Use of stilbene compounds in the preparation of antitumor drugs

Abstract

To provide the use of stilbene compounds in the preparation of anti-tumor medicaments.SOLUTION: The present invention provides the use of compounds represented by formula (I), or salts thereof, or stereoisomers thereof, or solvates thereof in the preparation of medicaments for preventing and / or treating anti-tumor medicaments and / or eye diseases (where M is selected from hydrogen, sodium, potassium, calcium, ammonium or organic amine, and R1, R2, R3, R4, and R5 are independently selected from hydrogen, a C1-C6 alkyl group, a C1-C6 alkoxy group, a hydroxy group, an amino group, and halogen).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of medicine, specifically to the use of stilbene compounds in the preparation of antitumor drugs. [Background technology]

[0002] CD8 + T lymphocytes are cytotoxic T lymphocytes that can kill tumor cells and block the spread of tumors. After using drugs, CD8 + Significantly increased T lymphocyte cell concentration and CD8 + If T lymphocytes can be recruited in large numbers into solid tumor tissue, the anticancer effects of drugs can be achieved.

[0003] In addition, the blood supply inside and around malignant tumors is very abundant. If drugs can act on tumor vascular endothelial cells to induce an inflammatory response in active tumor blood vessels, causing them to expand and block the blood vessels, blocking blood flow within the tumor. Then, after "depriving the tumor cells of nutrition," they can gradually starve and die, thereby achieving an antitumor effect.

[0004] The inventor's previous research has found that styrene acid derivatives have a certain degree of specific targeting effect on tumor blood vessels and therefore can inhibit tumor growth, but they cannot kill tumor cells, especially tumor cells at the edge of solid tumor tissue, which causes tumor tissue to rapidly proliferate through the edge, resulting in poor tumor treatment effects.

[0005] CD8 + If it were possible to provide a drug that could not only attract a large number of T lymphocytes to solid tumor tissue but also block blood flow within the tumor, this would be of great promise for antitumor applications. Summary of the Invention

[0006] An object of the present invention is to provide a use of a stilbene compound in the preparation of an antitumor drug.

[0007] The present invention provides the use of a compound as shown in formula I, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of an anti-tumor medicament and / or a medicament for preventing and / or treating eye diseases. TIFF0007674429000001.tif51170 where M is selected from hydrogen, sodium, potassium, calcium, ammonium, or an organic amine; R1, R2, R3, R4, and R5 are each independently selected from hydrogen, a C1 to C6 alkyl group, a C1 to C6 alkoxy group, a hydroxy group, an amino group, and a halogen.

[0008] Further, the organic amine is an amino acid, a peptide, t-butylamine or n-butylamine; and / or R1, R2, R3, R4, and R5 are each independently selected from hydrogen, a C1-C3 alkyl group, a methoxy group, an ethoxy group, a hydroxyl group, an amino group, fluorine, chlorine, or bromine; Preferably, the organic amine is selected from meglumine, pyrazine, alanine, n-butylamine, lysine, and t-butylamine; and / or R1, R2, R3, R4, and R5 are each independently selected from hydrogen, methoxy, ethoxy, hydroxy, fluorine, chlorine, or bromine.

[0009] Further, the compound is a compound of formula II, TIFF0007674429000002.tif51170 where M is selected from hydrogen, sodium, potassium, calcium, ammonium, meglumine, pyrazine, alanine, n-butylamine, lysine, or t-butylamine; R1, R2, R3, and R4 are each independently selected from hydrogen, a methoxy group, an ethoxy group, a hydroxy group, fluorine, chlorine, or bromine.

[0010] Further, the compound is a compound of formula III, TIFF0007674429000003.tif51170 where M is selected from hydrogen, sodium, potassium, calcium, ammonium, meglumine, pyrazine, alanine, n-butylamine, lysine, or t-butylamine; R1, R2, and R3 are each independently selected from hydrogen, a methoxy group, an ethoxy group, a hydroxy group, fluorine, chlorine, or bromine.

[0011] Further, the compound is a compound according to formula IV: TIFF0007674429000004.tif51170 where M is selected from hydrogen, sodium, potassium, calcium, ammonium, meglumine, pyrazine, alanine, n-butylamine, lysine, or t-butylamine; R2 and R3 are each independently selected from hydrogen, a methoxy group, an ethoxy group, a hydroxy group, fluorine, chlorine, or bromine.

[0012] Furthermore, the antitumor drug is capable of expressing CD8 + It is a drug that has the effect of attracting a large number of T lymphocytes and / or destroying the inner walls of tumor blood vessels. Preferably, the drug is one that induces necrosis of tumor cells in the central region of a solid tumor.

[0013] Furthermore, the antitumor drug is a drug that inhibits tumor metastasis.

[0014] Furthermore, when the antitumor drug is a drug that inhibits tumor metastasis, the structure of the compound shown in formula I is as shown in formula Va: TIFF0007674429000005.tif59170 Preferably, the drug that inhibits metastasis of a tumor is a drug that inhibits metastasis of melanoma.

[0015] Furthermore, the antitumor drug is a drug for preventing and / or treating lung cancer, liver cancer, gastric cancer, ovarian cancer, prostate cancer or renal cell cancer.

[0016] Furthermore, when the antitumor drug is a drug for preventing and / or treating lung cancer, liver cancer, gastric cancer or ovarian cancer, the structure of the compound shown in formula I is as shown in formula Va. TIFF0007674429000006.tif59170

[0017] Furthermore, when the anti-tumor drug is a drug for preventing and / or treating prostate cancer or renal cell carcinoma, the structure of the compound shown in formula I is as shown in formula Vb. TIFF0007674429000007.tif59170

[0018] Furthermore, the drug for preventing and / or treating the eye disease is a drug that inhibits the growth of blood vessels on the ocular surface.

[0019] Further, the eye disease is retinopathy, fundus hemangioma, fundus hemorrhage, dacryocystitis, glaucoma, cataract, vitreous opacification, optic nerve atrophy, macular degeneration and / or retinal detachment.

[0020] Furthermore, the eye disease is a diabetic eye disease, Preferably, the diabetic eye disease is diabetes-related retinopathy, fundus hemangioma, fundus hemorrhage, dacryocystitis, glaucoma, cataract, vitreous opacification, optic nerve atrophy, macular degeneration and / or retinal detachment.

[0021] Furthermore, when the drug is a drug for preventing and / or treating an eye disease, the structure of the compound is as shown in Formula Va. TIFF0007674429000008.tif53170

[0022] The present invention further provides a pharmaceutical formulation for treating tumors, the pharmaceutical formulation being prepared by using the aforementioned compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof as an active ingredient, and adding pharma- ceutically acceptable auxiliary materials or auxiliary ingredients; Preferably, the pharmaceutical formulation is an injectable formulation, an oral formulation or an external formulation.

[0023] The present invention further provides a pharmaceutical formulation for treating eye diseases, the pharmaceutical formulation being prepared by using the aforementioned compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof as an active ingredient, and adding pharma- ceutically acceptable auxiliary materials or auxiliary ingredients; Preferably, the drug formulation is a formulation for administration via the eye, More preferably, the formulation is in the form of eye drops, eye ointments, eye gels, nano formulations, microsphere formulations, liposome formulations; More preferably, the eye drop is an aqueous solution, a suspension, or an emulsion.

[0024] The present invention further provides the use of the aforementioned compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in combination with a tumor immunotherapy drug to prepare an antitumor combination drug; Preferably, the tumor immunotherapy drug is PD1, PD-L1, T cells, NK cells or CART cells.

[0025] Further, the use of a compound of formula Vc, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in combination with PD-L1 in the preparation of a combination medicament for preventing and / or treating lung cancer. TIFF0007674429000009.tif58170

[0026] Further, the present invention relates to the use of a compound of formula Va, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in combination with T cells in the preparation of a combination drug for preventing and / or treating liver cancer. TIFF0007674429000010.tif52170

[0027] Although the styrene acid derivatives that the inventors of the present invention have been studying have a certain degree of specific targeting effect on tumor blood vessels, they can only destroy tumor blood vessels and inhibit tumor growth, but cannot kill tumor cells, especially tumor cells at the edge of solid tumor tissues, which causes tumor tissues to rapidly proliferate from the edge. Based on the previous work, the inventors have demonstrated through structure optimization and in vivo screening that a series of compounds of the present invention can effectively target CD8+ in solid tumors. + Research has shown that this compound has the dual effect of attracting a large number of T lymphocytes and destroying adhesion proteins on endothelial cells of newly formed blood vessels, and can be used to prepare antitumor drugs.

[0028] The stilbene compound of the formula (I) structure is used as an active ingredient, and can be combined with the corresponding auxiliary additives such as medicinal auxiliary materials or carriers that are acceptable in medicine, and processed according to the corresponding conventional pharmaceutical method, and then prepared as a vascular targeting drug of the corresponding form such as oral, injection or external use.For example, it can be mixed with the commonly used auxiliary additives such as disintegrants, excipients, lubricants, binders, fillers, etc. that are acceptable in oral drug formulations, and then processed according to the corresponding conventional process method, and prepared as an oral drug in the form of solid preparations such as tablets, pills, granules, capsules, or suitable forms of sustained release agents, release control agents, etc. After mixing with the appropriate solvents and additives that are permitted to be used in injection drug formulations and processed according to the corresponding process operation, it can be prepared as an injection drug formulation of the corresponding intramuscular or intravenous form such as aqueous injection or powder injection. They can be mixed with commonly used solvents, stabilizers, or lipophilic and / or water-soluble substrates such as sodium polyacrylate, polyvinyl alcohol, polyethylene glycol, glycerin, vegetable oil, petrolatum, lanolin, etc., and prepared according to the processing of corresponding external drug preparations into eye drops or ointments, ointments, gels, ointments, liniments, liniments, etc. These injection preparations such as sterile powder injections and sterile freeze-dried powder injections, oral preparations such as tablets, capsules, various sustained-release preparations, and drugs in the form of external preparations such as eye drops and ointments, etc. can be used for the treatment of anti-solid tumor drugs.

[0029] The present invention provides the use of stilbene compounds in the preparation of antitumor drugs, and the present invention has been reported to show that some stilbene compounds induce CD8 expression in solid tumors. + It has the dual effect of attracting large numbers of T lymphocytes and targeting and destroying the inner walls of tumor blood vessels. + It was found that the killer T lymphocytes can eliminate tumor cells, while at the same time cutting off the blood supply to the tumor tissue, causing rapid necrosis inside the solid tumor, greatly improving the killing power against the tumor, and further having an inhibitory effect against tumor metastasis. The stilbene compounds of the present invention can be used to prepare antitumor drugs, and can also be used in combination with tumor immunotherapy drugs to exert a synergistic antitumor effect. These stilbene compounds can be used to prepare various antitumor drugs, since the antitumor effects of the compounds differ depending on the structure. In addition, the stilbene compounds of the present invention can inhibit the growth of blood vessels on the ocular surface, and therefore can be used to prepare drugs for treating various ocular diseases. The applications of the stilbene compounds of the present invention are promising.

[0030] Naturally, based on the above content of the present invention, various other forms of modifications, substitutions or changes can be made in light of general technical knowledge and conventional means in this field, on the premise that they do not deviate from the above basic technical idea of ​​the present invention.

[0031] The above content of the present invention will be described in more detail below by way of specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention. Any technology realized based on the above content of the present invention belongs to the scope of the present invention. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 is a flow chart of a quantitative proteomics experiment. [Diagram 2] FIG. 2 shows the results of HE staining of human lung cancer 95D. [Diagram 3] FIG. 3 shows the results of HE staining of human hepatoma Bel7404. [Figure 4] FIG. 4 shows the results of PCNA immunohistochemical staining of human lung cancer 95D. [Diagram 5] FIG. 5 shows the results of PCNA immunohistochemical staining of human hepatic cancer Bel7404. [Figure 6] FIG. 6 shows the state of blood vessel growth on the ocular surface on the 10th day (d10) after administration, where A is the model group, B is the blank control group, C is administration group 1, and D is administration group 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] All materials and equipment used in the Detailed Description of the Invention are conventional and purchased from commercial sources.

[0034] The structure of the stilbene compound of the present invention is as shown in formula (I): TIFF0007674429000011.tif51170In the formula, M is selected from hydrogen, sodium, potassium, calcium, ammonium, or an organic amine including an amino acid, t-butylamine, n-butylamine, or a peptide, and R1, R2, R3, R4, and R5 are each selected from hydrogen, an alkyl group, a hydroxyl group, a methoxy group, an ethoxy group, an amino group, a fluorine group, a chlorine group, or a bromine group.

[0035] Specific compounds of the present invention are shown in Table 1. Table 1. Specific structures of compounds of the present invention TIFF0007674429000012.tif127170

[0036] Example 1 Preparation of Compound 1 of the Invention TIFF0007674429000013.tif53170 Preparation method: Add 2.26g (0.010mol) of 3,4,5-trimethoxyphenylacetic acid, 2.02g (0.012mol) of 2,3-dihydroxy-4-methoxybenzaldehyde to a 50ml three-neck flask, add 3.2ml of acetic anhydride and 1.6ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath to 150℃. The mixture was heated to 50°C, kept at 50°C for 5 hours to react, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, and the mixture was reacted for 1 hour while maintaining the temperature at 80°C. The mixture was cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, and the reaction was continued for 1 hour. Then, 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour to continue the reaction. The mixture was filtered, and the filter cake was washed with ice-cooled absolute ethanol and dried under reduced pressure to obtain 2.17g of off-white crystalline solid (compound 1) (yield 57.7%). 1 H NMR (400 MHz, DMSO) 3.672(d 9H 3',4',5'-OCH3),3.821(d 3H 4''-OCH3),5.265(s 1H 2''-OH),5.318(s 1H 3''-OH),6.273(s 1H 2'-H),6.348(d 1H 6'-H),6.386(d 1H 5''-H),7.415(s 1H 3-H);10.982(s 1H CO-OH); 13 C NMR (100 MHz, DMSO) δ 57.692 ,57.826 ,62.837 ,109.313 ,112.713 ,118.627 ,125.392 ,129.883 ,137.423 ,138.153 ,138.232 ,138.772 ,146.391 ,149.167 ,154.482,178.277Total of 16 groups of carbon.

[0037] Example 2 Preparation of Compound 2 of the Invention TIFF0007674429000014.tif64170 Preparation method: 1.82g (4.84mmol) of (E)-3-(2,3-dihydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (compound 1) and 0.99g (5.08mmol) of meglumine were added to a 10ml three-neck flask, and 15ml of ethanol was added. The mixture was shaken to homogenize, and a magnetic stirrer was added. The mixture was heated in a water bath to 60℃. When the mixture was dissolved and became transparent, it was cooled to room temperature, and the solid was precipitated. The mixture was stirred for 1 hour, filtered, and the filter cake was washed with ice-cold anhydrous ethanol and dried under reduced pressure to obtain 2.21g of off-white crystalline solid (compound 2) (yield 80.0%). 1 H NMR (400 MHz, DMSO) 2.791(d,3H a-CH3),3.187(d,2H a-CH2),3.297(d,1H a-CH),3.365(d,1H a-CH),3.382(d,1H a-CH),3.516(d,2H a-CH2),3.683(d,4H a-OH),3.698(d,1H a-OH),3.713(d 9H 3',4',5'-OCH3),3.796(d 3H 4''-OCH3),5.159(s 1H 2''-OH),5.435(s 1H 3''-OH),6.368(s 1H 2'-H),6.521(d 1H 6'-H),6.472(d 1H 5''-H), 7.477(s 1H 3-H); 13 C NMR (100 MHz, DMSO) δ34.286,51.217, 57.692,57.826,62.837,63.295,68.786,71.198,73.136,73.982,109.313,112.713,118.627,125.392,129.883,137.423,138.153,138.232,138.772,146.391,149.167,154.482,178.277Total of 23 groups of carbon.

[0038] Example 3 Preparation of Compound 3 of the Invention TIFF0007674429000015.tif64170 Preparation method: Add 2.26g (0.010mol) of 3,4,5-trimethoxyphenylacetic acid and 1.99g (0.012mol) of 3,5-dimethoxybenzaldehyde to a 50ml three-neck flask, then add 3.2ml of acetic anhydride and 1.6ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath to 150℃. The mixture was heated to 50°C, kept at 50°C for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the reaction was continued for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the reaction was continued with stirring for 1 hour, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.25g of off-white crystalline solid (yield 60.1%). The off-white crystalline solid was (E)-3-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0039] Into a 10 ml three-neck flask, 1.81 g (4.84 mmol) of (E)-3-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 10 ml of anhydrous ethanol, and 0.5 ml of 40% sodium hydroxide solution were added, and the mixture was shaken to homogenize. A magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, the mixture was stirred continuously for 1 hour, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.62 g of an off-white crystalline solid (compound 3) (yield 84.4%). 1 H NMR (400 MHz, DMSO) 3.727(d 9H 3',4',5'-OCH3),3.869(d 6H 3'',5''-OCH3), 6.174(d 1H 4''-H),6.273(s 1H 2'-H),6.348(d 1H 6'-H),6.386(d 1H 5''-H),6.723(d 2H 2'',5''-H)),7.329(s 1H 3-H); 13C NMR (100 MHz, DMSO) δ55.823,57.314,62.729,108.476,113.626,117.196,126.181,128.347,136.973,137.826,137.951,137.865,147.127,148.331,154.672,171.323Total of 16 groups of carbon.

[0040] Example 4 Preparation of Compound 4 of the Invention TIFF0007674429000016.tif58170 Preparation method: Add 2.26g (0.010mol) of 3,4,5-trimethoxyphenylacetic acid, 1.85g (0.012mol) of 3-fluoro-4-methoxybenzaldehyde to a 50ml three-neck flask, add 3.2ml of acetic anhydride and 1.6ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath to 150℃. The temperature was raised, and the mixture was reacted for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution (1+5) was dropped, and the mixture was reacted for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, and the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 2.15g of off-white crystalline solid (yield 59.3%). The off-white crystalline solid was (E)-3-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0041] Into a 10 ml three-neck flask, 1.75 g (4.84 mmol) of (E)-3-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 10 ml of anhydrous ethanol, and 0.41 g of pyrazine were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, stirring was continued for 1 hour, followed by filtration. The filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.76 g of an off-white crystalline solid (compound 4) (yield 82.2%). 1H NMR (400 MHz, DMSO)δ3.612(d 9H 3',4',5'-OCH3),3.824(d 3H 4''-OCH3),6.287(s 2H 2',6'-H),6.413(s 1H 2''-H),6.578(d 1H 6''-H),6.612(d 1H 5''-H), 9.583(s 2H CH),9.689(s 2H CH), 9.823(s 1H 3-H); 13 C NMR (100 MHz, DMSO) δ 56.276 ,57.327 ,63.196 ,108.893,113.746,117.845 ,125.637 ,130.762 ,138.524 ,138.267 ,139.241 ,139.482 ,142.158,146.783,148.298 ,152.930,174.128Total of 17 groups of carbon.

[0042] Example 5 Preparation of Compound 5 of the Invention TIFF0007674429000017.tif70170 Preparation method: Add 2.26g (0.010mol) of 3,4,5-trimethoxyphenylacetic acid, 2.24g (0.012mol) of 3-chloro-5-hydroxy-4-methoxybenzaldehyde to a 50ml three-neck flask, add 3.2ml of acetic anhydride and 1.6ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath. The mixture was heated to 150°C, kept at 40°C for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the mixture was reacted for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.13g of off-white crystalline solid (yield 53.9%). The off-white crystalline solid was (E)-3-(3-chloro-5-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0043] In a 10ml three-neck flask, 1.75g ​​(4.84mmol) of (E)-3-(3-chloro-5-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 0.45g (5.08mmol) of alanine, and 10ml of anhydrous ethanol were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, the mixture was stirred for 1 hour, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.42g of off-white crystalline solid (compound 5) (yield 60.6%). [M+H] + :443.16. 1 H-NMR (CDCl3, δ(ppm)): 9.972(2H), 9.712(2H), 7.326(1H), 6.887(1H), 6.865(1H), 6.853(1H), 3.812(3H), 3.924(9H), 6.615(2H).

[0044] Example 6 Preparation of Compound 6 of the Invention TIFF0007674429000018.tif58170 Preparation method: Add 2.26 g (0.010 mol) of 3,4,5-trimethoxyphenylacetic acid and 1.83 g (0.012 mol) of isovanillin to a 50 ml three-neck flask, then add 3.2 ml of acetic anhydride and 1.6 ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, heat in an oil bath to 150 °C, and then add 5 ml of ethyl alcohol. The reaction was continued for 1 hour while keeping the temperature at 40°C, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, and the reaction was continued for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 12ml of 40% sodium hydroxide solution was dropped, and the reaction was continued for 1 hour, then 10ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the reaction was continued with stirring for 1 hour, filtered, and the filter cake was washed with ice-cooled absolute ethanol and dried under reduced pressure to obtain 2.56g of off-white crystalline solid (yield 71.0%). The off-white crystalline solid was (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0045] Into a 10 ml three-neck flask, 1.74 g (4.84 mmol) of (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 0.37 g (5.08 mmol) of n-butylamine, and 10 ml of absolute ethanol were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, stirring was continued for 1 hour, followed by filtration. The filter cake was washed with ice-cooled absolute ethanol and dried under reduced pressure to obtain 1.68 g of an off-white crystalline solid (Compound 6) (yield 80.1%). 1 H NMR (400 MHz, DMSO)δ0.832(d,3H a-CH3),1.329(d 2H b-CH3),1.546(d 2H c-CH2),2.914(d 2H,d-CH3),3.593(d 9H 3',4',5'-OCH3),3.749(d 3H 4''-OCH3),6.375(s 2H 2'-H),6.386(s 1H 2''-H),6.462(d 1H 6''-H),6.499(d 1H 5''-H),7.346(s 1H 3-H); 13 C NMR (100 MHz, DMSO) δ 15.103 ,21.343 ,31.101 ,41.526 ,57.788 ,58.186 ,63.341 ,109.224 ,113.626 ,118.900 ,125.518 ,130.918 ,137.718 ,137.812 ,138.175 ,139.291 ,146.588 ,149.858 ,154.930,178.099Total of 20 groups of carbon.

[0046] Example 7 Preparation of Compound 7 of the Invention TIFF0007674429000019.tif58170 Preparation method: Add 2.26 g (0.010 mol) of 3,4,5-trimethoxyphenylacetic acid and 1.83 g (0.012 mol) of vanillin to a 50 ml three-neck flask, then add 3.2 ml of acetic anhydride and 1.6 ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, heat in an oil bath to 150 °C, and keep at room temperature for 5 hours. The mixture was then cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, and the mixture was allowed to react for 1 hour while maintaining the temperature at 80°C. The mixture was then cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, and the reaction was continued for 1 hour. Then, 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued. The mixture was filtered, and the filter cake was washed with ice-cooled absolute ethanol and dried under reduced pressure to obtain 2.74g of off-white crystalline solid (yield 76.0%). The off-white crystalline solid was (E)-3-(4-hydroxy-3-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0047] In a 10 ml three-neck flask, 1.74 g (4.84 mmol) of (E)-3-(4-hydroxy-3-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid and 0.74 g (5.08 mmol) of lysine were added, and 15 ml of ethanol was added. The mixture was shaken to homogenize, a magnetic stirrer was added, and the mixture was heated in a water bath to 60°C. When the mixture was dissolved and became transparent, it was cooled to room temperature, and the solid was precipitated. The mixture was then stirred for 1 hour, filtered, and the filter cake was washed with ice-cold anhydrous ethanol and dried under reduced pressure to obtain 1.75 g of off-white crystalline solid (compound 7) (yield 71.4%). [M+H] + :507.23. 1 H-NMR(CDCl3,δ(ppm)):1.187(2H),1.742(2H),2.116(2H),3.281(2H),3.373(1H),5.095 (2H),7.386(1H),3.659(3H),5.287(1H),7.137(1H),7.349(1H),3.876(9H),6.517(2H).

[0048] Example 8 Preparation of Compound 8 of the Invention TIFF0007674429000020.tif64170 Preparation method: Add 2.26g (0.010mol) of 3,4,5-trimethoxyphenylacetic acid, 1.83g (0.012mol) of 5-fluoro-3-hydroxy-4-methoxybenzaldehyde to a 50ml three-neck flask, add 3.2ml of acetic anhydride and 1.6ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath. The mixture was heated to 150°C, kept at 40°C for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the reaction was continued for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the reaction was continued with stirring for 1 hour, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.45g of off-white crystalline solid (yield 64.7%). The off-white crystalline solid was (E)-3-(5-fluoro-3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0049] In a 10ml three-neck flask, 1.74g (4.84mmol) of (E)-3-(5-fluoro-3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 0.37g (5.08mmol) of t-butylamine, and 10ml of anhydrous ethanol were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, the mixture was stirred for 1 hour, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.52g of off-white crystalline solid (compound 8) (yield 69.6%). [M+H] + :452.20. 1 H-NMR (CDCl3, δ(ppm)): 1.287(9H), 3.635(3H), 3.831(9H), 6.228(2H), 7.267(1H), 7.338(1H), 7.643(1H).

[0050] Example 9 Preparation of Compound 9 of the Invention TIFF0007674429000021.tif58170 Preparation method: Add 2.26g (0.010mol) of 3,4,5-trimethoxyphenylacetic acid, 1.83g (0.012mol) of 2-fluoro-3-hydroxy-4-methoxybenzaldehyde to a 50ml three-neck flask, add 3.2ml of acetic anhydride and 1.6ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath. The mixture was heated to 150°C and reacted for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, and the mixture was reacted for 1 hour while maintaining the temperature at 80°C. The mixture was cooled to room temperature, 12ml of 40% sodium hydroxide solution was dropped, and the reaction was continued for 1 hour. Then, 10ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued. The mixture was filtered, and the filter cake was washed with ice-cooled absolute ethanol and dried under reduced pressure to obtain 2.56g of off-white crystalline solid (yield 67.6%). The off-white crystalline solid was (E)-3-(2-fluoro-3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0051] In a 10ml three-neck flask, 1.74g (4.84mmol) of (E)-3-(2-fluoro-3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 0.37g (5.08mmol) of n-butylamine, and 10ml of anhydrous ethanol were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, the mixture was stirred for 1 hour, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.78g of off-white crystalline solid (compound 9) (yield 81.5%). [M+H] + :452.20. 1 H-NMR (CDCl3, δ(ppm)): 0.912(3H), 1.276(2H), 2.314(2H), 3.251(2H), 3.586(3H), 3.773(9H), 6.643(2H), 7.084(1H), 7.237(1H), 7.539(1H).

[0052] Example 10 Preparation of Compound 10 of the Invention TIFF0007674429000022.tif64170 Preparation method: Add 2.26 g (0.010 mol) of 3,4,5-trimethoxyphenylacetic acid and 1.99 g (0.012 mol) of 4-fluoro-3,5-dimethoxybenzaldehyde to a 50 ml three-neck flask, then add 3.2 ml of acetic anhydride and 1.6 ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath for 15 minutes. The mixture was heated to 0°C, kept at 40°C for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the mixture was reacted for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.47g of off-white crystalline solid (yield 62.9%). The off-white crystalline solid was (E)-3-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0053] In a 10ml three-neck flask, 1.81g (4.84mmol) of (E)-3-(3,5-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 10ml of anhydrous ethanol, and 0.7ml of 40% potassium hydroxide solution were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, the mixture was stirred for 1 hour, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.45g of off-white crystalline solid (compound 10) (yield 69.6%). [M+H] + :432.08. 1 H-NMR (CDCl3, δ (ppm)): 3.541 (3H), 3.682 (3H), 3.773 (9H), 6.327 (2H), 6.495 (1H), 6.676 (1H), 7.329 (1H).

[0054] Example 11 Preparation of Compound 11 of the Invention TIFF0007674429000023.tif64170 Preparation method: Add 2.26 g (0.010 mol) of 3,4,5-trimethoxyphenylacetic acid and 2.02 g (0.012 mol) of 3,5-dihydroxy-4-methoxybenzaldehyde to a 50 ml three-neck flask, then add 3.2 ml of acetic anhydride and 1.6 ml of triethylamine. Shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath for 1 The mixture was heated to 50°C, kept at 40°C for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the mixture was reacted for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.14g of off-white crystalline solid (yield 56.8%). The off-white crystalline solid was (E)-3-(3,5-dihydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0055] In a 10ml three-neck flask, 1.82g (4.84mmol) of (E)-3-(3,5-dihydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid, 0.37g (5.08mmol) of n-butylamine, and 10ml of anhydrous ethanol were added, shaken to homogenize, a magnetic stirrer was added, and the mixture was stirred. After a solid precipitated, the mixture was stirred for 1 hour, filtered, and the filter cake was washed with ice-cooled anhydrous ethanol and dried under reduced pressure to obtain 1.56g of off-white crystalline solid (compound 11) (yield 71.7%). [M+H] + :450.21. 1 H-NMR (CDCl3, δ(ppm)): 0.821(3H), 1.327(2H), 2.172(2H), 3.319(2H), 3.591(3H), 3.836(9H), 5.243(2H), 6.385(2H), 7.326(2H), 7.613(1H).

[0056] Example 12 Preparation of Compound 12 of the Invention TIFF0007674429000024.tif58170 Preparation method: Add 2.26 g (0.010 mol) of 3,4,5-trimethoxyphenylacetic acid, 2.19 g (0.012 mol) of 3-hydroxy-2,4-dimethoxybenzaldehyde to a 50 ml three-neck flask, add 3.2 ml of acetic anhydride and 1.6 ml of triethylamine, shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath for 1 The mixture was heated to 50°C, kept at 40°C for 5 hours, cooled to 80°C, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the mixture was reacted for 1 hour while maintaining the temperature at 80°C, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the mixture was stirred for 1 hour and the reaction was continued, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.38g of off-white crystalline solid (yield 61.0%). The off-white crystalline solid was (E)-3-(3-hydroxy-2,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0057] In a 10 ml three-neck flask, 1.89 g (4.84 mmol) of (E)-3-(3-hydroxy-2,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid and 0.74 g (5.08 mmol) of lysine were added, and 15 ml of ethanol was added. The mixture was shaken to homogenize, a magnetic stirrer was added, and the mixture was heated in a water bath to 60°C. When the mixture was dissolved and became transparent, it was cooled to room temperature, and the solid was precipitated. The mixture was then stirred for 1 hour, filtered, and the filter cake was washed with ice-cold anhydrous ethanol and dried under reduced pressure to obtain 1.85 g of off-white crystalline solid (compound 12) (yield 75.5%). [M+H] + :537.24. 1 H-NMR(CDCl3,δ(ppm)):1.183(2H),1.649(2H),2.173(2H),3.286(2H),3.513(1H),3.597 (3H), 3.876(9H), 5.237(2H), 5.459(1H), 6.525(2H), 7.193(1H), 7.315(1H), 7.613(1H).

[0058] Example 13 Preparation of Compound 13 of the Invention TIFF0007674429000025.tif59170 Preparation method: Add 2.26 g (0.010 mol) of 3,4,5-trimethoxyphenylacetic acid and 1.99 g (0.012 mol) of 4-ethoxy-3-hydroxybenzaldehyde to a 50 ml three-neck flask, then add 3.2 ml of acetic anhydride and 1.6 ml of triethylamine. Shake to homogenize, add a magnetic stirrer, attach a reflux condenser, introduce nitrogen gas for protection, and heat in an oil bath to 150°C. The mixture was heated to ℃, kept at ℃ for 5 hours to react, cooled to 80℃, the reflux condenser was replaced with a dropping funnel, 8ml of hydrochloric acid solution was dropped, the reaction was continued for 1 hour while maintaining 80℃, cooled to room temperature, 2ml of 40% sodium hydroxide solution was dropped, the reaction was continued for 1 hour, then 1.8ml of concentrated hydrochloric acid was dropped, and after the solid precipitated, the reaction was continued with stirring for 1 hour, filtered, the filter cake was washed with ice-cooled absolute ethanol, and dried under reduced pressure to obtain 2.02g of off-white crystalline solid (yield 53.9%). The off-white crystalline solid was (E)-3-(4-ethoxy-3-hydroxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid.

[0059] In a 10 ml three-neck flask, 1.81 g (4.84 mmol) of (E)-3-(4-ethoxy-3-hydroxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid and 0.74 g (5.08 mmol) of lysine were added, and 15 ml of ethanol was added. The mixture was shaken to homogenize, a magnetic stirrer was added, and the mixture was heated in a water bath to 60°C. When the mixture was dissolved and became transparent, it was cooled to room temperature, and the solid was precipitated. The mixture was then stirred for 1 hour, filtered, and the filter cake was washed with ice-cold anhydrous ethanol and dried under reduced pressure to obtain 2.05 g of off-white crystalline solid (compound 13) (yield 81.4%). [M+H] + :521.25. 1H-NMR(CDCl3,δ(ppm)):1.187(2H),1.341(3H),1.843(2H),2.117(2H),3.415(2H),3.523(1H), 3.786(9H),4.217(2H),5.302(2H),5.476(1H),6.558(2H),7.181(1H),7.328(1H),7.613(1H).

[0060] Example 14 Preparation of 0.9% Sodium Chloride Injection Using Compounds of the Invention A 0.9% sodium chloride intravenous injection was prepared.

[0061] Formulation 1: TIFF0007674429000026.tif53170

[0062] Formulation 2: TIFF0007674429000027.tif39170

[0063] Formulation 3: TIFF0007674429000028.tif39170

[0064] In the above formulation, in order to improve the stability of the compound, when preparing the injection solution, the stabilizer, antioxidant, and pH adjuster that are commonly used in drugs can be added according to the general method.The stabilizer is, for example, cyclodextrin clathrate, surfactant (anionic surfactant, cationic surfactant, amphoteric surfactant, nonionic surfactant), etc., the antioxidant is, for example, sodium sulfite, sodium hydrogen sulfite, sodium pyrosulfite, sodium thiosulfate, ascorbic acid, cysteine, etc., and the pH adjuster is, for example, citric acid, fumaric acid, glutamic acid, L-aspartic acid, lactic acid, lactobionic acid, galacturonic acid, glucuronic acid, ascorbic acid, hydrochloric acid, acetic acid, etc.

[0065] Example 15 Preparation of sterile powder injection using the compound of the present invention Formulation 1: TIFF0007674429000029.tif33170

[0066] Formulation 2: TIFF0007674429000030.tif33170

[0067] Formulation 3: TIFF0007674429000031.tif26170

[0068] Example 16 Preparation of 5% glucose injection using compounds of the present invention A 5% glucose solution for intravenous injection was prepared.

[0069] Formulation 1: TIFF0007674429000032.tif53170

[0070] Formulation 2: TIFF0007674429000033.tif39170

[0071] Formulation 3: TIFF0007674429000034.tif46170

[0072] Example 17 Preparation of tablets using compounds of the present invention Formulation: TIFF0007674429000035.tif53170

[0073] When preparing tablets, the filler may be selected, for example, starch, dextrin, powdered sugar, pregelatinized starch, lactose, glucose, microcrystalline cellulose, calcium carbonate, calcium sulfate, calcium bicarbonate, etc.; the binder may be selected, for example, hydroxypropylmethylcellulose, povidone, starch slurry, dextrin slurry, syrup, gelatin slurry, sodium alginate, polyethylene glycol, peach gum, gum arabic, etc.; and the disintegrant may be selected, for example, cross-carboxymethylcellulose sodium, crospovidone, carboxymethylstarch sodium, hydroxypropylstarch, low-substituted hydroxypropylcellulose, citric acid, tartaric acid, acid anhydride, sodium bicarbonate, sodium carbonate, etc.

[0074] Example 18 Preparation of capsules using the compounds of the present invention Formulation: TIFF0007674429000036.tif53170

[0075] When preparing capsules, the filler may be selected, for example, starch, dextrin, powdered sugar, pregelatinized starch, lactose, glucose, microcrystalline cellulose, calcium carbonate, calcium sulfate, calcium bicarbonate, etc., and the binder may be selected, for example, hydroxypropylmethylcellulose, povidone, starch slurry, dextrin slurry, syrup, gelatin slurry, sodium alginate, polyethylene glycol, peach gum, gum arabic, etc.

[0076] The following specific test examples demonstrate the beneficial effects of the present invention.

[0077] Test Example 1. Antigen CD8 in a mouse Lewis non-small cell lung cancer model using the compound of the present invention + Studies on the concentration of killer T lymphocytes LLC (mouse lung carcinoma cells) are mouse Lewis lung carcinoma cells. The method for modeling LLC cell lung cancer in mice is as follows.

[0078] LLC cells were cultured in a general-purpose medium containing 10% fetal bovine serum and 100 μg / ml penicillin-streptomycin solution, and were routinely cultured in a cell culture incubator at 37°C and 5% CO2. LLC cells grown to the logarithmic phase were digested with 0.25% trypsin (containing EDTA) to prepare a cell suspension, centrifuged at 1000 rpm / min for 3 min, the supernatant was discarded, and the cell pellet was collected and resuspended in serum-free and penicillin-streptomycin solution-free medium, centrifuged again under the same conditions as for cell counting to adjust the volume, and the cell concentration was 1 × 10 7 The tumor cell inoculation was completed by injecting 200 μl of the cell suspension into the right flank of each mouse. The tumors were allowed to grow for approximately 10 days before reaching a volume of 100 mm 3 When this was reached, administration was started.

[0079] Preparation of drugs: Compounds 1 to 13 of the present invention were prepared using water for injection to prepare injection solutions.

[0080] Experimental method: Using intraperitoneal injection, mice with LLC cell lung cancer that had been successfully modeled were treated with an intraperitoneal injection of 400mg / kg of an injection solution containing the compound of the present invention. A negative group was set up in which no treatment was given (model mice were intraperitoneally injected with an equal volume of water for injection only) and an experimental group in which treatment was given (model mice were intraperitoneally injected with an injection solution prepared with the compound of the present invention). The intraperitoneal injection method was to administer the compound once a day at a fixed time, weigh the mice before administration, and administer the compound in a volume of 10ml / kg for 10 consecutive days. On the 10th day after treatment, the mice were killed and the subcutaneous tumors were excised. Antigen CD8 in tumor tissues of the negative and administration groups + The proportion of killer T lymphocytes was measured by flow cytometry.

[0081] CD8 in tumor tissue + Tumor-infiltrating lymphocytes were measured by flow cytometer. The specific steps are as follows:

[0082] The mouse was killed, the abdominal epidermis of the mouse was incised with ophthalmic scissors to fully expose the subcutaneous tumor, and the subcutaneous tumor tissue on the right flank of the mouse was gradually peeled off using forceps and ophthalmic scissors. After washing the tumor tissue with PBS, the tumor tissue peeled off with ophthalmic scissors was cut into small pieces of about 1 mm, and the cut tumor tissue was placed in 3 ml of PBS buffer solution containing 100 μg / ml hyaluronidase. The centrifuge tube containing the digestion solution was placed in a water bath pot and water bathed at 37 °C for 20 min, and the tumor tissue was thoroughly digested by spraying and shaking with a pipette every 5 min. The digestion solution containing the tumor tissue that was not completely digested was filtered through a 70 μm stainless steel cell strainer, and the remaining undigested tumor tissue was crushed on the 70 μm cell strainer using the plunger of a 20 ml syringe, and washed five times with 1 ml of PBS while being crushed. The cell suspension that passed through the cell strainer was transferred to a 15 ml centrifuge tube and centrifuged at 4000 rpm for 3 min. The cell precipitate was collected and 2 ml of red blood cell lysis buffer was added. After standing for 10 min, the cells were centrifuged again at 4000 rpm for 3 min. After washing with PBS, 2 to 3 ml of PBS was added to resuspend the cells.

[0083] An appropriate amount of cells was used as a sample for flow analysis, and the cell suspension was counted and the cell concentration was adjusted to 5 × 10 6 The cell suspension was adjusted to 100 μl / ml, and 100 μl of the cell suspension was aspirated using a pipette gun and added to a flow cytometry tube. FFITC anti mouse CD3, PE-Cy5 anti-mouse CD8, and PE / Cyanine7 anti-mouse CD45 were added to each flow cytometry tube, and the first two were added simultaneously to one flow cytometry tube. After the addition of the fluorescent antibodies, the flow cytometry tube was placed on ice again and incubated for 20 min in the dark. 2 ml of sterile PBS was then added to the flow cytometry tube, which was then centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 0.5 ml of sterile PBS was added to resuspend the cells, and the total number of cells was adjusted to 1 × 10 6 were selected, measured on a flow cytometer, and the results were analyzed using BD-FACSDiVa.

[0084] CD45 + , CD3 + and CD8 + CD8 in cells + The proportion of cytotoxic T lymphocytes containing the antigen was compared.

[0085] Tumor immunohistochemistry microscopic images were processed by the IHC Profiler plugin in ImageJ, and color deconvolution was performed on the stained images to separate the blue negative and brown positive areas and obtain the percentage of positive areas relative to the total stained area.

[0086] Drugs and CD8 in tumor tissue + The effect on the distribution of killer T lymphocytes and the tumor cell killing effect were measured by immunohistochemistry. The specific procedure is as follows:

[0087] Mice were sacrificed 10 days after treatment, and the abdominal epidermis of the mice was incised with ophthalmic scissors, the tumors were excised, and placed in formalin for fixation. After fixation, paraffin embedding was performed, and two consecutive 3 μm sections were cut and placed on glass slides and dried (60 °C, 1 h). After addition of primary antibodies, the sections were left overnight at 4 °C, and then secondary antibodies were added. The sections were developed with diaminobenzidine (DAB), restained with hematoxylin, dehydrated, cleared, and mounted.

[0088] For whole slide images taken at 20x magnification, stained section images were processed in ImageJ to separate blue negative and brown positive areas and calculate the percentage of positive areas relative to the total area of ​​the section.

[0089] The study results are shown in Table 2. Table 2 Compounds of the present invention inhibit CD8 + The effect of α on the concentration in tumor tissue and the distribution area ratio TIFF0007674429000037.tif138170

[0090] Description of experimental results: The compounds of the present invention bind to the antigen CD8 + It has the dual effect of attracting killer T lymphocytes to tumor tissue and targeting and destroying the inner walls of tumor blood vessels. + It can significantly improve the concentration rate and distribution area of ​​killer T lymphocytes in tumor tissue, and can also detect antigen CD8 + The killer T lymphocytes were able to eliminate tumor cells while simultaneously cutting off the blood supply to the tumor tissue, causing rapid necrosis inside the solid tumor.

[0091] Test Example 2. Effect of the compound of the present invention on adhesion proteins of non-fused human umbilical vein endothelial cells (HUVEC) Confluent HUVECs: HUVECs cells in the logarithmic growth phase were taken and diluted with 8 × 10 4 The cells were inoculated into a 96-well culture plate at 100 μl / ml and 100 μl / well (i.e., 8000 cells / well), and the cells were grown in a culture medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO2, and after 24 h, the test compound was added. The test sample was diluted with PBS(-) and then added to each well (10 μL / well), and 3 wells were set up at the same concentration. After culturing in the incubator for 24 hours, 10 μl of CCK-8 solution was added to each well, taking care not to generate bubbles. After continuing to culture in the incubator for about 2.5 hours, it became possible to measure the absorbance. The OD value and IC50 value were measured at a wavelength of 450 nm using an MK-2 fully automatic microplate reader.

[0092] Non-confluent HUVECs: HUVECs cells in the logarithmic growth phase were taken and diluted with 2 × 10 4The cells were inoculated into a 96-well culture plate at 100 μl / ml and 100 μl / well (i.e., 2000 cells / well), and the cells were grown in a culture medium containing 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO2, and after 24 h, the test compound was added. The test sample was diluted with PBS(-) and then added to each well (10 μL / well), and 3 wells were set up at the same concentration. After culturing in the incubator for 24 hours, 10 μl of CCK-8 solution was added to each well, taking care not to generate bubbles. After continuing to culture in the incubator for about 2.5 hours, it became possible to measure the absorbance. The OD value and IC50 value were measured at a wavelength of 450 nm using an MK-2 fully automatic microplate reader.

[0093] An experimental model was prepared using human umbilical vein endothelial cells (HUVECs) in a non-fused state to mimic neo-tumor blood vessels, and HUVECs in a fused state to mimic normal blood vessels. Human umbilical vein endothelial cells in two different states were treated with the same dose of 10 μM of the compound to mimic the fused cell state, and proteome samples were prepared. Peptide fragments were prepared and then labeled with TMT for quantitative proteomics studies. The efficiency of TMT labeling was over 99.4%, and with a stringent score of 1% FDR, 28,372 peptide fragments from a total of 4,934 proteins were identified, of which 4,909 were quantifiable proteins, and the quality was reliable. The drug-treated groups in the non-fused and fused states were compared with their respective control groups, and the control group was used as the reference, and a difference of more than 1.5-fold or less than 0.67-fold difference was used as the significant difference protein score criterion.

[0094] The total cellular protein of each non-fused cell group and fused cell group sample was extracted, and 2 μg of the protein sample was taken for mass measurement, and quantitative proteomics experiments were performed, and protein concentration measurement was completed using grayscale quantification. The specific flow chart is shown in Figure 1.

[0095] Analysis of the obtained data: The human protein database (Scientific name: Homo sapiens, 9606) was downloaded from UniProt. The database was searched using the MaxQuant (v 1.5.5.1) search engine. The TMT quantitative proteomics experiment identified a total of 4934 proteins (4909 quantified proteins) and 28372 peptide fragments, with a labeling efficiency of over 99% and a total enzyme cleavage ratio of 86.3%.

[0096] The results of proteomic testing of the compounds of the present invention on non-confluent human umbilical vein endothelial cells (HUVEC) are shown in Table 3. Table 3. Effects of the compounds of the present invention on proteins TIFF0007674429000038.tif130170

[0097] GO enrichment analysis of the differential proteins showed that the differentially expressed proteins in the non-fused group were mainly involved in the regulation of cell-cell adhesion, indicating that the above compounds targeted and destroyed the adhesive function of non-fused human umbilical vein endothelial cells (HUVECs), causing endothelial cells to detach from the vascular base, thereby inducing an inflammatory response in non-fused vessels and further blocking the vessels.

[0098] Test Example 3. Therapeutic effect of the compound on human lung cancer A549 transplanted tumors in nude mice This study investigated the antitumor effect of the test sample using a human lung cancer A549 model transplanted into nude mice, and the tumor inhibition rate against human lung cancer A549 was measured by orally administering the compound at a dose of 400 mg / kg for 14 days.

[0099] Preparation: Prior to administration, compounds were prepared in distilled water to the desired concentration. Animal strain: BALB / C nude mouse (SPF grade), body weight: 19-21 g, sex: male. Number of animals in each treatment group: 8; number of animals in the control group: >12.

[0100] Test Method: A well-grown lung cancer A549 tumor mass was taken, and uniform masses of 3 mm in size were excised under aseptic conditions, and one was inoculated subcutaneously into the right axilla of each mouse using a trocar to set up the following 9 groups, with 8 mice in each treatment group and more than 12 mice in the control group.

[0101] Fourteen days after inoculation, the average volume of the tumor mass was approximately 100 mm 3 Once this was confirmed, the animals were regrouped according to tumor size, and animals with tumors that were too large or too small were eliminated to make the average tumor volume of each group approximately equal, and then administration was initiated. The treatment group received the drug orally once a day for 14 consecutive days, with a single administration volume of 0.5 ml / 20 g body weight, while the control group received the same volume of saline. From the 14th day after inoculation, the tumor's major axis a (mm) and the tumor's minor axis b (mm) perpendicular to it were measured twice a week using a digital electronic caliper. The formula for calculating tumor volume is TV=ab 2 / 2, the formula for calculating relative tumor volume is RTV=Vt / Vo, where Vo is the tumor volume measured at the time of caging (i.e., d1), and Vt is the tumor volume at each measurement.

[0102] The results were determined according to the following formula: TIFF0007674429000039.tif24170

[0103] The test results are shown in Table 4. Table 4. Tumor-inhibitory effect of compounds on lung cancer A549 tumor masses TIFF0007674429000040.tif95170Comparison with control group: * P < 0.05, ** P<0.01. Comparison of other compounds with compound 6: # P < 0.05, ## P<0.01.

[0104] Explanation of the above test results: Some compounds of the present invention have significant inhibitory effects on lung cancer, and in particular, compound 6 has the best inhibitory effect on lung cancer, and can be used to prepare drugs for treating lung cancer.

[0105] Test Example 4. Therapeutic effect of the compound on human hepatoma QGY7703 transplanted tumors in nude mice This study investigated the antitumor effect of the test sample using a human hepatic cancer QGY7703 model transplanted into nude mice, and administered a dose of 100 mg / kg by intravenous injection for 14 days to measure the tumor inhibition rate against human hepatic cancer QGY7703.

[0106] Preparation: Prior to administration, compounds were prepared in sterile saline to the desired concentration. Animal strain: BALB / C nude mouse (SPF grade), body weight: 19-21 g, sex: male. Number of animals in each treatment group: 8; number of animals in the control group: >12.

[0107] Test Method: A mass of well-grown human hepatocarcinoma QGY7703 tumor was taken, and uniform masses of 3 mm in size were excised under aseptic conditions, and one was inoculated subcutaneously into the right axilla of each mouse using a trocar to set up the following 9 groups, with 8 mice in each treatment group and more than 12 mice in the control group.

[0108] Fourteen days after inoculation, the average volume of the tumor mass was approximately 100 mm 3 Once this was confirmed, the animals were regrouped according to tumor size, and animals with tumors that were too large or too small were eliminated to make the average tumor volume of each group approximately equal, and then administration was initiated. The treatment group received the drug via intravenous injection once a day for 14 consecutive days, with a single administration volume of 0.1 ml / 20 g body weight, while the control group received the same volume of sterile saline. From the 14th day after inoculation, the tumor's major axis a (mm) and the tumor's minor axis b (mm) perpendicular to it were measured twice a week using a digital electronic caliper. The formula for calculating tumor volume is TV=ab 2 / 2, the formula for calculating relative tumor volume is RTV=Vt / Vo, where Vo is the tumor volume measured at the time of caging (i.e., d1), and Vt is the tumor volume at each measurement.

[0109] The results were determined according to the following formula: TIFF0007674429000041.tif24170

[0110] The test results are shown in Table 5. Table 5. Tumor-inhibitory effect of compounds on hepatoma QGY7703 tumor mass TIFF0007674429000042.tif95170Comparison with control group: * P < 0.05, ** P<0.01. Comparison of other compounds with compound 6: # P < 0.05, ## P<0.01.

[0111] Explanation of the above test results: Some compounds of the present invention have significant inhibitory effects on liver cancer, and in particular, compound 6 has the best inhibitory effect on liver cancer, and can be used to prepare drugs for treating liver cancer.

[0112] Test Example 5. Therapeutic effect of the compound on human gastric cancer MGC803 transplanted tumors in nude mice This study investigated the antitumor effect of the test sample using a human gastric cancer MGC803 model transplanted into nude mice, and administered a dose of 200 mg / kg by intraperitoneal injection for 14 days to measure the tumor inhibition rate against human gastric cancer MGC803.

[0113] Preparation: Prior to administration, compounds were prepared in sterile saline to the desired concentration. Animal strain: BALB / C nude mouse (SPF grade), body weight: 19-21 g, sex: male. Number of animals in each treatment group: 8; number of animals in the control group: >12.

[0114] Test Method: A well-grown human gastric cancer MGC803 tumor mass was taken, and 3 mm uniform masses were excised under aseptic conditions. One mass was inoculated subcutaneously into the right axilla of each mouse using a trocar to set up the following 9 groups, with 8 mice in each treatment group and more than 12 mice in the control group.

[0115] Fourteen days after inoculation, the average volume of the tumor mass was approximately 100 mm 3 After confirming that this was the case, the animals were regrouped according to tumor size, and animals with tumors that were too large or too small were eliminated to make the average tumor volume of each group approximately equal, and then administration was started. Administration was performed once a day by intraperitoneal injection for 14 consecutive days, with a volume of 0.2 ml per injection per 20 g of body weight. From the 14th day after inoculation, the tumor's major axis a (mm) and the tumor's minor axis b (mm) perpendicular to this were measured twice a week using a digital electronic caliper. The formula for calculating tumor volume was TV=ab 2 / 2, the formula for calculating relative tumor volume is RTV=Vt / Vo, where Vo is the tumor volume measured at the time of caging (i.e., d1), and Vt is the tumor volume at each measurement.

[0116] The results were determined according to the following formula: TIFF0007674429000043.tif24170

[0117] The test results are shown in Table 6. Table 6. Tumor-inhibitory effect of compounds on gastric cancer MGC803 tumor mass TIFF0007674429000044.tif86170Comparison with control group: * P < 0.05, ** P<0.01. Comparison of other compounds with compound 6: # P < 0.05, ## P<0.01.

[0118] Explanation of the above test results: Some compounds of the present invention have significant inhibitory effects on gastric cancer, and in particular, compound 6 has the best inhibitory effect on gastric cancer, and can be used to prepare drugs for treating gastric cancer.

[0119] Test Example 6. Therapeutic effect of the compound on human ovarian cancer A2780 transplanted tumors in nude mice This study investigated the antitumor effect of the test sample using a human ovarian cancer A2780 model transplanted into nude mice, and administered a dose of 200 mg / kg by intraperitoneal injection for 14 days to measure the tumor inhibition rate against human ovarian cancer A2780.

[0120] Preparation: Prior to administration, compounds were prepared in sterile saline to the desired concentration. Animal strain: BALB / C nude mouse (SPF grade), body weight: 19-21 g, sex: male. Number of animals in each treatment group: 8; number of animals in the control group: >12.

[0121] Test Method: A well-grown ovarian cancer A2780 tumor mass was taken, and uniform masses of 3 mm in size were excised under aseptic conditions, and one mass was inoculated subcutaneously into the right axilla of each mouse using a trocar to set up the following 9 groups, with 8 mice in each treatment group and more than 12 mice in the control group.

[0122] Fourteen days after inoculation, the average volume of the tumor mass was approximately 100 mm 3 After confirming that this was the case, the animals were regrouped according to tumor size, and animals with tumors that were too large or too small were eliminated to make the average tumor volume of each group approximately equal, and then administration was initiated. The treatment group was administered the drug once a day by intraperitoneal injection for 14 consecutive days, with a volume of 0.2 ml per injection per 20 g of body weight, while the control group was administered the same volume of sterile saline. From the 14th day after inoculation, the tumor's major axis a (mm) and the tumor's minor axis b (mm) perpendicular to this were measured twice a week using a digital electronic caliper. The formula for calculating tumor volume was TV=ab 2 / 2, the formula for calculating relative tumor volume is RTV=Vt / Vo, where Vo is the tumor volume measured at the time of caging (i.e., d1), and Vt is the tumor volume at each measurement.

[0123] The results were determined according to the following formula: TIFF0007674429000045.tif24170

[0124] The test results are shown in Table 7. Table 7. Tumor-inhibitory effect of compounds on ovarian cancer A2780 tumor mass TIFF0007674429000046.tif86170Comparison with control group: * P < 0.05, ** P<0.01. Comparison of other compounds with compound 6: # P < 0.05, ## P<0.01.

[0125] Explanation of the above test results: Some compounds of the present invention have significant inhibitory effect on ovarian cancer, and in particular, compound 6 has the best inhibitory effect on ovarian cancer, and can be used in the preparation of drugs for treating ovarian cancer.

[0126] Test Example 7. Therapeutic effect of the compound on human prostate cell LNCaP transplanted tumor in nude mice This study investigated the antitumor effect of the test sample using a human prostate cell LNCaP model transplanted into nude mice, and administered a dose of 200 mg / kg by intraperitoneal injection for 14 days to measure the tumor inhibition rate against human prostate cell LNCaP.

[0127] Preparation: Prior to administration, compounds were prepared in sterile saline to the desired concentration. Animal strain: BALB / C nude mouse (SPF grade), body weight: 19-21 g, sex: male. Number of animals in each treatment group: 8; number of animals in the control group: >12.

[0128] Test Method: A mass of well-grown human prostate cell LNCaP tumor was taken, and under aseptic conditions, uniform masses of 3 mm in size were excised, and one mass was inoculated subcutaneously into the right armpit of each mouse using a trocar to set up the following 9 groups, with 8 mice in each treatment group and more than 12 mice in the control group.

[0129] Fourteen days after inoculation, the average volume of the tumor mass was approximately 100 mm 3After confirming that this was the case, the animals were regrouped according to tumor size, and animals with tumors that were too large or too small were eliminated to make the average tumor volume of each group approximately equal, and then administration was initiated. The treatment group was administered the drug once a day by intraperitoneal injection for 14 consecutive days, with a volume of 0.2 ml per injection per 20 g of body weight, while the control group was administered the same volume of sterile saline. From the 14th day after inoculation, the tumor's major axis a (mm) and the tumor's minor axis b (mm) perpendicular to this were measured twice a week using a digital electronic caliper. The formula for calculating tumor volume was TV=ab 2 / 2, the formula for calculating relative tumor volume is RTV=Vt / Vo, where Vo is the tumor volume measured at the time of caging (i.e., d1), and Vt is the tumor volume at each measurement.

[0130] The results were determined according to the following formula: TIFF0007674429000047.tif24170

[0131] The test results are shown in Table 8 below. Table 8. Tumor-inhibitory effect of compounds on prostate LNCaP tumor mass TIFF0007674429000048.tif78170Comparison with control group: * P < 0.05, ** P<0.01. Comparison of other compounds with compound 4: # P < 0.05, ## P<0.01.

[0132] Explanation of the above test results: Some compounds of the present invention have significant inhibitory effects on prostate cancer, and in particular, compound 4 has the best inhibitory effect on prostate cancer, and can be used to prepare drugs for treating prostate cancer.

[0133] Test Example 8. Therapeutic effect of compounds on human renal cell carcinoma AGHN transplanted tumors in nude mice This study investigated the antitumor effect of the test sample using a human renal cell carcinoma AGHN model transplanted into nude mice, and administered a dose of 200 mg / kg by intraperitoneal injection for 14 days to measure the tumor inhibition rate against human renal cell carcinoma AGHN.

[0134] Preparation: Prior to administration, compounds were prepared in sterile saline to the desired concentration. Animal strain: BALB / C nude mouse (SPF grade), body weight: 19-21 g, sex: male. Number of animals in each treatment group: 8; number of animals in the control group: >12.

[0135] Test Method: A well-grown human renal cell carcinoma AGHN tumor mass was taken, and under aseptic conditions, uniform masses of 3 mm in size were excised, and one mass was inoculated subcutaneously into the right axilla of each mouse using a trocar to set up the following 9 groups, with 8 mice in each treatment group and more than 12 mice in the control group.

[0136] Fourteen days after inoculation, the average volume of the tumor mass was approximately 100 mm 3 After confirming that this was the case, the animals were regrouped according to tumor size, and animals with tumors that were too large or too small were eliminated to make the average tumor volume of each group approximately equal, and then administration was initiated. The treatment group was administered the drug once a day by intraperitoneal injection for 14 consecutive days, with a volume of 0.2 ml per injection per 20 g of body weight, while the control group was administered the same volume of sterile saline. From the 14th day after inoculation, the tumor's major axis a (mm) and the tumor's minor axis b (mm) perpendicular to this were measured twice a week using a digital electronic caliper. The formula for calculating tumor volume was TV=ab 2 / 2, the formula for calculating relative tumor volume is RTV=Vt / Vo, where Vo is the tumor volume measured at the time of caging (i.e., d1), and Vt is the tumor volume at each measurement.

[0137] The results were determined according to the following formula: TIFF0007674429000049.tif24170

[0138] The test results are shown in Table 9. Table 9. Tumor-inhibitory effect of compounds on renal cell carcinoma AGHN tumor mass TIFF0007674429000050.tif86170Comparison with control group: * P < 0.05, ** P<0.01. Comparison of other compounds with compound 4: # P < 0.05, ## P<0.01.

[0139] Explanation of the above test results: Some compounds of the present invention have significant inhibitory effect on renal cell carcinoma, and in particular, compound 4 has the best inhibitory effect on renal cell carcinoma, and can be used to prepare drugs for treating renal cell carcinoma.

[0140] Test Example 9. In vivo tumor necrosis by compound 6 1. Test method: Well-grown solid tumors (human lung cancer 95D and human hepatoma Bel7404) were taken, and uniform masses of 2-3 mm in size were cut out under aseptic conditions, and each was inoculated subcutaneously into the right axilla of each nude mouse with a trocar. 14 days after inoculation, the compound was orally administered at a dose of 400 mg / kg, with a volume of 0.5 ml / body weight of 20 g (compound 6), and the drug was prepared using sterile saline. At different times after administration (4 h, 24 h), the nude mice were killed, dissected, and tumor tissues were removed, fixed in 10% formalin, and pathological sections were prepared, stained with HE, and photographed to observe the necrotic state inside the tumor and the cell proliferation state.

[0141] 2. Test results: HE staining is shown in Figure 2 and Figure 3. After 4 hours of oral administration of 400 mg / kg of compound 6, necrosis inside the tumor tissue began to appear, and within 24 hours, extensive necrosis (light-colored areas) was caused inside the tumor. This indicates that compound 6 of the present invention can effectively induce tumor necrosis and achieve the purpose of anti-tumor.

[0142] Test Example 10. In vivo tumor intravascular necrosis by compound 6 1. Test method: Well-grown solid tumors (human lung cancer 95D and human hepatoma Bel7404) were taken, and under aseptic conditions, uniform masses of 2-3 mm in size were cut out, and each was inoculated subcutaneously into the right axilla of each nude mouse with a trocar. 14 days after inoculation, the compound was orally administered at a dose of 400 mg / kg, with a volume of 0.5 ml / body weight of 20 g (compound 6), and the drug was prepared using sterile saline. At different times (4 h, 24 h) after administration, the nude mice were killed, dissected to remove the tumor tissues, fixed in 10% formalin, and pathological sections were prepared, PCNA immunohistochemical staining was performed, and photographs were taken to observe the necrotic state of the tumor blood vessels inside the tumor.

[0143] 2. Test results: PCNA immunohistochemistry (Figures 4 and 5) shows that 4 hours after oral administration of 400 mg / kg of compound 6, the proliferation of vascular endothelial cells in the tumor tissue was only partial, and 24 hours after administration, the tumor tissue was extensively blocked by blood flow and necrotized. The decreased expression of PCNA in the tumor tissue indicates a good tumor vascular destruction effect in vivo.

[0144] Test Example 11. Inhibitory effect of the combined use of Compound 3 and Lenvatinib on the growth of human hepatoma Bel7404 xenograft tumors in nude mice Test method: Human hepatoma Bel7404 tumors were transplanted into nude mice by subcutaneously inoculating the human hepatoma Bel7404 cell line into the axilla of nude mice. The cell inoculation dose was 1×10 6 After inoculation to form a transplanted tumor, the tumor was passaged for three generations in nude mice before use. 3 After excision and homogenization under sterile conditions, 1 × 10 7 A cell suspension of 0.1 ml was prepared and 0.1 ml was inoculated subcutaneously into the right axilla of nude mice. The diameter of the tumor implanted in the nude mice was measured with a caliper. 3When the mice had grown to 100 mg / kg, they were randomly divided into groups, with 6 mice per group. The antitumor effect of the test substances was dynamically observed using a method for measuring tumor diameter. Compound 3 was orally administered at 400 mg / kg once a day for 3 weeks, and lenvatinib was orally administered at 10 mg / kg once a day for 2 weeks. The tumor diameter was measured once every 3 days. The negative control group was orally administered the same amount of saline solution. The effect of combined administration was calculated using the Q value based on Kim's formula.

[0145] Q = Ea+b / (Ea+Eb-Ea×Eb) Ea+b is the tumor inhibition rate of the combined administration, and Ea and Eb are the tumor inhibition rates of drug A and drug B, respectively. A Q value between 0.85 and 1.15 was considered additive (+), and a Q value >1.15 was considered potentiating (++).

[0146] The test results are shown in Table 10. Table 10. Inhibitory effect of the combination of compound 3 and lenvatinib on the growth of human hepatocellular carcinoma Bel7404 xenograft tumors in nude mice. TIFF0007674429000051.tif56170Compared to the blank control group, * P<0.05

[0147] Test conclusions: The Q value of compound 3 in combination with lenvatinib was 0.970, indicating a significant additive antitumor effect, but no synergistic antitumor effect. There was no significant decrease in animal body weight, and no significant additive toxic effect was observed with the combination of lenvatinib.

[0148] Test Example 12. Inhibitory effect of the combination of Compound 6 and paclitaxel on the growth of human gastric cancer SGC7901 xenograft tumors in nude mice Test method: Human gastric cancer SGC7901 tumors were transplanted into nude mice by subcutaneously inoculating the human gastric cancer SGC7901 cell line into the axilla of nude mice. The cell inoculation dose was 1×10 6After inoculation to form a transplanted tumor, the tumor was passaged for three generations in nude mice before use. 3 After excision and homogenization under sterile conditions, 1 × 10 7 A cell suspension of 0.1 ml was prepared and 0.1 ml was inoculated subcutaneously into the right axilla of nude mice. The diameter of the tumor implanted in the nude mice was measured with a caliper. 3 When the mice had grown to 100 mg / kg, they were randomly divided into groups, with 6 mice per group. The antitumor effect of the test substances was dynamically observed using a method for measuring tumor diameter. Compound 6 was administered intraperitoneally at 200 mg / kg, once every other day for 3 weeks, and paclitaxel was administered intravenously at 10 mg / kg, once every other day for 3 weeks. The tumor diameter was measured once every 3 days. The administration volume was 0.1 ml / 20 g. The negative control group was administered the same amount of saline solution via intravenous injection. The effect of combined administration was calculated using the Q value based on Kim's formula.

[0149] Q = Ea+b / (Ea+Eb-Ea×Eb) Ea+b is the tumor inhibition rate of the combined administration, and Ea and Eb are the tumor inhibition rates of drug A and drug B, respectively. A Q value between 0.85 and 1.15 was considered additive (+), and a Q value >1.15 was considered potentiating (++).

[0150] The test results are shown in Table 11. Table 11. Inhibitory effect of the combination of compound 6 and paclitaxel on the growth of human gastric cancer SGC7901 xenograft tumors in nude mice. TIFF0007674429000052.tif56170Compared to the blank control group, * P<0.05

[0151] Test conclusions: The Q value of compound 6 in combination with paclitaxel was 0.927, which showed a significant additive antitumor effect, but no synergistic antitumor effect. There was no significant decrease in animal weight, and no significant additive toxic effect was observed with the combination of paclitaxel.

[0152] Test Example 13. Inhibitory effect of the combination of Compound 8 and carboplatin on the growth of human lung cancer A549 xenograft tumors in nude mice Test method: Human lung cancer A549 tumors were transplanted into nude mice by subcutaneously inoculating the human lung cancer A549 cell line into the axilla of nude mice. The cell inoculation dose was 1 × 10 6 After inoculation to form a transplanted tumor, the tumor was passaged for three generations in nude mice before use. 3 After excision and homogenization under sterile conditions, 1 × 10 7 A cell suspension of 0.1 ml was prepared and 0.1 ml was inoculated subcutaneously into the right axilla of nude mice. The diameter of the tumor implanted in the nude mice was measured with a caliper. 3 When the mice had grown to 100 mg / kg, they were randomly divided into groups, with 6 mice per group. The antitumor effect of the test substances was dynamically observed using a method for measuring tumor diameter. Compound 8 was administered by intraperitoneal injection at 200 mg / kg, once every other day for 3 weeks, and carboplatin was administered by intraperitoneal injection at 10 mg / kg, once every other day for 3 weeks. The tumor diameter was measured once every 3 days. The administration volume was 0.1 ml / 20 g. The negative control group was administered the same amount of saline solution by intravenous injection. The effect of combined administration was calculated by the Q value based on Kim's formula.

[0153] Q = Ea+b / (Ea+Eb-Ea×Eb) Ea+b is the tumor inhibition rate of the combined administration, and Ea and Eb are the tumor inhibition rates of drug A and drug B, respectively. A Q value between 0.85 and 1.15 was considered additive (+), and a Q value >1.15 was considered potentiating (++).

[0154] The test results are shown in Table 12. Table 12. Inhibitory effect of the combination of compound 8 and carboplatin on the growth of human lung cancer A549 xenograft tumors in nude mice. TIFF0007674429000053.tif56170Compared to the blank control group, * P<0.05

[0155] Test conclusions: The Q value of compound 8 in combination with carboplatin was 0.941, which showed a significant additive antitumor effect, but no synergistic antitumor effect. There was no significant decrease in animal body weight, and no significant additive toxic effect was observed with the combination of carboplatin.

[0156] Test Example 14. Tumor suppression effect of the combination of compound 11 and PD-L1 on Lewis non-small cell lung cancer model in mice Test method: The transplanted tumors in C57BL / 6 mice were created by subcutaneously inoculating the Lewis non-small cell lung cancer line into the axilla of C57BL / 6 mice. The cell inoculation dose was 1 × 10 6 The tumor tissues were inoculated to form transplanted tumors, which were then passaged for three generations in C57BL / 6 mice before use. 3 After excision and homogenization under sterile conditions, 1 × 10 7 The cell suspension was prepared at 100 μg / ml, and 0.1 ml of the suspension was inoculated subcutaneously into the right axilla of C57BL / 6 mice. The diameter of the tumor implanted in the C57BL / 6 mice was measured with a caliper. 3 When the mice reached 100 mm Hg, they were randomly divided into groups, with 6 mice per group. The antitumor effect of the test substances was dynamically observed by measuring the tumor size. Compound 11 was administered by intraperitoneal injection at 200 mg / kg, once every other day for 3 weeks, and PD-L1 was administered by intraperitoneal injection at 10 mg / kg, once every other day for 3 weeks. The tumor size was measured once every 3 days. The administration volume was 0.1 ml / 20 g. The negative control group was administered the same amount of saline solution by intravenous injection.

[0157] The effect of combined administration was calculated using the Q value based on Kim's formula.

[0158] Q = Ea+b / (Ea+Eb-Ea×Eb) Ea+b is the tumor inhibition rate of the combined administration, and Ea and Eb are the tumor inhibition rates of drug A and drug B, respectively. A Q value between 0.85 and 1.15 was considered additive (+), and a Q value >1.15 was considered potentiating (++).

[0159] The test results are shown in Table 13. Table 13. Tumor suppression effect of the combination of compound 11 and PD-L1 on Lewis non-small cell lung cancer model in mice. TIFF0007674429000054.tif53170Compared to the blank control group, * P<0.05

[0160] Test conclusions: The Q value of compound 11 in combination with PD-L1 was 1.16, indicating that there was a significant synergistic anti-lung cancer effect when compound 11 was combined with PD-L1. There was no significant decrease in the body weight of the animals, and no significant additive toxic effect was observed with the drug combination.

[0161] Test Example 15. Tumor suppression effect of the combination of Compound 6 and T cells in the human liver cancer QGY7703 model in NOG mice Test method: QGY7703 tumor cells were washed twice with PBS and then resuspended in PBS:Matrigel (mixed at a volume ratio of 1:1) to a cell concentration of 2.5 × 10 6 The cells were adjusted to 5 × 10 cells / ml and injected subcutaneously into the right axilla of the experimental animals at 200 μl per mouse, i.e., 5 × 10 cells per mouse. 5 The tumor was 50 mm 3 Once they had grown to a certain age, they were divided into groups and administered the drugs.

[0162] T cell culture and harvest: Donor PBMCs were activated and expanded in vitro according to standard operating procedures for T cell culture. Logarithmic growth phase cells were harvested and cultured at a medium dose (2 × 10 7 The cells were then suspended in a solvent according to the requirement (cells / ml) and prepared for use.

[0163] When the tumors grew to an appropriate size, T cells were injected and compound 6 was administered, and this time was recorded as D0. Compound 6 was administered intraperitoneally at a dose of 200 mg / kg. Administration was started on D0 and continued daily for 14 days. T cells were administered subcutaneously, and the administration site was the same as the tumor cell inoculation site, and they were administered by injection three times on D0. The injection schedule is shown in Table 14 below. Each administration of T cells was performed about 4 hours after administration of compound 6.

[0164] The general condition of the mice, the body weight of the mice, the tumor volume and the tumor weight were recorded every 3 days. Based on the tumor condition, it was decided whether to perform additional EAL injections. Table 14. Dosing regimen TIFF0007674429000055.tif29170

[0165] The effect of combined administration was calculated using the Q value based on Kim's formula. Q = Ea+b / (Ea+Eb-Ea×Eb) Ea+b is the tumor inhibition rate of the combined administration, and Ea and Eb are the tumor inhibition rates of drug A and drug B, respectively. A Q value between 0.85 and 1.15 was considered additive (+), and a Q value >1.15 was considered potentiating (++).

[0166] The test results are shown in Table 15. Table 15. Tumor suppression effect of the combination of compound 6 and T cells in the human liver cancer QGY7703 model in NOG mice TIFF0007674429000056.tif68170Compared to the blank control group, * P<0.05

[0167] Test conclusions: The Q value of compound 6 in combination with T cells was 1.20, indicating that there was a significant synergistic anti-liver cancer effect when compound 6 was combined with T cells. There was no significant decrease in the body weight of the animals, and no significant additive toxic effect was observed when T cells were combined.

[0168] Test Examples 14 and 15 demonstrate that the stilbene compound of the present invention can exert a synergistic antitumor effect when used in combination with a drug for tumor immunotherapy.

[0169] Test Example 16. Inhibitory effect of compound 6 on tumor metastasis Under sterile conditions, 2.8 x 10 cultures of B16 mouse melanoma cells in logarithmic growth phase were taken. 5 The cells were prepared into a cell suspension of 0.2 ml per mouse and inoculated into the tail vein of C57BL / 6 mice. The next day, the mice were administered the drug according to the administration method shown in Table 16. After 3 weeks, the animals were killed, the lungs of each group were removed, and the number of colonies metastasized to the lungs of the mice in each group was counted. The tumor inhibition rate was calculated based on the average number of colonies in the tumors of each group according to the formula: tumor inhibition rate % = [(average number of colonies in the control group - average number of colonies in the treatment group) / average number of colonies in the control group] x 100%. The results are shown in Table 16.

[0170] Table 16. Anti-metastatic effect of compound 6 on B16 mouse melanoma lung metastasis model TIFF0007674429000057.tif68170 ** P<0.01 vs. negative control.

[0171] The results indicate that compound 6 has an inhibitory effect on melanoma metastasis in B16 mice and shows a dose-dependent relationship, and the pathway may be related to the inhibition of cancer-associated thrombus movement.

[0172] Test Example 17. Study on the inhibition of neovascularization on the ocular surface of rats by compound 6 Four SD rats, regardless of sex, were modeled monocularly, for a total of four eyes, including one blank control (untreated group), one model group, and one treatment group 1 and one treatment group 2, respectively.

[0173] The eye was anesthetized with pentobarbital sodium (25-30 mg / kg, intraperitoneal injection), and two drops of oxybuprocaine hydrochloride eye drops were dropped into both eyes at the same time to anesthetize the ocular surface. 30 μL of NaOH (1 mol / L) solution was taken with a pipette gun and placed on a dry filter paper with a diameter of about 3 mm for 10 seconds. The wet circular filter paper was lightly attached to the center of the cornea of ​​the model eye (covering the edge of the cornea and sclera), and the adhesion was maintained for an appropriate time. The filter paper was then removed, and then saline was used to wash the eye for 1 minute using a needle. Ofloxacin eye ointment was applied after surgery to prevent infection (3 times / day, continued for 5 days).

[0174] On the 1st, 3rd, 5th, 7th and 10th days (D1, D3, D5, D7 and D10) after the modeling, the conjunctival congestion in the anterior part of the eye, corneal edema and the growth of corneal neovascularization were observed with a slit lamp.

[0175] On the fifth day (D5), neovascularization was observed, which was designated as the first day (d1) of administration of the test drug. The drug was administered three times a day for 10 consecutive days (d10). After the administration on day 10 was completed, the conjunctival congestion of the anterior eye, corneal edema, and the growth of corneal neovascularization were observed with a slit lamp. The test was completed, and the rats were euthanized.

[0176] Blank control (untreated group): No treatment, no modeling, no administration.

[0177] Model group: a circular filter paper (approximately 3 mm in diameter) moistened with 30 μL of NaOH (1 mol / L) was lightly attached to the cornea to model it, and anti-infection treatment was performed after modeling, but no administration was performed.

[0178] Treatment group 1: The administration was performed after modeling. Compound 6 was dissolved in saline to a concentration of 10 mg / mL, and 50 μL was administered per eye. The compound was instilled into the conjunctival sac of the lower eye three times per day for 10 consecutive days.

[0179] Treatment group 2: The administration was performed after modeling. Compound 6 was dissolved in saline to a concentration of 25 mg / mL, and 50 μL per eye was administered. The compound was instilled into the conjunctival sac of the lower eye three times per day for 10 consecutive days.

[0180] The results are shown in Figure 6. The ocular surface neovascularization in all of the administration groups was reduced compared to the model group, and the vascular proliferation area in the high-dose group was significantly reduced. Compound 6 has an inhibitory effect on the growth of neovascularization on the ocular surface and has a dose-response relationship.

[0181] As described above, the present invention provides the use of stilbene compounds in the preparation of antitumor drugs. The present invention has been reported to show that some stilbene compounds can inhibit the expression of CD8 in solid tumors. + It has the dual effect of attracting large numbers of T lymphocytes and targeting and destroying the inner walls of tumor blood vessels. + It was found that the killer T lymphocytes can eliminate tumor cells, while at the same time cutting off the blood supply to the tumor tissue, causing rapid necrosis inside the solid tumor, greatly improving the killing power against the tumor, and further having an inhibitory effect against tumor metastasis. The stilbene compounds of the present invention can be used to prepare antitumor drugs, and can also be used in combination with tumor immunotherapy drugs to exert a synergistic antitumor effect. These stilbene compounds can be used to prepare various antitumor drugs, since the antitumor effects of the compounds differ depending on the structure. In addition, the stilbene compounds of the present invention can inhibit the growth of blood vessels on the ocular surface, and therefore can be used to prepare drugs for treating various ocular diseases. The applications of the stilbene compounds of the present invention are promising.

Claims

1. Use of a compound of formula Va, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of a medicament for preventing and / or treating lung cancer: 。

2. Use of a compound represented by formula Va, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of a medicament for preventing and / or treating liver cancer: 。

3. Use of a compound of formula Va, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of a medicament for preventing and / or treating gastric cancer: 。

4. Use of a compound of formula Va, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of a medicament for preventing and / or treating ovarian cancer: 。

5. Use of a compound of formula Vb, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of a medicament for preventing and / or treating prostate cancer: 。

6. Use of a compound of formula Vb, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in the preparation of a medicament for preventing and / or treating renal cell carcinoma: 。

7. Use of a compound of formula Vc, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in combination with PD-L1 in the preparation of a combination drug for preventing and / or treating lung cancer: 。

8. Use of a compound of formula Va, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, in combination with T cells in the preparation of a combination drug for preventing and / or treating liver cancer: 。

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