately in advance or afterwards.

[0076] The present application is based on the structural design and modification of natural product coumarin, and a novel anti-tumor small molecule compound is synthesized in a simple and efficient method, and the molecular weight and structure are confirmed by mass spectrometry and nuclear magnetic resonance hydrogen and carbon spectrum. Through in vitro and in vivo experimental model research, the results show that it can effectively inhibit the proliferation of LLC cells in vitro; has good tumor inhibition effect in vivo; at the animal level, clinical observation, hematological indexes and HE pathological results all show that the small molecule compound has no obvious toxicity to mice. In addition, when used in combination with the PD-L1 antibody, in addition to no obvious toxicity to mice, it can also significantly improve the therapeutic effect of the PD-L1 antibody on non-small cell tumors: significantly reduce the tumor volume / tumor weight of tumor-bearing mice, and the flow cytometry results, IHC results and IF results all show that the small molecule compound can significantly promote the expression of CD8-positive T cells, reduce the expression of Treg cells, and promote the polarization of macrophages from M2 to M1. These research results show that the novel anti-tumor small molecule compound according to the present application has a significant therapeutic effect on non-small cell lung cancer, and can significantly improve the sensitivity of the PD-L1 antibody to non-small cell lung cancer, and the combined administration of the two can significantly improve the therapeutic effect and is safe and effective.

[0077] The present application will be further described in detail below in combination with specific embodiments.

[0078] Example 1 Synthesis of Anti-tumor Small Molecule Compound

[0079]

[0080] Take 20 mmol of 2-hydroxybenzaldehyde 1.22 g with an electronic analytical balance, place it in a 50 mL round-bottom flask, add 25 mL of anhydrous ethanol, stir to dissolve, then slowly add diethyl malonate (99%, 30 mmol, 4.6 ml) dropwise under stirring, piperidine 0.5 mL, react at room temperature for 1 h, detect the reaction progress by thin layer chromatography, and then cool to room temperature after the reaction is completed. Add H2O to quench the reaction, extract with CH2Cl2, collect the organic phase, dry over anhydrous Na2SO4, concentrate under reduced pressure, and separate by column chromatography to obtain coumarin-3-carboxylic acid ethyl ester 4.19 g with a yield of 96%.

[0081] 1 H NMR (600 MHz, DMSO) δ 8.72 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.74-7.70 (m, 1H), 7.43-7.36 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13 CNMR (151 MHz, DMSO) δ 162.56, 155.95, 154.50, 148.62, 134.45, 130.25, 124.81, 117.77, 117.65, 116.12, 61.23, 14.05.

[0082] Take 15 mmol of the above intermediate coumarin-3-carboxylic acid ethyl ester 3.27 g with an electronic analytical balance, place it in a 100 mL round-bottom flask, add 25 mL of anhydrous ethanol and 50 mL of 4 mol / L NaOH aqueous solution, fill the reaction system with nitrogen, and react at 75°C for 1 h. Then pour the reaction mixture into 70 mL of 1 mol / L hydrochloric acid aqueous solution under stirring, filter and dry the crystals precipitated, and recrystallize with 50% ethanol to obtain coumarin-3-carboxylic acid 2.71 g with a yield of 95%.

[0083] 1 H NMR (600 MHz, DMSO) δ 8.73 (d, J = 0.7 Hz, 1H), 7.90 (dd, J = 7.8, 1.6 Hz, 1H), 7.72 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.44-7.38 (m, 2H). 13 CNMR (151 MHz, DMSO) δ 164.00, 156.72, 154.47, 148.32, 134.27, 130.19, 124.83, 118.42, 118.00, 116.13.

[0084] The intermediate coumarin-3-carboxylic acid 1.9 g was weighed by an electronic analytical balance, and was placed in a 25 mL round-bottom flask. Thiophosgene (99.5%, 15 mmol, 750 uL) was added, and nitrogen was filled into the reaction system. The reaction was heated at 70 °C for 1 h. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the coumarin-3-carbonyl chloride 2.04 g was obtained by distillation under reduced pressure, with a yield of 98%.

[0085] 1 H NMR (600 MHz, DMSO) δ 8.73 (s, 1H), 7.90 (dd, J = 7.8, 1.7 Hz, 1H), 7.72 (ddd, J = 8.6, 7.3, 1.7 Hz, 1H), 7.43-7.37 (m, 2H). 13 C NMR (151 MHz, DMSO) δ 164.00, 156.75, 154.51, 148.38, 134.33, 130.25, 124.87, 118.43, 118.03, 116.17.

[0086] The intermediate coumarin-3-carbonyl chloride 208 mg and N-hydroxy-4-methoxybenzamide 200 mg (1.2 mmol) were weighed by an electronic analytical balance, and were placed in a 50 mL round-bottom flask. Nitrogen was filled into the reaction system, 25 mL of dichloromethane was added, and the reaction was stirred at room temperature to make it uniform. Then 2-3 drops of triethylamine were slowly added dropwise, and the reaction was stirred at room temperature for 1 h. The reaction progress was detected by thin layer chromatography. After the reaction was completed, the reaction was cooled to room temperature, and then H2O was added to quench the reaction. The organic phase was collected by extraction with CH2Cl2, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The anti-tumor small molecule compound 287.3 mg was obtained by column chromatography, with a yield of 85%, and was named ZYZ338.

[0087] The nuclear magnetic resonance spectrum of compound ZYZ338 is shown in Figure 1 and Figure 2 1 H NMR (600 MHz, DMSO) δ 8.73 (s, 1H), 7.90 (dd, J = 7.8, 1.7 Hz, 1H), 7.72 (ddd, J = 8.6, 7.3, 1.7 Hz, 1H), 7.43-7.37 (m, 2H). ​

[0088] Molecular weight characterization was performed by HPLC-SEC using a Figure 3 molecular formula of C 18 H 14 N2O5.

[0089] Example 2: Detection of the inhibitory effect of compound ZYZ338 on the in vitro proliferation of LLC non-small cell lung cancer

[0090] Method: LLC cells were cultured at 37°C, 5% CO2 to the logarithmic growth phase, and when the cell density reached about 80-90%, the cells were collected and plated at a cell number of 5000 per well. After the cells adhered overnight, different concentrations of compound ZYZ338 were added for incubation. After 24 h, the absorbance value of the 96-well plate was detected by CCK-8 detection kit, and the cell viability per well was calculated.

[0091] Results: As shown in Table 1, compound ZYZ338 had a good inhibitory effect on the proliferation of LLC cells. Figure 4

[0092] Example 3: Detection of the inhibitory effect of compound ZYZ338 and the combination of compound ZYZ338 and PD-L1 antibody on the tumor growth of tumor-bearing mice

[0093] This example constructs a C57BL / 6 mouse LLC non-small cell lung cancer CDX model to detect the inhibitory effect of compound ZYZ338 and the combination of compound ZYZ338 and PD-L1 antibody on the tumor growth of tumor-bearing mice. The specific method is as follows:

[0094] Mouse-derived non-small cell lung cancer cells LLC were cultured in 1640 medium containing 10% FBS in a CO2-free 37°C incubator. Before the cells were continuously cultured for ten generations, about 2x10 6 LLC cells (200 μl PBS resuspended) were inoculated subcutaneously in 40 C57 mice at a volume of 200 μl per mouse. Mice with tumor volumes of about 50 mm 3 left were randomly divided into control group, PD-L1 group, ZYZ338 group, PD-L1+ZYZ338 group, 8 mice per group, and the tumor volumes and mouse weights of each group were uniform. The dosages and administration frequencies of each group were as follows: the PD-L1 group was injected intraperitoneally with 200 μg of PD-L1 antibody (Atezolizumab) 3 times a day; the ZYZ338 group was injected intraperitoneally with 10 mg / kg 3 times a day; the PD-L1+ZYZ338 group was injected with PD-L1 antibody and ZYZ338 simultaneously, and the dosages and administration frequencies were the same as those of the PD-L1 group and the ZYZ338 group. Tumor volume and mouse weight were measured every 3 days, and the tumor volume was calculated as follows: tumor volume = 1 / 2 (length x width​2 ) When the tumor diameter of the control group mice was more than 1.5 cm, and the volume was more than 2000 mm 3 , the mice were sacrificed. The mouse tumors and organs (heart, liver, spleen, lung, kidney) were stripped, and blood samples and urine were collected for subsequent detection.

[0095] The inhibitory effect of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor growth is shown in Figure 5 and Figure 6 : Compound ZYZ338 can effectively inhibit the tumor growth of tumor-bearing mice, and when it is combined with PD-L1 antibody, the inhibitory effect is the best.

[0096] The effect of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on the body weight of mice is shown in Figure 7 : When PD-L1 antibody is administered alone, the body weight of mice is reduced compared with the control group; compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody can improve the body weight of mice compared with the control group.

[0097] Example 4: Detection of the effect of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on the liver function and kidney function of mice

[0098] Method: The blood samples and urine of each group of mice collected in Example 3 were detected by glutamic-pyruvic transaminase (GPT / ALT) activity detection kit, glutamic-oxaloacetic transaminase (GOT / AST) activity detection kit, serum (plasma) creatinine (Scr) detection kit, urine nitrogen (BUN) test detection kit and urine protein quantitative detection kit (CBB method).

[0099] The results are shown in Figure 8 : There was no significant difference in ALT, AST, BUN, Scr and urine protein (Urine protein) levels of each group of mice. It is shown that the treatment of ZYZ338 administration group and ZYZ338 combined with PD-L1 antibody group does not significantly affect the liver and kidney function of mice.

[0100] Example 5: Effect of compound ZYZ338 combined with PD-L1 antibody on the immune status of tumor-bearing mice

[0101] Methods: Spleens and tumors obtained in Example 3 were collected from animals. After single-cell processing of the spleen and tumors, the levels of macrophage polarization (M1 and M2), CD84 T cells, CD8+ T cells, and Treg cells were detected by flow cytometry. The distribution of M1 and M2 macrophages (CD206, CD86), CD84 T cells (CD8), CD8+ T cells (CD8), and Treg cells (FOXP3) in the tissues was analyzed by immunohistochemistry. The distribution of M1 and M2 macrophages (CD206, CD86), CD8+ T cells (CD8), and Treg cells (FOXP3) in the tissues was analyzed by immunofluorescence.

[0102] Figure 9 The results showed that compound ZYZ338, PD-L1 antibody, and the combination of compound ZYZ338 and PD-L1 antibody could increase the expression of CD8 positive T cells in the spleen of tumor-bearing C57BL / 6 mice and decrease the expression of Treg cells in the spleen of tumor-bearing C57BL / 6 mice; the combination of compound ZYZ338 and PD-L1 antibody had a better effect.

[0103] Figure 10 The results showed that compound ZYZ338, PD-L1 antibody, and the combination of compound ZYZ338 and PD-L1 antibody could promote the expression of M1 macrophages in the spleen of tumor-bearing C57BL / 6 mice and reduce the expression of M2 cells in the spleen of tumor-bearing C57BL / 6 mice; the combination of compound ZYZ338 and PD-L1 antibody had a better effect.

[0104] Figure 11 Flow cytometry results Figure 13 Immunohistochemical analysis results of tissues and Figure 15 The results of tissue immunofluorescence analysis showed that compound ZYZ338, PD-L1 antibody, and the combination of compound ZYZ338 and PD-L1 antibody could increase the infiltration of tumor CD8 positive T cells and decrease the infiltration of tumor Treg cells in tumor-bearing C57BL / 6 mice; the effect of compound ZYZ338 combined with PD-L1 antibody was better.

[0105] Figure 12 The results showed that compound ZYZ338, PD-L1 antibody, and the combination of compound ZYZ338 and PD-L1 antibody could all promote the polarization of tumor M2 macrophages into M1 macrophages in tumor-bearing C57BL / 6 mice, with the combination of compound ZYZ338 and PD-L1 antibody showing a better promoting effect.

[0106] Figure 14 Immunohistochemical analysis results of tissues and Figure 16The results of the histological immunofluorescence analysis showed that the compound ZYZ338, the PD-L1 antibody, and the combination of the compound ZYZ338 and the PD-L1 antibody can promote the tumor M1 macrophage infiltration and reduce the M2 cell infiltration of the tumor-bearing C57BL / 6 mice, and the combination of the compound ZYZ338 and the PD-L1 antibody has a better effect.

[0107] Example 6 Histopathological detection of the tumor and the main organs of the mice treated with the compound ZYZ338 and the combination of the compound ZYZ338 and the PD-L1 antibody

[0108] Method: The tumor and other tissue blocks taken in Example 3 were fixed in 10% formalin for 48 hours. The fixed tissues were washed with running water to remove the residual fixing solution and impurities. The tissues were dehydrated with different concentrations of ethanol, 50%, 70%, 85%, 95% and pure alcohol (anhydrous ethanol) for 2 hours each time. The tissue blocks were placed in an equal volume mixture of pure ethanol and dimethylbenzene for 2 hours, then in pure dimethylbenzene for 2 hours, and then in pure dimethylbenzene for another 2 hours. The tissue blocks soaked in wax were wrapped in paraffin, and the tissue sections were cut by a microtome with a thickness of 4-7 μm. The cut slides were placed in a constant temperature oven at 65°C for 30 min; they were immersed in dimethylbenzene I for 15 min, and then in dimethylbenzene II for 15 min. The deparaffinized sections were immersed in 100% alcohol, 95% alcohol, 85% alcohol, and 75% alcohol for 5 min, respectively, washed with tap water for 10 min, and then subjected to hematoxylin staining and eosin staining, respectively. Finally, the slides were transparentized in dimethylbenzene for 3 min twice, sealed with neutral balsam, placed in a 65°C oven for 15 min, and then the results were collected.

[0109] The results are shown in Table 2. Figure 17 As shown in Table 2, there was no significant difference in the pathological results of the main organs such as heart, liver, spleen, lung and kidney between the compound ZYZ338, the PD-L1 antibody, and the combination of the compound ZYZ338 and the PD-L1 antibody and the control group. This indicates that the compound ZYZ338 and the combination of the compound ZYZ338 and the PD-L1 antibody have no obvious toxic side effects on the main organs of the animals and have good safety.

[0110] Tumor pathological results: The tumor cancer cells of the control group were diffusely distributed, some of which were arranged in a cord-like manner to form an adenoid structure, and the cancer cells had obvious atypia, and no necrotic foci were observed. The tumor pathology of the PD-L1 administration group had no obvious change compared with the control group. The local cancer cells of the ZYZ338 group had unclear fusion boundaries, and obvious necrotic foci were observed. The tumor of the combination of ZYZ338 and PD-L1 administration group had a large number of necrotic foci, and the cancer cells had cell death characteristics such as karyopyknosis, lysis and chromatin margination. This indicates that the combination of ZYZ338 and PD-L1 has an obvious inhibitory effect on the in vivo proliferation of LLC cells.

[0111] The above results prove that the novel antitumor small molecule compound prepared by the application has a significant therapeutic effect on cancer diseases such as non-small cell lung cancer, is safe and effective, and has a better anticancer effect when used in combination with a PD-L1 antibody, and can be used for preparing a drug for treating cancer diseases such as non-small cell lung cancer.

[0112] The above-described embodiments only express several embodiments of the application, which are described in a more specific and detailed manner, but cannot be understood as a limitation on the scope of the patent. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the application, which all belong to the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A small molecule compound having the structure shown in Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof. I 2. The use of the small molecule compound of claim 1, or its pharmaceutically acceptable salt, or its stereoisomer, in the preparation of an antitumor drug, wherein the tumor is non-small cell lung cancer.

3. The use of the small molecule compound of claim 1, or its pharmaceutically acceptable salt, or its stereoisomer, in combination with a PD-L1 antibody in the preparation of an antitumor drug, wherein the tumor is non-small cell lung cancer; and the PD-L1 antibody is atezolizumab.

4. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the small molecule compound of claim 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

5. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. Its active ingredients include drug component 1 and PD-L1 antibody, wherein drug component 1 is the small molecule compound of claim 1 or its pharmaceutically acceptable salt or its stereoisomer; and the PD-L1 antibody is atezolizumab. The drug component 1 and the PD-L1 antibody can each be an independent drug delivery unit, or the drug component 1 and the PD-L1 antibody can be combined to form a drug delivery unit.

6. A method of preparing the small molecule compound of claim 1, wherein, Includes the following steps: (1) 2-hydroxybenzaldehyde reacts with diethyl malonate to give ethyl coumarin-3-carboxylic acid; (2) Coumarin-3-carboxylic acid ethyl ester undergoes hydrolysis to obtain coumarin-3-carboxylic acid; (3) Coumarin-3-carboxylic acid reacts with thionyl chloride to give coumarin-3-acyl chloride; (4) Coumarin-3-acyl chloride reacts with N-hydroxy-4-methoxyphenylcarboxylimide to give a small molecule compound with the structure shown in Formula I below; The reaction formula is as follows:

7. The preparation method according to claim 6, characterized in that, In step (1), the reaction is carried out under the action of a base; The solvent for the reaction is anhydrous ethanol; The molar ratio of 2-hydroxybenzaldehyde to diethyl malonate is 1:1.2-1.8; The reaction temperature is 15℃-30℃, and the reaction time is 0.5h-3h.

8. The preparation method according to claim 7, characterized in that, The base used in step (1) is piperidine.

9. The preparation method according to claim 6, characterized in that, In step (2), the hydrolysis reaction is carried out in an alkaline solution; The hydrolysis reaction is carried out at a temperature of 60℃-80℃ for a time of 0.5h-3h. The hydrolysis reaction is carried out in an inert gas atmosphere; After the hydrolysis reaction is complete, the following post-processing steps are also included: pour the reaction mixture into an aqueous hydrochloric acid solution, filter, dry, and recrystallize the precipitated crystals to obtain coumarin-3-carboxylic acid.

10. The preparation method according to claim 9, characterized in that, The alkali in the alkaline solution is NaOH.

11. The preparation method according to claim 9, characterized in that, The alkaline solution is composed of anhydrous ethanol and NaOH aqueous solution in a volume ratio of 1:1.5-2.5, and the concentration of NaOH in the NaOH aqueous solution is 3mol / mL-5mol / mL.

12. The preparation method according to claim 9, characterized in that, The concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 0.8 mol / mL to 1.5 mol / mL.

13. The preparation method according to any one of claims 6-12, characterized in that, The molar ratio of coumarin-3-carboxylic acid to thionyl chloride in step (3) is 1:1.2-1.8; The reaction temperature in step (3) is 60℃-80℃, and the reaction time is 0.5h-3h; The reaction described in step (3) is carried out in an inert gas atmosphere; In step (4), the reaction is carried out under the action of a base; The solvent for the reaction in step (4) is dichloromethane; The molar ratio of coumarin-3-acyl chloride to N-hydroxy-4-methoxyphenylcarboxylimide in step (4) is 1:1-1.5; The reaction temperature in step (4) is 15℃-30℃, and the reaction time is 0.5h-3h; The reaction described in step (4) is carried out in an inert gas atmosphere.

14. The preparation method according to claim 13, characterized in that, Its features are, The base used in step (4) is triethylamine.

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