Anti-tumor small molecule compound as well as preparation method and application thereof

Abstract

The invention relates to an anti-tumor small molecule compound as well as a preparation method and application thereof. The small molecule compound has a structure as shown in a formula I in the specification. The novel anti-tumor small molecule compound can inhibit LLC cell proliferation in vitro, has good druggability, has no obvious toxicity characterization on mice on the animal level, has a good tumor inhibition effect, can sensitize the inhibition effect of a PD-L1 antibody on non-small cell lung cancer, can synergistically improve the treatment effect through combined administration of the compound and the PD-L1 antibody, is safe and effective, and has wide application prospects. The compound can be used for preparing medicines for treating non-small cell lung cancer. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to anti-tumor drugs, in particular to an anti-tumor small molecule compound and a preparation method and application thereof. Background Art

[0002] Lung cancer is the most common cancer worldwide and the leading cause of cancer death worldwide. Approximately 85% of lung cancer patients have non-small cell lung cancer (NSCLC), with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) being the most common subtypes. The treatment of NSCLC has advanced with the introduction of tyrosine kinase inhibitors for patients harboring EGFR, ALK, ROS1, and NTRK mutations. In recent years, the use of immune checkpoint inhibitors (ICIs), primarily targeting PD-1 / PD-L1, has significantly transformed the treatment landscape for NSCLC. Unfortunately, numerous factors within the tumor microenvironment (TME) can inhibit the efficacy of immune checkpoint inhibitors such as PD-1 / PD-L1. These factors include antigen deficiency (low mutational load), inefficient antigen presentation (downregulation of the major histocompatibility complex (MHC-I) or the transporter protein TAP involved in antigen processing), and T cell insensitivity (PD-L1 expression and mutations in the interferon γ pathway). Conventional chemotherapy combined with immunotherapy has an efficacy rate of only 50%. Furthermore, multiple clinical trials have shown that the incidence of ICI-related ILD (such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis) after ICI treatment is approximately 3.5-14.5%. This can lead to ICI treatment interruption and tumor progression. Therefore, there is an urgent need to explore combination therapy studies that can overcome ICI resistance and alleviate ICI-induced pulmonary complications, thereby improving the overall efficacy and safety of anti-PD-1 / PD-L1 therapies in NSCLC treatment. Summary of the Invention

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

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

[0005] In a first aspect, the present invention provides a small molecule compound having a structure as shown in Formula I below, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof,

[0006]

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

[0008] In a third aspect, the present invention provides the 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 anti-tumor drug.

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

[0010] In a fourth aspect, the present invention provides an anti-tumor drug prepared from an active ingredient and acceptable excipients in the drug, wherein the active ingredient comprises a small molecule compound of the structure shown in Formula I or a pharmaceutically acceptable salt or stereoisomer thereof.

[0011] In a fifth aspect, the present invention provides another anti-tumor drug, whose active ingredients include a drug component 1 and a PD-L1 antibody, wherein the drug component 1 is a small molecule compound with a structure represented by Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; the drug component 1 and the PD-L1 antibody each constitute an independent administration unit, or the drug component 1 and the PD-L1 antibody together form a combined administration unit.

[0012] In a sixth aspect, the present invention provides a method for preparing the small molecule compound, comprising the following steps:

[0013] (1) 2-Hydroxybenzaldehyde reacts with diethyl malonate to obtain ethyl malonate-3-carboxylate;

[0014] (2) hydrolyzing coumarin-3-carboxylic acid ethyl ester to obtain coumarin-3-carboxylic acid;

[0015] (3) coumarin-3-carboxylic acid reacts with thionyl chloride to obtain coumarin-3-acyl chloride;

[0016] (4) coumarin-3-yl chloride reacts with N-hydroxy-4-methoxybenzenecarboximide to obtain a small molecule compound having a structure shown in the following formula I;

[0017] The reaction formula is as follows:

[0018]

[0019] This invention synthesizes a novel anti-tumor small molecule compound by structurally modifying coumarin using a simple and efficient method. This novel anti-tumor small molecule compound can inhibit LLC cell proliferation in vitro. In animal models, it exhibits no significant toxicity to mice, exhibits a good tumor-suppressing effect, and can enhance the tumor-suppressing effect of PD-L1 antibodies on LLC tumor-bearing C57BL / 6 mice. This novel anti-tumor small molecule compound can improve tumor immune infiltration in LLC tumor-bearing C57BL / 6 mice, increasing CD8-positive T cell infiltration, reducing Tre cells, and promoting the polarization of M2 macrophages to M1 macrophages. While effectively inhibiting tumor growth in tumor-bearing mice, it does not affect animal body weight, clinical symptoms, or blood parameters, demonstrating a good safety profile. When this anti-tumor small molecule compound is used in combination with PD-L1 antibody, the inhibitory effect on tumor growth is more obvious, proving that this new anti-tumor small molecule compound has a significant therapeutic effect on tumor diseases such as non-small cell lung cancer, and can sensitize the therapeutic effect of PD-L1 antibody on LLC non-small cell lung cancer. The combined administration of the two has a significant synergistic effect, can significantly improve the therapeutic effect, is safe and effective, and can be used to prepare drugs for the treatment of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the H NMR spectrum of the anti-tumor small molecule compound ZYZ338;

[0021] Figure 2 This is the carbon NMR spectrum of the anti-tumor small molecule compound ZYZ338;

[0022] Figure 3 Schematic diagram of mass spectrometry for molecular weight characterization of anti-tumor small molecule compounds;

[0023] Figure 4 This is the test result of compound ZYZ338 inhibiting the proliferation of LLC non-small cell lung cancer in vitro;

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

[0025] Figure 6 Schematic diagram of the inhibitory effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on in vivo and ex vivo tumors in LLC tumor-bearing C57BL / 6 mice;

[0026] Figure 7 Effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on body weight of LLC tumor-bearing C57BL / 6 mice;

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

[0028] Figure 9 To detect the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on the expression of splenic CD8-positive T cells and splenic Treg cells in tumor-bearing C57BL / 6 mice by flow cytometry;

[0029] Figure 10 To detect the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on the expression of splenic M1 macrophages and splenic M2 cells in tumor-bearing C57BL / 6 mice by flow cytometry;

[0030] Figure 11 To detect the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor CD8-positive T cell infiltration and tumor Treg cell infiltration in tumor-bearing C57BL / 6 mice by flow cytometry;

[0031] Figure 12 To detect the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on the polarization of tumor M2 macrophages to M1 macrophages in tumor-bearing C57BL / 6 mice by flow cytometry;

[0032] Figure 13 To analyze the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor CD8-positive T cell infiltration and Treg cell infiltration in tumor-bearing C57BL / 6 mice using tissue immunohistochemistry;

[0033] Figure 14 To analyze the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor M1 macrophage infiltration and M2 cell infiltration in tumor-bearing C57BL / 6 mice using tissue immunohistochemistry;

[0034] Figure 15 To analyze the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor CD8-positive T cell infiltration and Treg cell infiltration in tumor-bearing C57BL / 6 mice by tissue immunofluorescence;

[0035] Figure 16 To analyze the effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor M1 macrophage infiltration and M2 cell infiltration in tumor-bearing C57BL / 6 mice by tissue immunofluorescence;

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

[0037] Control in each figure refers to the control group. DETAILED DESCRIPTION

[0038] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention 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 understanding of the present disclosure more thorough and comprehensive.

[0039] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0041] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0042] In some embodiments of the present invention, it relates to a small molecule compound having a structure as shown in Formula I below, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof,

[0043]

[0044] In some embodiments of the present invention, a method for preparing the small molecule compound comprises the following steps:

[0045] (1) 2-Hydroxybenzaldehyde reacts with diethyl malonate to obtain ethyl malonate-3-carboxylate;

[0046] (2) hydrolyzing coumarin-3-carboxylic acid ethyl ester to obtain coumarin-3-carboxylic acid;

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

[0048] (4) coumarin-3-yl chloride reacts with N-hydroxy-4-methoxybenzenecarboximide to obtain a small molecule compound having a structure shown in the following formula I;

[0049] The reaction formula is as follows:

[0050]

[0051] In some preferred embodiments, the reaction in step (1) is carried out under the action of a base, and the base 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 reaction temperature in step (1) is 15° C.-30° C., and the reaction time is 0.5 h-3 h, preferably 1 h-2 h.

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

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

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

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

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

[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 aqueous hydrochloric acid solution, filtering, drying, and recrystallizing the precipitated crystals to obtain coumarin-3-carboxylic acid.

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

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

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

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

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

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

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

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

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

[0070] In some embodiments of the present invention, it relates to the use of the small molecule compound of the structure shown in Formula I or its pharmaceutically acceptable salt or its stereoisomer in the preparation of anti-tumor drugs.

[0071] In some embodiments of the present invention, the present invention relates to the use of the small molecule compound of the structure shown in Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof, in combination with a PD-L1 antibody in the preparation of an anti-tumor drug.

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

[0073] In some embodiments of the present invention, an anti-tumor drug is provided, which is prepared from an active ingredient and acceptable excipients in the drug, wherein the active ingredient comprises the small molecule compound described in the present invention or a pharmaceutically acceptable salt or stereoisomer thereof.

[0074] In some embodiments of the present invention, an anti-tumor drug is provided, the active ingredients of which include a drug component 1 and a PD-L1 antibody, wherein the drug component 1 is the small molecule compound described in the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; the drug component 1 and the PD-L1 antibody each constitute an independent administration unit, or the drug component 1 and the PD-L1 antibody together form a combined administration unit.

[0075] The small molecule compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer and the PD-L1 antibody can be administered together in a combined form; can also be administered separately as separate dosing units; can be administered simultaneously or separately before and after.

[0076] This invention, based on the structure of the natural product coumarin, designed and modified the compound to synthesize a novel anti-tumor small molecule compound using a simple and efficient method. The molecular weight and structure were confirmed by mass spectrometry and nuclear magnetic resonance hydrogen and carbon spectra. In vitro and in vivo experimental models showed that the compound can effectively inhibit the proliferation of LLC cells in vitro and has a good tumor inhibitory effect in vivo. At the animal level, clinical observations, hematological indicators, and HE pathological results all showed that the small molecule compound had no obvious toxicity in mice. In addition, when combined with PD-L1 antibodies, in addition to having no obvious toxicity in mice, it can also significantly improve the therapeutic effect of PD-L1 antibodies on non-small cell tumors: significantly reducing the tumor volume / tumor weight of tumor-bearing mice. Flow cytometry, IHC results, and IF results all showed that the small molecule compound, when administered in combination with PD-L1 antibodies, 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 new anti-tumor small molecule compound described in the present invention has a significant therapeutic effect on non-small cell lung cancer, and can significantly increase the sensitivity of PD-L1 antibodies to non-small cell lung cancer. The combined administration of the two can significantly improve the therapeutic effect and is safe and effective.

[0077] The present invention is further described in detail below with reference to specific embodiments.

[0078] Example 1 Synthesis of antitumor small molecule compounds

[0079]

[0080] 1.22 g of 2-hydroxybenzaldehyde (20 mmol) was weighed using an electronic analytical balance and placed in a 50 mL round-bottom flask. 25 mL of anhydrous ethanol was added and stirred to dissolve the mixture. Diethyl malonate (99%, 30 mmol, 4.6 ml) and 0.5 mL of piperidine were then slowly added dropwise with stirring. The mixture was allowed to react at room temperature for 1 h. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the mixture was cooled to room temperature. H2O was then added to the reaction solution for quenching. The mixture was extracted with CH2Cl2, and the organic phase was collected, dried over anhydrous Na2SO4, concentrated under reduced pressure, and separated by column chromatography to obtain 4.19 g of coumarin-3-carboxylic acid ethyl ester in a yield of 96%.

[0081] 1 H NMR (600MHz, DMSO) δ8.72 (s, 1H), 7.89 (d, J = 7.7Hz, 1H), 7.74-7.70 (m, 1H), 7.43-7.36 (m, 2H), 4.29 (q, J = 7.1Hz, 2H), 1.31 (t, J = 7.1Hz, 3H). 13 CNMR(151MHz,DMSO)δ162.56,155.95,154.50,148.62,134.45,130.25,124.81,117.77,117.65,116.12,61.23,14.05.

[0082] 3.27 g of the intermediate coumarin-3-carboxylic acid ethyl ester (15 mmol) was weighed using an electronic analytical balance and placed in a 100-mL round-bottom flask. 25 mL of anhydrous ethanol and 50 mL of a 4 mol / L aqueous NaOH solution were added. Nitrogen was then added to the reaction system. The reaction was heated at 75°C for 1 h. The reaction mixture was then poured into 70 mL of a 1 mol / L aqueous hydrochloric acid solution with stirring. The precipitated crystals were filtered, dried, and recrystallized from 50% ethanol to obtain 2.71 g of coumarin-3-carboxylic acid in a yield of 95%.

[0083] 1 H NMR (600MHz, DMSO) δ8.73 (d, J=0.7Hz, 1H), 7.90 (dd, J=7.8, 1.6Hz, 1H), 7.72 (ddd, J=8.7, 7.3, 1.6Hz, 1H), 7.44-7.38 (m, 2H). 13 C NMR (151MHz, DMSO) δ164.00,156.72,154.47,148.32,134.27,130.19,124.83,118.42,118.00,116.13.

[0084] 1.9 g of the intermediate coumarin-3-carboxylic acid (10 mmol) was weighed out using an electronic analytical balance and placed in a 25 mL round-bottom flask. Thionyl chloride (99.5%, 15 mmol, 750 uL) was added and the reaction system was purged with nitrogen. The reaction was heated at 70°C for 1 h. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the mixture was concentrated by vacuum distillation to obtain 2.04 g of coumarin-3-yl chloride in a yield of 98%.

[0085] 1 H NMR (600MHz, DMSO) δ8.73 (s, 1H), 7.90 (dd, J=7.8, 1.7Hz, 1H), 7.72 (ddd, J=8.6, 7.3, 1.7Hz, 1H), 7.43-7.37 (m, 2H). 13 C NMR (151MHz, DMSO) δ164.00,156.75,154.51,148.38,134.33,130.25,124.87,118.43,118.03,116.17.

[0086] 208 mg of the intermediate coumarin-3-yl chloride and 200 mg (1.2 mmol) of N-hydroxy-4-methoxybenzenecarboximide (1 mol) were weighed out using an electronic analytical balance and placed in a 50 mL round-bottom flask. The reaction system was purged with nitrogen, and 25 mL of dichloromethane was added. The mixture was stirred at room temperature to allow for a uniform reaction. 2-3 drops of triethylamine were then slowly added dropwise. The reaction was stirred at room temperature for 1 h. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the mixture was cooled to room temperature, quenched by the addition of H2O, and extracted with CH2Cl2. The organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The anti-tumor small molecule compound, 287.3 mg, was separated by column chromatography to obtain an 85% yield and was named ZYZ338.

[0087] The NMR spectrum of compound ZYZ338 is as follows Figure 1 and Figure 2 As shown: 1 H NMR (600MHz, DMSO) δ8.93(s,1H),7.94(d,J=7.8Hz,1H),7.79-7.70(m,3H),7.51-7.40(m,2H),7.04(d,J=8.9Hz,2H),3.82(s,3H).13C NMR (126MHz, DMSO) δ161.21,160.48,157.53,156.73,154.44,148.78,134.4 7,130.24,128.31,124.96,123.10,118.02,117.55,116.23,113.83,55.34.

[0088] Molecular weight characterization Figure 3 As shown, its molecular formula is: C 18 H 14 N2O5.

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

[0090] Methods: LLC cells were cultured at 37°C and 5% CO2 until the logarithmic growth phase. When the cell density reached approximately 80-90%, the cells were harvested and plated at 5,000 cells per well. After overnight attachment, the cells were incubated with various concentrations of the compound ZYZ338. After 24 hours, the absorbance of the 96-well plate was measured using a CCK-8 assay kit, and the cell viability of each well was calculated.

[0091] The results are as follows Figure 4 It is shown that compound ZYZ338 has a good inhibitory effect on the proliferation of LLC cells.

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

[0093] In this example, a C57BL / 6 mouse LLC non-small cell lung cancer CDX model was constructed to detect the inhibitory effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on tumor growth in tumor-bearing mice. The specific methods are as follows:

[0094] Murine non-small cell lung cancer cells LLC were cultured in 1640 medium supplemented with 10% FBS in a CO2-free 37°C incubator. Before the tenth generation of cell culture, approximately 2×10 6 LLC cells (resuspended in 200 μl PBS) were subcutaneously injected into 40 C57 mice with an inoculation volume of 200 μL per mouse. 3 The mice were randomly divided into control group, PD-L1 group, ZYZ338 group, and PD-L1+ZYZ338 group, with 8 mice in each group. The tumor volume and weight of mice in each group were uniform. The dosage and frequency of administration of each group were as follows: 200 μg PD-L1 antibody (Atezolizumab) was injected intraperitoneally in the PD-L1 group for 3 days / time; 10 mg / kg was injected intraperitoneally in the ZYZ338 group for 3 days / time; PD-L1+ZYZ338 group was injected with PD-L1 antibody and ZYZ338 at the same time, and the dosage and frequency of administration were the same as those in the PD-L1 group and ZYZ338 group. Tumor volume and mouse weight were measured every 3 days. Tumor volume = 1 / 2 (length × width2 When the tumor diameter of the control group mice exceeded 1.5 cm and the volume exceeded 2000 mm 3 The mice were killed at 4 ℃ and 1 ℃, and their tumors and organs (heart, liver, spleen, lung, and kidney) were removed, and blood and urine samples were collected for subsequent testing.

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

[0096] Effects of compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody on mouse body weight Figure 7 As shown: When the PD-L1 antibody was administered alone, the body weight of the mice was reduced compared with the control group; compound ZYZ338 and compound ZYZ338 combined with PD-L1 antibody were able to increase the body weight of the mice compared with the control group.

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

[0098] Methods: The blood samples and urine of each group of mice collected in Example 3 were detected using alanine aminotransferase (GPT / ALT) activity detection kit, aspartate aminotransferase (GOT / AST) activity detection kit, serum (plasma) creatinine (Scr) detection kit, urea nitrogen (BUN) test kit and urine protein quantitative detection kit (CBB method).

[0099] The results are as follows Figure 8 As shown: There was no significant difference in the levels of ALT, AST, BUN, Scr, and urinary protein among the mice in each group, indicating that the ZYZ338-treated group and the ZYZ338 combined with PD-L1 antibody treatment did not significantly affect the liver and kidney function of the mice.

[0100] Example 5 Effect of Compound ZYZ338 Combined with PD-L1 Antibody on the Immune Status of Tumor-Bearing Mice

[0101] Methods: The spleens and tumors obtained from the animals in Example 3 above were collected and monoclonalized. Flow cytometry was used to analyze the levels of macrophage polarization (M1 and M2), CD84 T cells, CD8+ T cells, and Treg cells. Tissue immunohistochemistry was used to analyze the distribution of M1 and M2 macrophages (CD206, CD86), CD84 T cells (CD8), CD8+ T cells (CD8), and Treg cells (FOXP3). Tissue immunofluorescence was used to analyze the distribution of M1 and M2 macrophages (CD206, CD86), CD8+ T cells (CD8), and Treg cells (FOXP3).

[0102] Figure 9 The results showed that compound ZYZ338, PD-L1 antibody, and the combination of compound ZYZ338 and PD-L1 antibody can increase the expression of CD8-positive T cells in the spleen of tumor-bearing C57BL / 6 mice and reduce the expression of Treg cells in the spleen of tumor-bearing C57BL / 6 mice; the combination of compound ZYZ338 and PD-L1 antibody has 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 can promote the expression of splenic M1 macrophages in tumor-bearing C57BL / 6 mice and reduce the expression of splenic M2 cells in tumor-bearing C57BL / 6 mice; the combination of compound ZYZ338 and PD-L1 antibody has a better effect.

[0104] Figure 11 The flow cytometry results Figure 13 The results of tissue immunohistochemical analysis 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 can increase tumor CD8-positive T cell infiltration and reduce tumor Treg cell infiltration in tumor-bearing C57BL / 6 mice; the combination of compound ZYZ338 and PD-L1 antibody has a better effect.

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

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

[0107] Example 6 Histopathological Detection of Mouse Tumors and Major Organs by Compound ZYZ338 and Compound ZYZ338 Combined with PD-L1 Antibody

[0108] Methods: Tumor and other tissue blocks obtained in Example 3 were fixed in 10% formalin for 48 hours. The fixed tissues were rinsed with running water to remove residual fixative and impurities. Dehydration was performed in a series of ethanol concentrations, from 50%, 70%, 85%, and 95% to pure ethanol (anhydrous ethanol), with each level lasting 2 hours. The tissue blocks were placed in an equal volume mixture of pure ethanol and xylene for 2 hours, then in pure xylene for 2 hours, and then in pure xylene again for 2 hours. The wax-soaked tissue blocks were encapsulated in paraffin and sectioned using a microtome to a thickness of 4-7 μm. The cut slides were baked in a 65°C oven for 30 minutes, then immersed in xylene I for 15 minutes, and then immersed in xylene II for 15 minutes. The dewaxed sections were immersed in 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each. After rinsing with tap water for 10 minutes, the sections were stained with hematoxylin and eosin. Finally, the slides were placed in xylene for 3 min × 2 times for transparency, sealed with neutral gum, and placed in a 65°C oven for 15 min before collecting the results.

[0109] The results are as follows Figure 17 As shown, compared with the control group, the pathological results of the heart, liver, spleen, lung, and kidney in the animal groups treated with compound ZYZ338, PD-L1 antibody, and compound ZYZ338 combined with PD-L1 did not show significant differences. This indicates that compound ZYZ338 and its combination with PD-L1 antibody have no significant toxic side effects on major organs in animals and have a good safety profile.

[0110] Tumor pathology results: In the control group, tumor cells were more diffusely distributed, with some arranged in cords and glandular structures. Cancer cells showed significant atypia, but no necrosis was observed. Tumor pathology in the PD-L1 group showed no significant differences compared to the control group. In the ZYZ338 group, localized cancer cell fusions with unclear boundaries and distinct necrosis were observed. Tumors in the ZYZ338 plus PD-L1 group showed numerous necrotic foci, and cancer cells exhibited characteristics of cell death, including nuclear condensation, nuclear dissolution, and chromatin marginalization. This suggests that the combination of ZYZ338 and PD-L1 significantly inhibits LLC cell proliferation in vivo.

[0111] The above results demonstrate that the novel anti-tumor small molecule compound prepared by the present invention has a significant therapeutic effect on cancer diseases such as non-small cell lung cancer, is safe and effective, and has an even better anti-cancer effect when used in combination with PD-L1 antibodies. It can be used to prepare drugs for the treatment of cancer diseases such as non-small cell lung cancer.

[0112] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A small molecule compound having the structure shown in Formula I below, or a pharmaceutically acceptable salt or stereoisomer thereof, 2. Use of the small molecule compound according to claim 1 or a pharmaceutically acceptable salt or a stereoisomer thereof in the preparation of an anti-tumor drug.

3. Use of the small molecule compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in combination with a PD-L1 antibody in the preparation of an anti-tumor drug.

4. The use according to claim 2 or 3, characterized in that The tumor is non-small cell lung cancer.

5. An anti-tumor drug, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient comprises the small molecule compound according to claim 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

6. An anti-tumor drug, characterized in that: Its active ingredients include drug component 1 and PD-L1 antibody, wherein the drug component 1 is the small molecule compound according to claim 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; the drug component 1 and the PD-L1 antibody each become an independent administration unit, or the drug component 1 and the PD-L1 antibody together form a combined administration unit.

7. A method for preparing the small molecule compound according to claim 1, characterized in that: The steps include: (1) 2-Hydroxybenzaldehyde reacts with diethyl malonate to obtain ethyl malonate-3-carboxylate; (2) hydrolyzing coumarin-3-carboxylic acid ethyl ester to obtain coumarin-3-carboxylic acid; (3) coumarin-3-carboxylic acid reacts with thionyl chloride to obtain coumarin-3-acyl chloride; (4) coumarin-3-yl chloride reacts with N-hydroxy-4-methoxybenzenecarboximide to obtain a small molecule compound having a structure shown in the following formula I; The reaction formula is as follows:

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

9. The preparation method according to claim 7, characterized in that In step (2), the hydrolysis reaction is carried out in an alkaline solution; And / or, the hydrolysis reaction temperature is 60°C-80°C, and the reaction time is 0.5h-3h; and / or, the hydrolysis reaction is carried out under an atmosphere of inert gas; And / or, after the hydrolysis reaction is completed, the following post-treatment step is further included: pouring the reaction mixture into a hydrochloric acid aqueous solution, filtering, drying, and recrystallizing the precipitated crystals to obtain coumarin-3-carboxylic acid; Preferably, the base in the alkaline solution is NaOH; preferably, the alkaline 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 mol / mL-5 mol / mL; Preferably, the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 0.8 mol / mL-1.5 mol / mL.

10. The preparation method according to any one of claims 7 to 9, characterized in that: The molar ratio of coumarin-3-carboxylic acid to thionyl chloride in step (3) is 1:1.2-1.8; And / or, the reaction temperature in step (3) is 60°C-80°C, and the reaction time is 0.5h-3h; and / or, the reaction in step (3) is carried out under an atmosphere of inert gas; And / or, in step (4), the reaction is carried out under the action of a base, and the base is preferably triethylamine; and / or, the solvent for the reaction in step (4) is dichloromethane; and / or, the molar ratio of coumarin-3-yl chloride to N-hydroxy-4-methoxybenzenecarboximide in step (4) is 1:1-1.5; And / or, the reaction temperature in step (4) is 15°C-30°C, and the reaction time is 0.5h-3h; And / or, the reaction in step (4) is carried out under an inert gas atmosphere.

Images