An anti-tumor drug

Abstract

The present invention provides a novel anti-tumor drug HL-1, which can significantly inhibit the activity of tumor cells, and inhibit the proliferation, migration and cloning ability of tumor cells in a dose-dependent manner, induce KYSE30 cell cycle arrest in the G1 phase in a dose-dependent and time-dependent manner, and induce cell apoptosis. The compound HL-1 exerts anti-tumor effects in different tumors through different mechanisms or pathways, and the anti-tumor activity of HL-1 is specific and safe. HL-1 can be used alone or in combination with a second therapeutic agent for the treatment of tumors.

Description

Technical Field

[0001] The invention relates to the fields of chemistry and biological sciences, and in particular to a novel anti-tumor drug. Background Art

[0002] Chemotherapy is one of the important methods for treating cancer, but most anti-tumor drugs will eventually become resistant. During anti-tumor treatment, the phenomenon that tumor cells are insensitive to anti-tumor drugs is called drug resistance. The emergence of anti-tumor drug resistance is one of the main reasons for the failure of cancer chemotherapy and one of the key issues facing cancer treatment. From the perspective of tumor cells, the mechanism of drug resistance may be the heterogeneity of tumor cells, changes in drug transport and metabolism, changes in the expression level of drug targets, activation of alternative pathways, epigenetic changes, epithelial-mesenchymal transition, etc. From the perspective of the tumor microenvironment, the occurrence of drug resistance is also closely related to other cells and components such as infiltrating immune and inflammatory cells, cancer-associated fibroblasts, and vascular endothelial cells in the tumor microenvironment.

[0003] Tumor resistance is common in small molecule targeted anticancer drugs, and it is challenging to design new small molecule drugs targeting their resistance mechanisms. Among the many strategies to deal with antitumor drug resistance, one of the most valuable strategies is to continuously screen and discover new drugs to interfere with specific signaling pathways of tumor growth, inhibit tumor cell proliferation, and improve treatment effects. After the tumor becomes resistant, new drugs can be replaced, new drugs can be used for combined or alternating treatments, and even new drugs can be used to reverse resistance, which is an important way to fight tumors and antitumor resistance. Therefore, new antitumor drugs are urgently needed to be discovered. Summary of the invention

[0004] In order to solve the above technical problems, the present invention uses cell models (esophageal squamous cell carcinoma cell KYSE30, human breast cancer cell MDA-MB-231) to obtain new anti-tumor drugs through cell proliferation detection and screening.

[0005] The first aspect of the present invention provides the use of a compound represented by the following formula I, a stereoisomer thereof or a physiologically acceptable salt thereof in the preparation of an anti-tumor drug:

[0006] Wherein, R1 is selected from one of hydrogen, methyl, ethyl and propyl, and R2 is selected from hydrogen or halogen.

[0007] In certain embodiments, R1 is methyl and R2 is fluoro.

[0008] In certain embodiments, the compound has the following structure:

[0009]

[0010] In certain embodiments, the physiologically acceptable salt comprises an organic salt and an inorganic salt, wherein the inorganic salt is selected from hydrochloride, hydrobromide, phosphate, sulfate and perchlorate, and the organic salt is selected from acetate, oxalate, maleate, tartrate, citrate, succinate and malonate.

[0011] In certain embodiments, the compound, its stereoisomer or a physiologically acceptable salt thereof inhibits tumor cell proliferation and migration, and promotes tumor cell apoptosis.

[0012] In certain embodiments, the tumor is breast cancer or esophageal cancer.

[0013] The second aspect of the present invention provides the use of a compound represented by Formula I or Formula II in combination with a second therapeutic agent in the preparation of an anti-tumor drug.

[0014] In certain embodiments, the second therapeutic agent is selected from a second chemotherapeutic agent, an immune checkpoint inhibitor, an oncolytic virus, CAR-T, or TCRT.

[0015] In certain embodiments, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0016] In certain embodiments, the anti-PD-1 antibody is selected from pembrolizumab, sintilimab, nivolumab, carrelizumab and toripalimab; and the anti-PD-L1 antibody is selected from durvalumab and atezolizumab.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention uses cell models (KYSE30 and MDA-MB-231) to screen the internal compound library based on cell proliferation phenotype, and discovers the highly effective anti-tumor drug HL-1 and its structural analogue HL-2. Studies have shown that compound HL-1 significantly inhibits the activity of tumor cells KYSE30 and MDA-MB-231, and inhibits the proliferation and cloning ability of KYSE30 and MDA-MB-231 tumor cells in a dose-dependent manner.

[0019] Further studies have shown that compound HL-1 inhibits the migration of KYSE30 and MDA-MB-231 tumor cells in a dose-dependent manner, induces KYSE30 cell cycle arrest in the G1 phase in a dose-dependent and time-dependent manner, and induces cell apoptosis. The HL-1 compound does not affect the cell cycle and apoptosis of MDA-MB-231, suggesting that compound HL-1 exerts anti-tumor effects through different mechanisms or pathways in different tumors. It is worth noting that HL-2, which is similar in structure to compound HL-1, has no obvious toxicity to normal cells, and HL-2 has no effect on tumor cell proliferation, clone formation, migration, cycle, apoptosis, etc., further illustrating the specificity and safety of the anti-tumor activity of compound HL-1, and HL-1 can be used for the treatment of tumors alone or in combination with a second therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0021] Figure 1 The structural formulas of HL-1 and HL-2 are shown.

[0022] Figure 2 Shows the results of CCK8 detection of cell proliferation. Figure 2 A shows the treatment of tumor cells KYSE30 and MDA-MB-231 with different concentrations of compounds HL-1 and HL-2. Figure 2 A is the cell viability and IC of normal cells Het1a, MCF-10A and NP460 50 .

[0023] Figure 3 Shown are the effects of HL-1 and HL-2 on the proliferation of KYSE30 (A) and MDA-MB-231 (B) cells detected by EdU.

[0024] Figure 4 The effects of compounds HL-1 and HL-2 on the clone formation ability of tumor cells KYSE30 and MDA-MB-231 are shown. Figure 4 A and 4B show the effects of HL-1 at low, medium, and high doses on the clone-forming ability of MDA-MB-231 and KYSE30 cells; Figure 4 C shows the effect of high dose of HL-2 on the clonogenicity of KYSE30 and MDA-MB-231 cells.

[0025] Figure 5 The cell scratch test shows the effect of compounds HL-1 and HL-2 on the migration ability of tumor cells MDA-MB-231 and KYSE30. Figure 5A shows the effects of high, medium and low doses of compound HL-1 and high dose HL-2 on the migration ability of tumor cells MDA-MB-231 for 36 hours and 72 hours; Figure 5 B shows the statistical analysis results of the healing percentage of MDA-MB-231 cells; Figure 5 C shows the effects of high, medium and low doses of compounds HL-1 and HL-2 on the migration ability of tumor cells KYSE30 for 36 hours and 72 hours; Figure 5 D shows the statistical analysis results of KYSE30 cell healing percentage.

[0026] Figure 6 The flow cytometry test shows the effect of compound HL-1 on the cell cycle of KYSE30 cells. Figure 6 A shows the effects of high, medium, low doses of HL-1 and high dose of HL-2 on the cell cycle of tumor cells KYSE30 for 36 hours and the statistical results; Figure 6 B shows the effects of high, medium, and low doses of HL-1 and high dose of HL-2 on the cell cycle of tumor cells KYSE30 for 72 hours and the statistical results; (*: comparison between each group and Control; #: comparison between medium dose and low dose; $: comparison between high dose and medium dose).

[0027] Figure 7 The flow cytometry test shows the effect of compound HL-1 on the cell cycle of MDA-MB-231 cells. Figure 7 A shows the effects of high, medium, low doses of HL-1 and high doses of HL-2 on the cell cycle of tumor cells MDA-MB-231 for 36 hours and the statistical results; Figure 7 B shows the effects of high, medium, and low doses of HL-1 and high dose of HL-2 treatment on the cell cycle of tumor cells MDA-MB-231 for 72 hours and the statistical results (*: comparison between each group and Control; #: comparison between medium dose and low dose; $: comparison between high dose and medium dose).

[0028] Figure 8 The effect of compound HL-1 on apoptosis of KYSE30 and MDA-MB-231 is shown. Figure 8 A shows flow cytometric analysis of KYSE30 cells treated with high, medium, and low doses of HL-1 for 36 and 72 hours; Figure 8 B shows flow cytometric analysis of KYSE30 cells treated with high doses of HL-2 for 36 and 72 hours; Figure 8 C shows the statistical analysis of compound-induced apoptosis of KYSE30; Figure 8 D shows flow cytometric analysis of MDA-MB-231 cells treated with high, medium, and low doses of HL-1 and HL-2 for 36 and 72 hours. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0031] Example 1 Screening of anti-tumor compounds using CCK-8 method

[0032] Cell proliferation is an important indicator for understanding the survival or death mechanism of certain genes, proteins or pathways after exposure to drugs. The CCK8 method is one of the methods for measuring cell proliferation. Its principle is: in living cells, WST-8 can be reduced to a water-soluble orange-yellow formazan dye by dehydrogenase in mitochondria. The absorbance at 450nm can be measured using an enzyme reader to indirectly reflect the number of living cells (dead cells do not have this function). Within a certain range, the amount of product formed is proportional to the number of living cells. The specific experimental steps are as follows: Collect cells: Collect KYSE30 and MDA-MB-231 cells in logarithmic growth; Count: Count using a Countstar cell counter and adjust the cell concentration to 2×10 4 -3×10 4 / mL; plating: add 100μL of cell suspension to each well of a 96-well plate, fill the edge wells with sterile PBS to eliminate edge effects, set up 3 replicate wells for each group, and culture overnight in a 5% CO2, 37℃ incubator; dilute compounds: dilute compounds with culture medium, high concentration 100μM, perform 2-fold gradient dilution, add 100μL to a 96-well plate in sequence, and culture in a 5% CO2, 37℃ incubator for 72h; CCK8 staining: add 10μL to each well CCK8 solution, continue to culture for 4h; data collection: microplate reader detects the absorbance of each well at a wavelength of 450nm; data processing: according to the formula: cell inhibition rate = [1-(experimental group-blank group) / (control group-blank group)]*100, calculate the inhibition rate, and import the data into the software Prism8, and convert the concentration (X) into X=log(X) through the "Analyze" option, and finally perform "nonlinear regression" on the converted data, and select the log (inhibitor) vs. response method to fit the results, and finally obtain the half inhibition concentration.

[0033] Figure 1It shows that a group of compounds HL-1 and HL-2 with extremely similar skeletons were obtained by extensive screening of the internal compound library using CCK8.

[0034] Figure 2 (A) IC values ​​of compounds HL-1 and HL-2 against tumor cells KYSE30 and MDA-MB-231, and normal cells Het1a, NP460, and MCF-10A 50 Value. Figure 2 It can be seen that compound HL-1 effectively inhibits the viability of tumor cells KYSE30 and MDA-MB-231, and its IC 50 The compound HL-2 had no effect on the activity of tumor cells KYSE30 and MDA-MB-231 (IC 50 >100μM). This suggests that compounds HL-1 and HL-2 may act on the same target, but due to subtle differences in structure, they cannot efficiently bind to or inhibit the function of the target. In addition, compound HL-1 had no effect on the activity of normal cells Het1a, MCF-10A, and NP460, suggesting that compound HL-1 inhibited tumor cell viability due to its specific action on a certain target, not due to compound toxicity. In subsequent studies, 2×IC 50 Defined as high dose (40 μM), 1×IC 50 Defined as a medium dose (20 μM), 0.5×IC 50 Defined as low dose (10 μM).

[0035] Example 2 EdU cell proliferation detection

[0036] EdU (5-Ethynyl-2'-deoxyuridine) is a thymidine analog that can replace T and be incorporated into the replicating DNA molecules in the cell during cell proliferation. It can also react covalently with the fluorescently labeled probe through the catalysis of monovalent copper ions to form a stable triazole ring, which is called a click reaction. Through the above reaction, the fluorescent probe will label the newly synthesized DNA of the cell, and the proliferating cells can be directly and accurately detected by detecting the fluorescence. Using EdU to detect the newly synthesized DNA of the cell, combined with Hoechest to label the cell nucleus, the cell location and the proportion of proliferating cells can be analyzed more comprehensively.

[0037] The specific operation of EdU cell proliferation detection experiment is as follows:

[0038] 1) EdU labeling, fixation, washing and permeabilization

[0039] Take logarithmically growing cells and inoculate 3×10 4 / well cells, culture overnight; treat cells with different concentrations of compounds for 72h; add equal volumes of 2× EdU working solution (20μM) to a 24-well plate to make a final concentration of 1×; continue to culture cells for 2 hours (the culture time is usually about 10% of the cell cycle); after EdU labeling of cells, remove the cell culture medium and add 4% paraformaldehyde that can cover the sample wells to fix for 15 minutes; remove the fixative and wash the cells 3 times with PBS containing 3% BSA, each time for 3 to 5 minutes; remove the washing solution, add 1mL of permeabilization solution to each well, and incubate at room temperature for 10 to 15 minutes; remove the permeabilization solution, and wash the cells 1 to 2 times with 1mL of washing solution to each well, each time for 3 to 5 minutes.

[0040] 2) EdU detection

[0041] Prepare Click Additive Solution: Dissolve Click Additive in 1.3 mL of deionized water; prepare Click reaction solution according to the instructions of the kit (must be used within 15 minutes after preparation); remove the washing solution and add 0.2 mL of Click reaction solution to each well to ensure that the sample can be evenly covered; incubate at room temperature in the dark for 30 minutes, then aspirate the reaction solution and wash three times with washing solution, each time for 3 to 5 minutes.

[0042] 3) Nuclear staining

[0043] Hoechst 33342 can pass through the cell membrane and bind to the minor groove in the DNA double strand, and emit indigo fluorescence at 461nm. The specific operation is as follows: dilute Hoechst 33342 (1000×) with PBS at a ratio of 1:1000; add 0.25mL of 1×Hoechst 33342 solution to each well, incubate at room temperature and away from light for 10 minutes; remove the Hoechst33342 solution, wash 3 times with washing solution, each time for 3 to 5 minutes; then perform fluorescence detection, and select 3 fields of view to count EdU and Hoechst stained cells respectively; take the EdU / Hoechst ratio of the control group as 100%, and calculate the percentage of the experimental group.

[0044] The results are as follows Figure 3 As shown, for KYSE30, HL-1 significantly reduced the number (nuclei, blue) and proliferation (proliferating cells, red) of KYSE30 cells at high and medium doses, while the low dose had little effect on the number and proliferation of KYSE30 cells ( Figure 3 A). For MDA-MB-231, HL-1 significantly reduced the number (nuclei, blue) and proliferation (proliferating cells, red) of KYSE30 cells at high and medium doses, and also had a significant effect on their proliferation at low doses ( Figure 3B). The above research results suggest that compound HL-1 can effectively inhibit tumor cell proliferation, and the target of compound HL-1 plays an important role in the proliferation of different tumor cells. However, compound HL-2 with a similar skeleton has no effect on the proliferation of KYSE30 and MDA-MB-231 tumor cells, further indicating that the inhibition of tumor cell proliferation by HL-1 is not caused by the toxicity of the compound.

[0045] Example 3 Clone formation

[0046] A clone refers to a colony of more than 50 cells that continues to proliferate in vitro. It can measure the proliferation capacity of a single cell, its sensitivity to killing factors, and reflect the dependence and proliferation capacity of a cell population. The specific operation is as follows: Take cells in the logarithmic growth phase, count and adjust the cells to an appropriate concentration (750 cells / well), inoculate them in a 12-well plate, and let the cells adhere to the wall for 24 hours after "cross" mixing; add the drug to be tested for 24 hours, and then replace the complete culture medium; culture in a 37℃ 5% CO2 incubator for 2 to 3 weeks, and change the medium every 3 days; observe frequently, and terminate the culture when visible clones appear in the culture dish; discard the supernatant, add 4% paraformaldehyde to fix for 15 minutes; discard the fixative and add an appropriate amount of crystal violet dye to cover the bottom of the well plate, stain for 30 minutes, wash with PBS 3 to 5 times, and air dry; directly observe and count the number of clones greater than 50 cells under a low-power optical microscope, and calculate the clone formation rate according to the formula: clone formation rate = clone formation number / inoculated cell number × 100.

[0047] After the tumor cells were treated with HL-1 for 24 hours, conventional cell culture was performed to explore the long-term effects of compound HL-1 on tumor cells. Figure 4 The effects of compounds HL-1 and HL-2 on the clone formation ability of tumor cells KYSE30 and MDA-MB-231 are shown (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001). Figure 4 It can be seen that high-dose HL-1 treatment for 24 hours completely inhibited the clone formation ability of KYSE30 cells, and medium-dose HL-1 treatment for 24 hours inhibited half of the KYSE30 cells from forming clones. Even at low doses, HL-1 can still effectively inhibit the clone formation of KYSE30 cells ( Figure 4 AB). For MDA-MB-231, high-dose HL-1 treatment for 24 hours inhibited the clone formation of most MDA-MB-231 cells. At medium doses, HL-1 could still effectively inhibit the clone formation of KYSE30 cells, but the effect was not as good as that of high doses ( Figure 4AB). The above results show that although compound HL-1 significantly inhibits the cloning of tumor cells KYSE30 and MDA-MB-231, its long-term effects are not consistent, which may be caused by the difference in the expression and function of HL-1 targets in KYSE30 and MDA-MB-231. It is worth noting that the compound HL-2, which has a very similar structure, has no significant effect on the cloning ability of KYSE30 and MDA-MB-231 cells ( Figure 4 C), further indicating that the inhibition of HL-1 clone formation is a specific action on a target in tumor cells and is not caused by the toxicity of the compound.

[0048] Example 4 Cell migration experiment

[0049] To further explore the effect on HL-1 tumor cell migration, the cell scratch assay was used to evaluate the HL-1 migration inhibition ability.

[0050] The cell scratch test is a technique for studying cell migration in vitro. The basic steps include culturing a monolayer of cells in a culture dish, and then using a micropipette tip to artificially create a blank scratch area on the monolayer of cells. The cells at the edge of the scratch will gradually migrate to the blank area, thereby judging the changes in cell migration ability under drug intervention. In order to eliminate the influence of cell proliferation on the experimental results, the experiment is usually carried out using a culture medium without serum or low serum (<2%). The basic operation is as follows: use a marker pen to evenly draw 5 lines on the back of a 6-well plate, approximately every 0.5 to 1 cm; take cells in the logarithmic growth phase, adjust the cell concentration and inoculate them in a 6-well plate at an appropriate cell density (it is best if the cells fuse into a monolayer after overnight culture), and culture them overnight at 37°C 5% CO2; use a 10ul pipette tip to forcefully and evenly scratch the culture dish perpendicular to the marked line; wash 3 times with PBS to remove the scratched cells, confirm under a microscope and take pictures; prepare different concentrations of compounds in a culture medium containing 2% serum and add them to the corresponding wells, and use 4 / 1000 DMSO as a control, and culture them in a 37°C 5% CO2 incubator; measure the distance of cell movement every 24 hours and take pictures.

[0051] The results are as follows Figure 5As shown, for the tumor cell KYSE30, high, medium and low doses of compound HL-1 can significantly inhibit the migration of KYSE30 cells within 24 hours, and the effect is dose-dependent. After 48 hours, high, medium and low doses of compound HL-1 can significantly inhibit the migration of KYSE30 cells, and the difference is statistically significant. More importantly, the healing area of ​​cells treated with high doses of HL-1 is almost the same as that at 24 hours, reflecting the sustained effectiveness of compound HL-1 in inhibiting cell migration. It is worth noting that high-dose treatment of HL-2, which has an extremely similar skeleton, has no significant effect on the migration of tumor cells MDA-MB-231, suggesting that HL-1 inhibits the migration of tumor cells KYSE30 due to its function rather than cytotoxicity (5A and 5B).

[0052] For MDA-MB-231 tumor cells, the results were as follows Figure 5 As shown in Figures C and 5D, within 24 hours, compound HL-1 can significantly inhibit the migration of MDA-MB-231 cells, and it is dose-dependent. After 48 hours, although there is no difference in the scratch healing rate between high and medium doses, HL-1 can still inhibit the migration of MDA-MB-231 cells, and the difference is significant. It is worth noting that HL-2, which has a very similar skeleton, has no significant effect on the migration of tumor cells MDA-MB-231, suggesting that HL-1 inhibits the migration of tumor cells MDA-MB-231 due to its function rather than cytotoxicity.

[0053] In summary, compound HL-1 can inhibit the migration of tumor cells KYSE30 and MDA-MB-231 in a dose-dependent manner regardless of high, medium or low doses, while compound HL-2 with an extremely similar skeleton does not have this function, indicating that compound HL-1 is a good anti-tumor cell migration drug.

[0054] Example 5 Flow cytometry to detect changes in cell cycle and cell apoptosis

[0055] 1. Detection of cell cycle changes

[0056] The cell cycle refers to the entire process that a cell goes through from the completion of one division to the end of the next division. The DNA content in different phases (G0, G1, S, G2, M) will change periodically. In the G1 phase, cells mainly synthesize RNA and proteins, but the DNA content remains unchanged; DNA synthesis begins in the S phase, and the DNA content is between G1 and G2; cell DNA replication ends and the cell enters the G2 phase; cells in the G2 phase continue to synthesize RNA and proteins until the cell enters the M phase. The G2 and M phases of a cell cannot be distinguished from the DNA content alone, and the same cannot be said for G0 and G1. Therefore, the cell cycle can be described as G0 / G1, S, and G2 / M.

[0057] Propidium iodide (PI) is a double-stranded DNA fluorescent dye. The fluorescence intensity generated after binding to double-stranded DNA is proportional to the double-stranded DNA content. Cells treated with propidium iodide can be measured for DNA content using a flow cytometer. Based on the distribution of DNA content obtained by the test, cell cycle (fluorescence intensity of each phase: if G0 / G1 phase = 1, then G2 / M = 2, S = 1-2) and cell apoptosis analysis can be performed. The DNA of apoptotic cells will be fragmented and lost, so PI staining will show obvious weak staining, that is, the fluorescence intensity is less than 1, and a sub-G1 peak (apoptotic cell peak) appears on the fluorescence graph.

[0058] The specific steps are as follows:

[0059] 1) Collect cells: Collect all cells in each experimental group and centrifuge at about 1000g for 3-5 minutes;

[0060] 2) Wash cells: Carefully remove the supernatant, add about 1 mL of ice-cold PBS to resuspend the cells, transfer to a 1.5 mL centrifuge tube, and centrifuge again to precipitate the cells;

[0061] 3) Fix the cells: add 1 mL of pre-cooled 70% ethanol and gently pipette to mix, then fix at 4°C for 24 hours;

[0062] 4) Wash cells: Centrifuge at 1000g for 3-5 minutes to precipitate cells, add about 1 mL of ice-cold PBS to resuspend the cells, and centrifuge again to collect the cell precipitate;

[0063] The preparation of propidium iodide staining solution is as shown in Table 1:

[0064] Table 1 Preparation of propidium iodide staining solution

[0065]

[0066] 5) Staining: Add 0.5 mL of PI staining solution to the sample and incubate at 37°C in the dark for 30 minutes;

[0067] 6) Flow cytometric detection and analysis: Red fluorescence was detected using a flow cytometer at an excitation wavelength of 535 nm.

[0068] The results are as follows Figure 6As shown, the high, medium and low doses of compound HL-1 were used to treat tumor cells KYSE30 and MDA-MB-231 for 36 hours and 72 hours, respectively. For KYSE30, the proportion of G1 phase cells was 48.2% after 36 hours of treatment. The high, medium and low doses of compound HL-1 significantly induced cell cycle G1 arrest for 36 hours, and showed a dose-dependent characteristic. The proportion of G1 phase cells was 63.7%, 76.7% and 80.9%, respectively. The high dose of compound HL-2 for 36 hours had no significant effect on the cell cycle of KYSE30. After 72 hours of treatment, the proportion of G1 phase cells was 68.7%. The high, medium and low doses of compound HL-1 for 72 hours significantly induced cell cycle G1 arrest, and showed a dose-dependent characteristic. The proportion of G1 phase cells was 71.9%, 84.1% and 99.2%, respectively. The high dose of compound HL-2 for 72 hours had no significant effect on the cell cycle of KYSE30 (6A and 6B).

[0069] For the tumor cell MDA-MB-231, the proportion of G1 phase cells was 51.3% after 36 hours of treatment. The high, medium and low doses of compound HL-1 had no significant effect on the cell cycle for 72 hours, and the proportion of G1 phase cells was 52.0%, 53.6% and 59.5%, respectively. The high dose of compound HL-2 had no significant effect on the cell cycle of MDA-MB-231 for 36 hours. The proportion of G1 phase cells was 51.3% after 72 hours of treatment. The high, medium and low doses of compound HL-1 had no significant effect on the cell cycle for 72 hours, and the proportion of G1 phase cells was 53.7%, 54.5% and 59.8%, respectively. Figure 7 A and 7B).

[0070] In conclusion, compound HL-1 has different effects on different tumor cells. It significantly induced KYSE30 cell cycle arrest at the G1 phase in a dose-dependent manner, but had no effect on the MDA-MB-231 cell cycle, suggesting that compound HL-1 can exert its anti-tumor activity through different pathways.

[0071] 2. Detection of cell apoptosis changes

[0072] Apoptosis is a spontaneous, programmed death process. Phosphatidylserine (PS) of apoptotic cells is translocated from the inner membrane to the outer layer. Annexin V is a calcium-dependent phospholipid binding protein with a high affinity for PS, which can bind to apoptotic cells and necrotic cells. PI cannot penetrate the intact cell membrane, but can penetrate the cell membrane of necrotic cells. Therefore, the use of Annexin V / PI double staining can effectively distinguish apoptotic cells from necrotic cells. Early apoptotic cells: Annexin V (+), PI (-); late apoptotic or necrotic cells: Annexin V (+), PI (+); early mechanical necrosis and nonspecific staining: Annexin V (-), PI (-); normal cells: Annexin V (-), PI (-).

[0073] Specific operation: Collect cells: Collect cells and wash cells as in cell cycle detection; take 5×10 4 ~1×10 5 The resuspended cells were centrifuged at 1000 g for 5 minutes, the supernatant was discarded, 195 μL Annexin V-APC binding solution was added to gently resuspend the cells; 5 μL Annexin V-APC was added and gently mixed; 10 μL PI staining solution was added and gently mixed; incubated at room temperature in the dark for 10 to 20 minutes, then placed on ice and tested on an instrument.

[0074] The results are as follows Figure 7 As shown, for KYSE30, after 36 hours of treatment with different doses of compound HL-1, only the medium and high doses induced apoptosis of tumor KYSE30 cells, and the apoptosis rates reached ~10% and ~27%, respectively. When the treatment time was extended to 72 hours, significant apoptosis occurred in the high, medium and low dose groups, and the percentage of apoptotic cells was ~10%, ~15% and ~72%, respectively ( Figure 8 A). It is worth noting that the compound HL-2, which is extremely similar in structure, did not induce cell apoptosis at high doses, whether for 36 hours or 72 hours. The above results suggest that the compounds induce apoptosis of tumor cells KYSE30 in a dose-dependent and time-dependent manner ( Figure 8 B and 8C). For MDA-MB-231 cells, no matter the high, medium or low doses of compounds HL-1 and HL-2 were treated for 36 or 72 hours, no apoptosis was induced in the cells, suggesting that the anti-tumor activity of compound HL-1 in MDA-MB-231 cells is not through inducing apoptosis, but through inhibiting cell proliferation and cell migration to exert anti-tumor effects ( Figure 8 D).

[0075] The above experimental results show that the anti-tumor compound HL-1 has different mechanisms of anti-tumor effects in different tumors, suggesting that it may act on different targets, or it may act on the same target but the target has different functions in different tumor cells. In short, compound HL-1 can exert anti-tumor effects through different pathways and is a compound worthy of further in-depth study.

[0076] Example 6 Synthesis of Compounds HL-1 and HL-2

[0077] 1. Synthesis of compound HL-1

[0078] Take piperidine-4-carboxylic acid ethyl ester and m-fluorobenzyl chloride as raw materials and react at room temperature for 48 hours to obtain intermediate compound HL-1a. Dissolve compound HL-1a in tetrahydrofuran, add lithium aluminum hydride at 0°C, react for 1 hour, and then move to room temperature and reflux for 4 hours to react to obtain intermediate compound HL-1b. At the same time, 3-amino-6-methylbenzenesulfonamide reacts with stannous chloride and nitrous acid to produce intermediate compound HL-1c. Intermediate compounds HL-1b and HL-1c are dissolved in dichloromethane in the presence of triethylamine and 4-(dimethylamino)piperidine and react at room temperature for 2.5 hours to produce intermediate compound HL-1d. HL-1d is then dissolved in water with 1,3-cyclohexanedione and reacted at 90°C overnight in the presence of trifluoroacetic acid to obtain the final compound HL-1.

[0079]

[0080] Synthesis of compound HL-1

[0081] Reagents and reaction conditions: (i) K2CO3, EtOH, room temperature, 48h; (ii) LiAlH4, THF, 0℃, 1h, RT reflux, 4h; (iii) SnCl2, HNO2; (iv) Et3N, 4-(dimethylamino)piperidine, DCM, room temperature, 2.5h; (v) TFA, water, 90℃, overnight.

[0082] 2. Synthesis of compound HL-2

[0083] 1,3-cyclohexanedione and compound HL-2a were used as raw materials and boric acid was used as a catalyst to react for 4 minutes to generate the intermediate compound HL-2b. HL-2b was dissolved in tetrahydrofuran, potassium tert-butoxide and potassium amide were slowly added at room temperature, and then the reaction temperature was moved to 60°C for 2 hours to obtain the intermediate compound HL-2c. The intermediate was reacted at 130°C for 24 hours in the presence of a catalyst to generate the intermediate compound HL-2d. HL-2d was dissolved in methanol and activated at room temperature for 40 minutes, then moved to 0°C, sodium borohydride was added for reaction for 20 minutes, and then purified and separated to obtain the final product HL-2.

[0084]

[0085] Synthesis of compound HL-2

[0086] Reagents and reaction conditions: (i) boric acid, 4 min; (ii) potassium tert-butoxide, potassium amide, tetrahydrofuran, room temperature, 60°C, 2 h; (iii) silver carbonate, 1-adamantanecarbonyl chloride, copper acetate monohydrate, tri-tert-butylphosphine tetrafluoroborate, 1,2-dichloroethane, 130°C, 24 h; (iv) sodium borohydride, methanol, room temperature, 0°C, 20 min.

[0087] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0088] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Use of a compound represented by formula II or a physiologically acceptable salt thereof in the preparation of an anti-tumor drug, (Formula II); the tumor is breast cancer or esophageal cancer.

2. The use according to claim 1, characterized in that: The physiologically acceptable salt is hydrochloride.

3. The use according to claim 1, characterized in that: The compound or a physiologically acceptable salt thereof inhibits tumor cell proliferation and migration and promotes tumor cell apoptosis.

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