Small molecule K-H03 and application thereof in antitumor drugs

Abstract

The invention belongs to the field of chemical synthesis, and particularly relates to a small molecule compound K-H03 with an anti-tumor effect and application of the small molecule compound K-H03 in anti-tumor drugs, and the small molecule compound has a structural formula as shown in a formula I. The compound has a good killing effect on renal cell carcinoma cells and is suitable for treating renal cell carcinoma. The invention also provides a method for preparing the compound and application of the compound in preparation of antitumor drugs. Experimental results show that the IC50 of the K-H03 to the 786-O kidney cancer cells is 203.0 [mu] M, and the IC50 of the K-H03 to the A498 kidney cancer cells is 175.5 [mu] M, which indicates that the K-H03 can obviously reduce the cell viability of the 786-O and A498 and shows obvious killing efficacy. The K-H03 provides a new treatment strategy for patients with renal cell carcinoma, and is expected to improve the treatment effect and the life quality of the patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical synthesis, and particularly relates to the preparation of an anti-tumor small molecule K-H03 and its application in anti-tumor drugs. Background Art

[0002] Renal cell carcinoma (RCC) is a malignant tumor originating from renal tubular epithelial cells, accounting for approximately 90% of malignant renal tumors. It is highly invasive, easily metastatic, and insensitive to traditional radiotherapy and chemotherapy. Globally, its incidence rate is increasing year by year, and the five-year survival rate of patients in the advanced stage is less than 15%. Current clinical treatment is mainly surgical resection, but approximately 30% of patients have metastasized at the time of diagnosis and need to rely on systemic treatment. In recent years, although targeted therapy and immunotherapy have significantly improved patient prognosis, they still face challenges such as drug resistance, toxic side effects, and high treatment costs. There is an urgent need to develop innovative therapies that are more specific, safe, and accessible.

[0003] Small molecule drugs have become an important area of ​​drug development for renal cell carcinoma due to their advantages, including small molecular weight, high oral bioavailability, easy structural modification, and low production costs. They directly block key nodes of tumor growth by inhibiting tyrosine kinases (such as VEGFR and PDGFR), regulating the HIF-VEGF signaling pathway, or intervening in metabolic reprogramming. Patented technologies often focus on optimizing drug targeting, such as enhancing selective inhibition of specific kinases through structural modifications, or developing dual / multi-kinase inhibitors to synergistically inhibit tumor angiogenesis and proliferation signals. However, small molecule drugs still have shortcomings such as toxic reactions caused by off-target effects, insufficient metabolic stability that affects the duration of efficacy, and the frequent development of drug resistance after long-term use. In recent years, patented technologies have gradually improved the targeted delivery efficiency of drugs and reduced systemic toxicity by introducing prodrug design, nanodelivery systems, and artificial intelligence-assisted compound screening, providing more competitive solutions for the treatment of renal cell carcinoma. Therefore, for modern cancer treatment, the development of compounds that can inhibit tumor cells is of great significance to the development of anti-tumor drugs. Summary of the Invention

[0004] In response to the above-mentioned existing technical problems, the present invention provides a class of small molecule compounds with anti-tumor activity and a preparation method thereof. The small molecule compound is synthesized mainly through a ten-step reaction. The structural formula of the small molecule compound with anti-tumor activity is shown in Formula (I);

[0005]

[0006] The present invention provides a method for preparing the small molecule compound having anti-tumor activity, comprising the following steps:

[0007] 1) 4-Aminobenzylamine was dissolved in dichloromethane, and CDI and DMAP were added in sequence. The reaction was completed at room temperature, and compound 1 was obtained after post-treatment.

[0008]

[0009] 2) Dissolve 2-bromothioanisole in ultra-dry tetrahydrofuran, slowly add n-butyl lithium n-hexane solution at -78°C, stir the reaction at -78°C for 20 minutes, add tert-butyl 2-oxopyrrolidine-1-carboxylate,

[0010] The reaction was carried out at -78°C for 2 hours. The reaction was allowed to proceed overnight at room temperature until the reaction was complete. Compound 2 was obtained after post-treatment.

[0011]

[0012] 3) Compound 2 was dissolved in ultra-dry dichloromethane, trifluoroacetic acid was added thereto, and the reaction was completed at room temperature. Compound 3 was obtained after post-treatment.

[0013]

[0014]

[0015] 4) Compound 3 was dissolved in a mixed solution of methanol and water, sodium borohydride was added thereto, and the reaction was completed at room temperature. Compound 4 was obtained after post-treatment.

[0016]

[0017] 5) Dissolve 3,6-diamino-acridine in tetrahydrofuran, add Boc anhydride, and react at 65°C until complete.

[0018] Compound 5 was obtained after post-treatment.

[0019]

[0020] 6) Compound 5 was dissolved in DMF, and 4-((tert-butyloxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid, EDCI, HOBT, and DIEA were added in sequence. The reaction was completed at room temperature, and compound 6 was obtained after post-treatment.

[0021]

[0022] 7) Compound 6 was dissolved in a mixed solvent of EtOH and H2O. Sodium hydroxide was dissolved in water, cooled, and then slowly added dropwise to the above system. The reaction was completed at room temperature, and compound 7 was obtained after post-treatment.

[0023]

[0024] 8) The compound was dissolved in DMF, and 2-(2-(methylthio)phenyl)pyrrolidine, EDCI, HOBT, and DIEA were added in sequence. The reaction was completed at room temperature, and compound 8 was obtained after post-treatment.

[0025]

[0026] 9) Compound 8 was dissolved in ethyl acetate, and an ethyl acetate HCl solution was added. The reaction was completed at room temperature, and compound 9 was obtained after post-treatment.

[0027]

[0028] 10) Compound 9 was dissolved in acetonitrile, and triethylamine and N-(4-aminobenzyl)-1H-imidazole-1-carboxamide were added to the suspension. The reaction was completed at room temperature, and the target compound K-H03 was obtained after post-treatment.

[0029] The present invention provides use of the small molecule compound or the small molecule compound obtained by the preparation method in the preparation of anti-tumor drugs.

[0030] Preferably, the tumor is renal cell carcinoma.

[0031] The beneficial effects of the present invention are as follows:

[0032] The cyclic peptide of the present invention has good inhibitory and killing effects on various tumor cells including renal cell carcinoma cells, and can be used to prepare corresponding anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The synthetic route of the small molecule compound K-H03 obtained in Example 1 of the present invention is shown;

[0034] Figure 2 : represents the H NMR spectrum of the small molecule compound K-H03 obtained in Example 1 of the present invention;

[0035] Figure 3 Graph showing the mass spectrometry results of the small molecule compound K-H03 obtained in Example 1 of the present invention;

[0036] Figure 4 Represents the cell status when different concentrations of K-H03 acted on 786-O cells for 72 h;

[0037] Figure 5 Represents the cell status when different concentrations of K-H03 acted on A498 cells for 72 h;

[0038] Figure 6 The fitting curve diagram represents the half-maximal inhibitory concentration IC50 of the small molecule compound K-H03 in 786-O cells;

[0039] Figure 7 The fitting curve diagram represents the half-maximal inhibitory concentration IC50 of the small molecule compound K-H03 in A498 cells; DETAILED DESCRIPTION

[0040] In response to the above-mentioned existing technical problems, the present invention provides a class of small molecule compounds with anti-tumor effects and a preparation method thereof. The small molecule compounds are synthesized mainly through a ten-step reaction. The general formula of the small molecule compounds with anti-tumor effects is shown in Formula (I);

[0041]

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] Example 1: Preparation of small molecule compounds

[0045] This embodiment provides a method for synthesizing a small molecule compound K-HO3, whose structural formula is shown in Formula 1, which can be used to prepare anti-tumor drugs.

[0046] The small molecule synthesis reaction route is as follows:

[0047]

[0048] The specific preparation steps are as follows:

[0049] 1) 4-Aminobenzylamine (5.5 g, 45 mmol) was dissolved in 60 mL of dichloromethane. CDI (5.5 g, 45 mmol) and DMAP (5.5 g, 45 mmol) were added sequentially and allowed to react at room temperature for 4 hours. The reaction was monitored by liquid chromatography-mass spectrometry (LC-MS / MS) to reveal the product. 300 mL of ethyl acetate was added and the mixture was stirred vigorously for 20 minutes. The reaction solution was washed with water (100 mL x 2) and saturated brine (100 mL), respectively. The organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to yield Compound 1: N-(4-aminobenzyl)-1H-imidazole-1-carboxamide (8 g, 82.3% yield).

[0050] 2) Dissolve 2-thioanisole (12 g, 59.7 mmol) in 120 mL of ultra-dry tetrahydrofuran. Cool the reactor to -78°C and slowly add n-butyllithium in n-hexane (2.5 M, 26.3 mL, 65.7 mmol) dropwise. Stir the reaction at -78°C for 20 minutes. Add 1-tert-butyloxycarbonyl-3-pyrrolidone (13.25 g, 71.6 mmol) dropwise and react at -78°C for 2 hours. Allow the reaction to warm to room temperature overnight. Monitor the reaction by liquid chromatography-mass spectrometry to confirm product formation. Saturated ammonium chloride solution (100 mL) was added to the mixture at 0°C to quench the reaction. The reaction solution was extracted with ethyl acetate (200 mL), and the organic phase was washed with water (100 mL x 2) and saturated brine (100 mL), dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain compound 2: tert-butyl (4-(2-(methylthio)phenyl)-4-oxobutyl)carbamate (17.273 g, 93.6% yield). The crude product was a white oil. The crude product was used directly in the next step without further purification.

[0051] 3) tert-Butyl (4-(2-(methylthio)phenyl)-4-oxobutyl)carbamate (17.273 g, 55.9 mmol) was dissolved in 100 mL of ultra-dry dichloromethane, and 50 mL of trifluoroacetic acid was added. The reaction was allowed to react at room temperature for 16 hours. Liquid chromatography-mass spectrometry (LC-MS / MS) was used to monitor the reaction, indicating product formation. Excess solvent was removed by distillation under reduced pressure, and the remaining product was diluted with ethyl acetate (200 mL). The organic phase was washed with water (100 mL x 2) and 100 mL of saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 15 g of crude white oil. This crude product was purified on a silica gel column using a 1:3 ratio of ethyl acetate to petroleum ether to afford compound 3: 5-(2-(methylthio)phenyl)-3,4-dihydro-2H-pyrrole (10 g, 93.5% yield). The product was obtained as a white solid.

[0052] 4) 5-(2-(Methylthio)phenyl)-3,4-dihydro-2H-pyrrole (10 g, 52.3 mmol) was dissolved in 100 mL of a 4:1 mixture of methanol and water. Sodium borohydride (2.3 g, 57.5 mmol) was added and allowed to react at room temperature for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry, indicating product formation. Excess solvent was removed by distillation under reduced pressure, and the remaining product was diluted with water (200 mL) and extracted with ethyl acetate (100 mL x 2). The organic phase was washed with 100 mL of saturated brine and dried over anhydrous sodium sulfate. After distillation under reduced pressure, compound 4: 2-(2-(methylthio)phenyl)pyrrolidine (10 g, 99.0% yield) was obtained. The product was a white, opaque oil.

[0053] 5) Dissolve 3,6-diaminoacridine (50 g, 177 mmol) in 300 mL of tetrahydrofuran, add Boc anhydride (77.58 g, 355 mmol), and react at 65°C for 8 hours. Monitor the reaction by liquid chromatography-mass spectrometry. After completion, remove excess solvent by vacuum distillation. The remaining solid is slurried with 200 mL of a 1:1 mixture of ethyl acetate and petroleum ether for 1 hour, filtered, and the filtrate is collected. Vacuum distillation of the filtrate yields 12 g of a crude brown solid. The crude product is purified on a silica gel column using a 1:3 ratio of ethyl acetate and petroleum ether to afford compound 5: tert-butyl (6-aminoacridin-3-yl)carbamate (12 g, 21.8% yield). The product is a yellow solid.

[0054] 6) tert-Butyl (6-aminoacridin-3-yl)carbamate (12 g, 38.7 mmol) was dissolved in 120 mL of DMF, and 4-((tert-butyloxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (10.1 g, 38.7 mmol), EDCI (7.39 g, 38.7 mmol), HOBT (5.22 g, 38.7 mmol) and DIEA (10.75 g, 77.4 mmol) were added in sequence. The reaction was allowed to proceed at room temperature for 8 hours, and the reaction was monitored by liquid chromatography. After the reaction was completed, 200 mL of water was added while stirring vigorously, filtered, and the filter cake was dissolved in 200 mL of ethyl acetate, washed with water (100 mL x 2) and saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, and the organic phase was distilled under reduced pressure to give 18 g of a dark solid crude product. The crude product was purified on a silica gel column and eluted with ethyl acetate:petroleum ether 1:4 to give compound 6: N2-(tert-butoxycarbonyl)-N5-(6-((tert-butoxycarbonyl)amino)acridin-3-yl)glutamine (14 g, 65.4% yield) as a yellow solid.

[0055] 7) Methyl N2-(tert-Butyloxycarbonyl)-N5-(6-((tert-Butyloxycarbonyl)amino)acridin-3-yl)glutamate (14 g, 25.3 mmol) was dissolved in 140 mL of a 1:1 mixture of EtOH:H2O. Sodium hydroxide (2 g, 50.6 mmol) was dissolved in 20 mL of water, cooled, and slowly added dropwise to the above system. The reaction was allowed to react at room temperature for 12 hours, with liquid chromatography-mass spectrometry monitoring. After completion of the reaction, the pH was adjusted to 7 while vigorously stirring for 1 hour. The reaction was filtered, the filter cake was washed with water, and dried under vacuum to obtain compound 7: N2-(tert-Butyloxycarbonyl)-N5-(6-((tert-Butyloxycarbonyl)amino)acridin-3-yl)glutamine (12 g, yield, 88.16%). The product was a crude brown-yellow solid, which was used directly in the next reaction without purification.

[0056] 8) N2-(tert-Butyloxycarbonyl)-N5-(6-((tert-Butyloxycarbonyl)amino)acridin-3-yl)glutamine (12 g, 22.3 mmol) was dissolved in 120 mL of DMF, and 2-(2-(methylthio)phenyl)pyrrolidine (4.30 g, 22.3 mmol), EDCI (4.25 g, 22.3 mmol), HOBT (3.0 g, 22.3 mmol) and DIEA (5.75 g, 44.6 mmol) were added in sequence. The reaction was allowed to react at room temperature for 12 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, 200 mL of water was added while stirring vigorously, filtered, and the filter cake was dissolved in 200 mL of ethyl acetate, washed with water (100 mL x 2) and saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, and the organic phase was distilled under reduced pressure to obtain 18 g of a dark solid crude product. The crude product was purified by silica gel column and eluted with ethyl acetate:petroleum ether 1:1 to give compound 8: tert-butyl 5-((6-((tert-butoxycarbonyl)amino)acridin-3-yl)amino)-1-(2-(2-(methylthio)phenyl)pyrrolidin-1-yl)-1,5-dioxolane-2-yl)carbamate (15 g, yield 94.3%) as a brown to yellow solid.

[0057] 9) tert-Butyl 5-((6-((tert-Butoxycarbonyl)amino)acridin-3-yl)amino)-1-(2-(2-(methylthio)phenyl)pyrrolidin-1-yl)-1,5-dioxolan-2-yl)carbamate (15 g, 21.0 mmol) was dissolved in 150 mL of ethyl acetate. A 4 M solution of ethyl acetate in hydrogen chloride (100 mL, 400 mmol) was added and the mixture was reacted at room temperature for 4 hours. Liquid chromatography-mass spectrometry indicated the formation of the product. The mixture was filtered and the filter cake dried under vacuum to afford Compound 9: 2-amino-N-(6-aminoacridin-3-yl)-5-(2-(2-(methylthio)phenyl)pyrrolidin-1-yl)-5-oxopentanamide hydrochloride (10 g, 92.8% yield). The product was a crude brown-red solid that was used directly in the next step without purification.

[0058] 10) 2-amino-N-(6-aminoacridin-3-yl)-5-(2-(2-(methylthio)phenyl)pyrrolidin-1-yl)-5-oxopentamide hydrochloride (5 g, 9.8 mmol) was dissolved in 50 mL of acetonitrile, and triethylamine (3.50 g, 29.6 mmol) and N-(4-aminobenzyl)-1H-imidazole-1-carboxamide (2.10 g, 9.8 mmol) were added to the suspension. The mixture was reacted at room temperature for 12 hours. The reaction was monitored by liquid chromatography-mass spectrometry. The product was generated and the excess solvent was distilled off under reduced pressure to obtain 5 g of a reddish-brown solid crude product and 2 g of The crude product was purified on a 330 g reverse-phase C18 column (solvent A: 0.5‰ formic acid in water, solvent B: acetonitrile; method: flow rate 100 mL / min, gradient 5% to 5% solvent A over 5 minutes, 5% to 70% solvent A over 15 minutes, 70% to 70% solvent A over 10 minutes, and 70% to 100% solvent A over 5 minutes) to afford compound K-H03: N-(6-aminoacridin-3-yl)-4-(3-(4-aminobenzyl)ureido)-5-(2-(2-(methylthio)phenyl)pyrrolidin-1-yl)-5-oxopentanamide (1 g, 38.2% yield). The molecular weight of the product was confirmed by LCMS.

[0059] Example 2: Cell viability after adding K-H03 to 786-O (renal cancer cells) by microscopic observation and CCK-8 kit

[0060] Main materials and instruments

[0061] The main experimental reagents (sources) of the present invention: 786-O (renal cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd., and the required experimental materials were: DMEM high-glucose medium; RPMI 1640 medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; Cell Counting Kit (CCK-8); CCK-8 kit (Yisheng Biological).

[0062] The main experimental instruments (sources) of the present invention are: fully automatic cell analyzer (Shanghai Ruiyu Biotechnology Co., Ltd.), inverted microscope (OLYMPUS), and BIOBASE carbon dioxide incubator (Jinan Xinbeisi Biotechnology Co., Ltd.).

[0063] Cell recovery: Remove the cryopreserved tube of 786-O cell line from the liquid nitrogen tank and place it in a 37°C water bath to shake until thawed. Pipette the thawed cell line into a 15mL sterile centrifuge tube in a biosafety cabinet, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. Add RPMI 1640 complete medium to the 786-O tube and transfer it to a 25cm 2 The cells were cultured in sterile culture bottles at 5% CO2 and 37°C.

[0064] Cell passaging: Observe the cells under a microscope. Passage the cells when the cell morphology is normal and the growth density reaches about 90%. First, take out the culture flask containing the cells and place it in the clean bench. Aspirate the original culture medium and wash the cells once with PBS buffer. After absorbing the PBS, add 2mL of trypsin so that it can completely cover the bottom of the culture flask. After tightening the bottle cap, place it in a 37℃ incubator for digestion. After digestion, blow the cells off the wall of the flask with fresh complete culture medium. Then, aspirate the cell suspension into a 15mL sterile centrifuge tube and centrifuge at 1000r / min for 5 minutes. Remove the supernatant, add fresh complete culture medium to resuspend the cells and perform cell passaging at a ratio of 1:3.

[0065] Cell cryopreservation: After centrifugation and removal of the supernatant according to the above cell passage process, add freezing solution (complete culture medium: DMSO = 9:1) to resuspend the cells, and add the suspension to 1.5 mL cell cryopreservation tubes (3 × 10 6 After labeling, cryopreservation was performed using the gradient cooling method.

[0066] In this experiment, 786-O cells were treated with different concentrations of K-H03 for 72 hours. During this period, the cell status after treatment was observed and photographed every day. At 72 hours, cell viability and IC were detected using CCK-8 reagent. 50 Drawing of fitting curve graph.

[0067] Cell status observation:

[0068] After treating 786-O cells with different concentrations of K-H03, the cell status after treatment was observed and photos were taken every day. Figure 4 shown.

[0069] Cell viability detection:

[0070] 1. Prepare 100 μL of cell suspension in a 96-well plate and place the plate in an incubator for 24 hours (37°C, 5% CO2).

[0071] 2. Add different concentrations of K-H03 to the culture plate.

[0072] 3. Incubate the culture plate in an incubator for 72 hours.

[0073] 4. Add 10 μL of CCK-8 solution to each well.

[0074] 5. Incubate the culture plate in the incubator for 1.5 hours.

[0075] 6. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0076] Data processing was performed according to the CCK-8 reagent instructions. Cell viability after treatment is shown in Table 1.

[0077] Table 1 Cell viability after adding K-H03 to 786-O

[0078]

[0079] IC 50 Plotting the fitting curve:

[0080] Perform IC in GraphPad Pirsm 50 Drawing of fitting curve. Figure 6 As shown. 786-O IC 50 =203.0μM.

[0081] Example 3: Cell viability of A498 (renal cancer cells) after addition of compound K-H03 was detected by microscopic observation and CCK-8 kit

[0082] Main materials and instruments

[0083] The main experimental reagents (sources) of the present invention: A498 (renal cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd., and the required experimental materials were: DMEM high-glucose medium; RPMI 1640 medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; Cell Counting Kit (CCK-8) CCK-8 kit (Yisheng Biological).

[0084] The main experimental instruments (sources) of the present invention are: fully automatic cell analyzer (Shanghai Ruiyu Biotechnology Co., Ltd.), inverted microscope (OLYMPUS), and BIOBASE carbon dioxide incubator (Jinan Xinbeisi Biotechnology Co., Ltd.).

[0085] Cell recovery: Take the A498 cell line cryotube out of the liquid nitrogen tank and place it in a 37°C water bath to shake until thawed. Pipette the thawed cell line into a 15mL sterile centrifuge tube in a biosafety cabinet, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. Add DMEM complete medium to the A498. Finally, transfer to a 25cm 2 The cells were cultured in sterile culture bottles at 5% CO2 and 37°C.

[0086] Cell passaging: Observe the cells under a microscope. Passage the cells when the cell morphology is normal and the growth density reaches about 90%. First, take out the culture flask containing the cells and place it in the clean bench. Aspirate the original culture medium and wash the cells once with PBS buffer. After absorbing the PBS, add 2mL of trypsin so that it can completely cover the bottom of the culture flask. After tightening the bottle cap, place it in a 37℃ incubator for digestion. After digestion, blow the cells off the wall of the flask with fresh complete culture medium. Then, aspirate the cell suspension into a 15mL sterile centrifuge tube and centrifuge at 1000r / min for 5 minutes. Remove the supernatant, add fresh complete culture medium to resuspend the cells and perform cell passaging at a ratio of 1:3.

[0087] Cell cryopreservation: After centrifugation and removal of the supernatant according to the above cell passage process, add freezing solution (complete culture medium: DMSO = 9:1) to resuspend the cells, and add the suspension to 1.5 mL cell cryopreservation tubes (3 × 10 6 After labeling, cryopreservation was performed using the gradient cooling method.

[0088] In this experiment, A498 cells were treated with different concentrations of K-HO3 for 72 hours. During this period, the cell status after treatment was observed and photographed every day. At 72 hours, cell viability and IC were detected using CCK-8 reagent. 50 Drawing of fitting curve graph.

[0089] Cell status observation:

[0090] After A498 cells were treated with different concentrations of K-H03, the cell status after treatment was observed and photos were taken every day. Figure 5 shown.

[0091] Cell viability detection:

[0092] 1. Prepare 100 μL of cell suspension in a 96-well plate and place the plate in an incubator for 24 hours (37°C, 5% CO2).

[0093] 2. Add different concentrations of K-H03 to the culture plate.

[0094] 3. Incubate the culture plate in an incubator for 72 hours.

[0095] 4. Add 10 μL of CCK-8 solution to each well.

[0096] 5. Incubate the culture plate in the incubator for 1.5 hours.

[0097] 6. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0098] Data processing was performed according to the CCK-8 reagent instructions. Cell viability after treatment is shown in Table 2.

[0099] Table 2 Cell viability after addition of K-H03 to A498 cells

[0100]

[0101] IC 50 Plotting the fitting curve:

[0102] Perform IC in GraphPad Pirsm 50 Drawing of fitting curve. Figure 7 As shown. A498 IC 50 =175.5μM.

[0103] Combined with the above experiments, it can be shown that K-H03 can significantly reduce the cell viability of 786-O and A498 cells.

Claims

1. A small molecule compound with anti-tumor activity and its use in anti-tumor drugs, Its characteristics are: The structure of the small molecule compound with anti-tumor activity is shown in formula (I).

2. The method for preparing the small molecule compound with anti-tumor activity according to claim 1, It is characterized by: The following steps are involved: 1) 4-Aminobenzylamine was dissolved in dichloromethane, and CDI and DMAP were added in sequence. The reaction was completed at room temperature, and compound 1 was obtained after post-treatment. 2) Dissolve 2-bromothioanisole in ultra-dry tetrahydrofuran, slowly add n-butyllithium (n-hexane) dropwise at -78°C, and stir for 20 minutes at -78°C. Then, add tert-butyl 2-oxopyrrolidine-1-carboxylate dropwise and react at -78°C for 2 hours. Warm to room temperature overnight and allow to react until complete. Post-processing yields Compound 2. 3) Compound 2 was dissolved in ultra-dry dichloromethane, trifluoroacetic acid was added thereto, and the reaction was completed at room temperature. Compound 3 was obtained after post-treatment. 4) Compound 3 was dissolved in a mixed solution of methanol and water, sodium borohydride was added thereto, and the reaction was completed at room temperature. Compound 4 was obtained after post-treatment. 5) 3,6-diamino-acridine was dissolved in tetrahydrofuran, and Boc anhydride was added. The reaction was completed at 65°C, and compound 5 was obtained after post-treatment. 6) Compound 5 was dissolved in DMF, and 4-((tert-butyloxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid, EDCI, HOBT, and DIEA were added in sequence. The reaction was completed at room temperature, and compound 6 was obtained after post-treatment. 7) Compound 6 was dissolved in a mixed solvent of EtOH and H2O. Sodium hydroxide was dissolved in water, cooled, and then slowly added dropwise to the above system. The reaction was completed at room temperature, and compound 7 was obtained after post-treatment. 8) The compound was dissolved in DMF, and 2-(2-(methylthio)phenyl)pyrrolidine, EDCI, HOBT, and DIEA were added in sequence. The reaction was completed at room temperature, and compound 8 was obtained after post-treatment. 9) Compound 8 was dissolved in ethyl acetate, and an ethyl acetate HCl solution was added. The reaction was completed at room temperature, and compound 9 was obtained after post-treatment. 10) Compound 9 was dissolved in acetonitrile, and triethylamine and N-(4-aminobenzyl)-1H-imidazole-1-carboxamide were added to the suspension. The reaction was completed at room temperature, and the target compound K-H03 was obtained after post-treatment.

3. Use of the small molecule compound according to claim 1 or the small molecule compound obtained by the preparation method according to claim 2 in the preparation of anti-tumor drugs.

4. The use according to any one of claims 1 or 3, characterized in that The tumor is renal cell carcinoma.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the small molecule compound according to claim 1.

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