Application of compound or salt thereof in preparation of medicine for treating cancer

Abstract

The invention belongs to the technical field of cancer drugs, and particularly relates to application of a compound or salt thereof in preparation of drugs for treating cancers. The structural formula of the compound is as follows: # imgabs0 #, wherein R is selected from halogen. When the compound is applied to treatment of cancers (such as triple negative breast cancer), tumor growth can be effectively inhibited, and obvious toxic and side effects do not exist.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer drugs, and in particular relates to the use of a compound or a salt thereof in preparing a drug for treating cancer. Background Art

[0002] The progression of malignant tumors depends on complex biological regulatory mechanisms, including abnormal cell proliferation, angiogenesis, invasion and metastasis, and immune escape. At present, clinical treatment is still mainly based on surgery, radiotherapy, chemotherapy, and targeted drugs, but there are limitations such as drug resistance, significant toxic side effects, and high treatment costs. In recent years, targeted therapy strategies targeting key regulatory factors in the tumor microenvironment have gradually become a research hotspot.

[0003] Studies have shown that tumor cells can activate G protein-coupled receptors in endothelial cells by secreting specific signaling molecules, triggering a cascade reaction of downstream signaling pathways (such as PI3K / Akt, MAPK / ERK, etc.), thereby promoting the proliferation and migration of vascular endothelial cells and accelerating tumor angiogenesis. This abnormal angiogenesis not only provides oxygen and nutrients for tumors, but also creates conditions for metastatic cells to break through the basement membrane barrier; in addition, tumor cells can also activate intracellular pro-survival pathways through autocrine signals, inhibit the expression of apoptosis-related proteins, and enhance their ability to survive in harsh microenvironments.

[0004] At the current treatment technology level, RNA interference (such as siRNA / shRNA) has been tried to specifically inhibit the expression of oncogenes, but it has defects such as low targeting efficiency, poor in vivo stability and insufficient drugability; although monoclonal antibody drugs can accurately block the function of specific proteins, they are limited by complex production processes, large dosages and heavy economic burden on patients, and their clinical application is restricted. In contrast, small molecule compounds have become an important direction for the development of targeted therapy due to their advantages such as small molecular weight, strong structural modifiability, convenient administration and controllable production costs. Summary of the invention

[0005] The present invention is based on a virtual screening method of molecular docking and an optimized pharmacophore model and ADMET (absorption, distribution, metabolism, excretion and toxicity) characteristics, and screens out a small molecule compound (hereinafter also referred to as small molecule compound AA768) that can target and bind to proteins in the tumor microenvironment, and the small molecule compound has been verified to effectively inhibit tumor growth.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a use of a compound or a salt thereof in the preparation of a drug for treating cancer. The structural formula of the compound is shown below: Wherein, R is selected from halogen.

[0008] Preferably, in the above application, R is selected from the element F.

[0009] Preferably, in the above application, the salt of the compound is hydrochloride.

[0010] Preferably, in the above application, the cancer is breast cancer.

[0011] More preferably, in the above application, the cancer is triple-negative breast cancer.

[0012] More preferably, in the above application, the effective amount of the compound or its salt in the medicine is 5 mg / kg-15 mg / kg.

[0013] Preferably, in the above application, the dosage form of the drug includes injection, liquid oral agent, spray, patch, tablet, powder, ointment or suppository.

[0014] The beneficial effects of the present invention include: applying the small molecule compound AA768 of the present invention to the treatment of cancer (such as triple-negative breast cancer) can effectively inhibit tumor growth without obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the effects of different concentrations of the small molecule compound AA768 on the MDA-MB-231 cell line at different action times;

[0016] Figure 2 This is a picture showing the actual tumor growth after intervention with different concentrations of the small molecule compound AA768;

[0017] Figure 3 This is the time-volume diagram of tumor growth after intervention with different concentrations of the small molecule compound AA768;

[0018] Figure 4 This is a graph showing the tumor weight after intervention with different concentrations of the small molecule compound AA768;

[0019] Figure 5 The figure is a graph showing the effects of different concentrations of the small molecule compound AA768 on the blood routine, AST and ALT of mice; wherein the horizontal axis in the figure is from left to right the Vehicle group (control group), 5 mg / kg group, 10 mg / kg group and 15 mg / kg group. DETAILED DESCRIPTION

[0020] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0021] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0022] An embodiment of the present invention provides a use of a compound or a salt thereof in preparing a drug for treating cancer. The structural formula of the compound is shown below: Wherein, R is selected from halogen.

[0023] It should be noted that the present invention has discovered and verified that the compound of the above structure or its salt form can effectively inhibit tumor growth and achieve the purpose of treating cancer; wherein R can more preferably be any halogen selected from F, Cl, Br or I; more preferably F, that is, the above compound can more preferably be

[0024] In some specific examples, in the above applications, the salt of the compound is selected as hydrochloride.

[0025] It should be noted that preparing the compound of the above structure into a salt form can increase its solubility, stability and bioavailability. The salt form can be a salt form known in the art, and generally a pharmaceutically acceptable hydrochloride can be selected.

[0026] In some specific examples, in the above applications, the cancer is breast cancer.

[0027] It should be noted that the present invention has discovered and verified that the compound of the above structure or its salt form can effectively inhibit the growth of breast tumors and achieve the purpose of treating breast cancer, especially triple-negative breast cancer, a subtype of breast cancer.

[0028] In some specific examples, in the above application, the effective amount of the compound or its salt in the medicine is 5 mg / kg-15 mg / kg.

[0029] It should be noted that, in the present invention, the effective amount of the compound of the above structure or its salt in the drug can be 5 mg / kg-15 mg / kg, such as 5 mg / kg, 7 mg / kg, 9 mg / kg, 12 mg / kg, 14 mg / kg or 15 mg / kg, etc. In addition, the effective amount of the compound or its salt in the drug refers to the effective amount for animals (such as mouse models) or humans.

[0030] In some specific examples, in the above applications, the dosage form of the drug includes injection, liquid oral agent, spray, patch, tablet, powder, ointment or suppository.

[0031] It should be noted that the compound of the above structure or its salt can be prepared into different dosage forms with different excipients, such as injections, liquid oral preparations, sprays, patches, tablets, powders, ointments or suppositories; the above dosage forms can be selected comprehensively according to the location of the tumor and the patient's condition; in addition, the excipients of the above different dosage forms are well known to those skilled in the art, and the methods for preparing drugs in different dosage forms are also well known to those skilled in the art.

[0032] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0033] 1. Cellular activity experiment of small molecule compound AA768

[0034] In the examples of the present invention, the method for testing the cytotoxicity of the small molecule compound AA768 is as follows:

[0035] (1) The cells were inoculated in a 96-well plate, 3000 cells / well, 100ul / well of cell suspension, and 3 replicates were set in each group, which was set as the blank control group (only cell culture medium, no cells);

[0036] (2) On the second day, different concentration gradients (0, 1 μM, 3 μM, 10 μM, 30 μM and 100 μM) of the small molecule compound AA768 were added and incubated in an incubator at 37°C and 5% CO2 for 24 h; 10 μl of CCK8 reagent (cell proliferation and toxicity detection reagent) was added to each well of the 96-well plate, and the plate was protected from light and bubbles;

[0037] (3) The 96-well plate was returned to the incubator and incubated for another 2 h. The absorbance at λ = 450 nm was measured by a full-wavelength microplate reader (TECAN);

[0038] (4) Cell survival rate (%) = [(test well - blank well) / (control well - blank well)] × 100%, wherein the test wells: culture medium containing cells and the test compound; control wells: culture medium containing cells (Gibco DMEM medium) without the test compound; blank wells: culture medium without cells and the test compound.

[0039] (5) Use GraphPad software to create a bar graph.

[0040] According to the above method, the effect of small molecule compound AA768 on the cell activity of MDA-MB-231 cell line was tested respectively. The results are as follows Figure 1As shown, the results showed that the cell activity of MDA-MB-231 cell line did not change significantly when AA768 of different concentrations was applied.

[0041] II. Experiment on the inhibition of tumor growth and toxic side effects of small molecule compound AA768

[0042] In the following examples, the single-reagent method and the two-reagent method are as follows:

[0043] Single reagent method:

[0044] The method of preparing working reagents mainly involves diluting the reagents in the kit according to a certain ratio. For example, for reagent five, it is necessary to dilute reagent five and double distilled water at a volume ratio of 1:9 and prepare it before use. This preparation method is suitable for single-reagent detection and can ensure the stability of the reagents and the accuracy of the reaction.

[0045] Double reagent method: Use R1 and R2 separately; the specific preparation steps are as follows: Preparation of washing solution: dilute with distilled water 1:20 (for example: add 19mL of distilled water to 1mL of concentrated washing solution); the concentrated washing solution taken out of the refrigerator may have crystals, which is a normal phenomenon. You can use a 40℃ water bath to slightly heat it to completely dissolve the crystals before preparing the washing solution. Use it on the same day. Preparation of standard: Take out the standard from the kit, add 1mL of standard & sample diluent to dissolve, cover it and let it stand at room temperature for about 10 minutes. Take 7 1.5mL centrifuge tubes, mark them as S6, S5, S4, S3, S2, S1 and S0 respectively, and add 250μL of standard & sample diluent to each tube; pipette 250μL of standard from S7 into the first centrifuge tube S6, and mix them by gently pipetting; prepare biotin-labeled antibody working solution: 20 minutes before use, dilute 100× concentrated biotin-labeled antibody to 1× working concentration with biotin-labeled antibody diluent (for example: 10μL concentrated solution + 990μL diluent); prepare according to the required amount, use it on the same day, and discard the rest; in addition, prepare the enzyme conjugate working solution: 20 minutes before use, dilute 100× concentrated enzyme conjugate to 1× working concentration with enzyme conjugate diluent (for example: 10μL concentrated solution + 990μL diluent). Prepare according to the required amount, use it on the same day and discard the rest; the preparation of TMB colorimetric solution is as follows: 10 minutes before use, mix TMB colorimetric solution A and solution B in a 1:1 ratio and keep it away from light for later use.

[0046] (I) Construction of a mouse model of spontaneous lung metastasis from orthotopic tumor formation

[0047] (1) Mouse triple-negative breast cancer cells EO771 in the logarithmic growth phase were routinely digested, centrifuged, the supernatant removed, and washed twice with PBS;

[0048] (2) Resuspend in PBS, count, and adjust the cell density to 1×10 7 / ml;

[0049] (3) Three days before inoculation, female C57BC / L mice aged 6-8 weeks were ear-tagged; the prepared cell suspension was blown evenly and then inoculated subcutaneously on one side of the buttocks at 50 μl / side, i.e., 40×10 4 / , a total of 20 mice were vaccinated;

[0050] (4) After inoculation, mice were kept in an SPF environment, and the length and width of the tumors were measured every other day starting from the third day;

[0051] (5) Starting from the 5th day, the mice were randomly divided into 4 groups: the first group was only given saline intervention and was set as the control group; the second group was given an oral administration of 5 mg / kg (small molecule compound AA768); the third group was given an oral administration of 10 mg / kg (small molecule compound AA768); and the fourth group was given an oral administration of 15 mg / kg (small molecule compound AA768). The drugs were given once every three days.

[0052] (5) On day 15, the mice were killed;

[0053] (6) The blood of the mice was taken out and divided into two parts, one for routine blood test and the other for AST and ALT detection (preparation of working reagents: single reagent method directly used; double reagent method using R1 and R2 separately; setting the corresponding parameters on the fully automatic biochemical microplate reader; loading the sample, and the fully automatic biochemical analyzer automatically measured; the experimental results were exported after being detected by the fully automatic biochemical analyzer, and graphpad was used to make a graph); the in situ tumor was completely removed and weighed, and the mouse growth curve and tumor volume (tumor volume (mm)) were prepared using graphpad software. 3 ) = long diameter × wide diameter (2 × 0.5) and tumor weight graph.

[0054] (II) Results

[0055] First, the tumors of mice in each group were Figure 2 As shown, the results showed that the tumor volume of EO771 subcutaneous in situ tumor formation in the AA768 group was significantly smaller than that in the control group, and as the concentration of AA768 continued to increase, the tumor volume continued to decrease.

[0056] Second, the changes in tumor volume of each group of mice from day 5 to day 15 are as follows: Figure 3 As shown in the figure (where each group's curve is the average within the group), the results showed that the tumor growth of EO771 subcutaneous in situ tumor formation in the AA768 group was significantly slower than that in the control group; and as the concentration of AA768 continued to increase, the tumor growth rate gradually decreased.

[0057] Third, the tumor volumes of mice in each group after 15 days are shown in Table 1.

[0058] Table 1 Tumor volume of mice in different groups

[0059]

[0060] It can be seen from Table 1 that the tumor volume of EO771 subcutaneous in situ tumor formation in the AA768 group was significantly lower than that in the control group, and as the concentration of AA768 continued to increase, the tumor volume gradually decreased.

[0061] Fourthly, the tumor weights of mice in each group after 15 days are shown in Table 2 and Figure 4 As shown (in the figure, the black dots are the data of each mouse, and the bar graph data are the average within the group).

[0062] Table 2 Tumor weights of mice in different groups

[0063]

[0064] From the above table 2 and Figure 4 It can be seen that the tumor weight after subcutaneous in situ tumor formation of EO771 in the AA768 group was significantly lower than that in the control group, and as the concentration of AA768 continued to increase, the tumor weight gradually decreased.

[0065] Fifth, the blood routine indexes (RBC, WBC, HCG and PLT) and AST and ALT indexes of each group of mice were tested as follows: Figure 5 As shown, the results showed that at different dosage concentrations, there were no obvious abnormalities in the blood routine and liver function of mice, that is, different concentrations of the small molecule compound AA768 had no obvious toxic side effects on the blood routine and AST, ALT of mice.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Use of a compound or its salt in the preparation of a drug for treating cancer, the structural formula of the compound is as follows: in, R is selected from halogen.

2. The use according to claim 1, characterized in that: R is selected from F, Cl, Br or I.

3. The use according to claim 1 or 2, characterized in that: The salt of the compound is preferably hydrochloride.

4. The use according to claim 1 or 2, characterized in that: The cancer is breast cancer.

5. The use according to claim 4, characterized in that: The cancer is triple-negative breast cancer.

6. The use according to claim 3, characterized in that: The cancer is breast cancer.

7. The use according to claim 6, characterized in that: The cancer is triple-negative breast cancer.

8. The use according to claim 1, 2, 5, 6 or 7, characterized in that: The effective amount of the compound or its salt in the medicine is 5 mg / kg-15 mg / kg.

9. The use according to claim 1, 2, 5, 6 or 7, characterized in that: The medicine comes in the form of an injection, oral liquid, spray, patch, tablet, powder, ointment or suppository.

10. The use according to 8, characterized in that The medicine comes in the form of an injection, oral liquid, spray, patch, tablet, powder, ointment or suppository.

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