CN114573468B

CN114573468B - Protein inhibitors and uses thereof

Info

Publication number: CN114573468B
Application number: CN202210156693.8A
Authority: CN
Inventors: 郭方; 陈峻崧; 徐文克
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2022-02-21
Filing date: 2022-02-21
Publication date: 2023-05-02
Anticipated expiration: 2042-02-21
Also published as: CN114573468A

Abstract

Legumain protein inhibitor and application thereof, and structural formula thereof is as follows

The inhibitor can specifically inhibit Legumain protein which is highly expressed in tumor cells, inhibit the activity of the Legumain protein and reduce the protein expression by combining with a binding domain of the Legumain protein, thereby inhibiting the migration and invasion of the tumor cells, inducing the reduction of the tolerance of the tumor cells to chemotherapy, and has the characteristics of good specificity, high bioavailability, no toxic or side effect of liver and kidney and the like, can be used for treating breast cancer, has definite action mechanism, and can effectively inhibit the metastasis and invasion of tumors.

Description

Protein inhibitors and uses thereof

Technical Field

The invention relates to a technology in the field of medicine, in particular to a small molecular asparaginyl endopeptidase (Legumain) protein inhibitor and application thereof

Background

The structure of the asparaginyl endopeptidase (also called AEP) is quite conserved, the asparaginyl endopeptidase is the only asparaginyl endopeptidase in a mammal body which is known at present, the asparaginyl endopeptidase can specifically hydrolyze aspartic peptide bonds, plays roles of shearing proteins and signal transduction in cells, is closely related to pathophysiological processes such as antigen presentation, tumor, alzheimer disease, liver injury, kidney injury and the like, and has a regulatory effect on an important pathway PI 3K-AKT-mTOR in tumors when Legumain is highly expressed, so that the expression of the pathway is up-regulated, cell adhesion is reduced, cell apoptosis is inhibited, the chemotherapeutical resistance of tumor cells is enhanced, tumor angiogenesis is promoted, and epithelial-mesenchymal transition (EMT) is regulated to promote invasion and metastasis of breast cancer. In addition to this pathway, legumain also exerts a tumor growth promoting effect through a regulatory effect on NF- κb pathway, erk pathway, and the like. Immunohistochemical experiments showed that Legumain is highly expressed in a variety of cancer species including breast cancer, and its high expression in the interior of tumors is considered to be highly correlated with poor prognosis, clinical staging, etc. Thus Legumain is expected to become a new tumor targeted therapeutic target.

The existing inhibitors developed for Legumain mainly comprise oligonucleotides, natural drug molecules, small molecular compounds and the like, but the oligonucleotide inhibitors have poor in vivo stability, easy degradation and low bioavailability; the natural medicine has complex molecular components, multi-target effect and undefined mechanism; some small molecule compounds have the problems of insufficient specificity, low bioavailability and the like.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a Legumain protein inhibitor and application thereof, which can specifically inhibit Legumain protein which is highly expressed in tumor cells, inhibit the activity and reduce the protein expression by combining with a binding domain of the Legumain protein, thereby inhibiting the migration and invasion of the tumor cells, inducing the reduction of the tolerance of the tumor cells to chemotherapy, and has the characteristics of good specificity, high bioavailability, no toxic and side effects of liver and kidney and the like, can be used for treating breast cancer, has definite action mechanism, and can effectively inhibit the metastasis and invasion of tumors.

The invention is realized by the following technical scheme:

the invention relates to a synthesis method of Legumain protein inhibitors, which uses CH ₂ CH(NH ₂ ) COOH as raw material and CH as added ₃ CHO, naBH3CN andby H ₂ O and CH ₃ Mixing OH as solvent and then reacting at room temperature to obtain CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]COOH; by addition of tert-butyl carbazate (NH) ₂ NHBoc), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), 1-hydroxybenzotriazole (HOBt), N-Diisopropylethylamine (DIEA) and in CH ₂ Cl ₂ The solvent is reacted at room temperature to obtain CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHBoc; further adding trifluoroacetic acid (TFA), dichloromethane (DCM) and adding CH ₂ Cl ₂ The solvent is reacted at room temperature to obtain CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNF ₂ TFA, further addition of NH ₂ COCH ₂ Br、 K ₂ CO ₃ And Tetrahydrofuran (THF) is used as solvent to react at room temperature to obtain CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHCH ₂ CONH ₂ Finally add ClCOCH ₂ R，Na ₂ CO ₃ And reacting at room temperature by using THF as a solvent to obtain CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CON(COCH ₂ R)CH ₂ CONH ₂ 。

The synthetic route of the method specifically comprises the following steps:

wherein: the radical R being H, C _1-6 Alkyl, C _2-6 Unsaturated fatty chain alkyl, C _2-6 Unsaturated fatty chain hydroxy, C _3-6 Cycloalkyl, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, halogenated C _2-6 Unsaturated fatty chain alkyl, C _1-6 alkyl-C _3-6 Cycloalkyl or halo C _3-6 Cycloalkyl groups.

The invention relates to a Legumain protein inhibitor, which has a chemical structural formula as follows:

the Legumain protein inhibitor is preferably any one of the following structures and pharmaceutically acceptable salts or deuterated products thereof:

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The invention relates to an application of a Legumain protein inhibitor, which is used for preparing an anti-tumor drug or an anti-Alzheimer disease drug.

The tumor is specifically human breast cancer cells MDA-MB-231 or mouse highly metastatic breast cancer cells 4T1.2.

The effective dose of the medicine is preferably 1mg-800 mg/day.

Technical effects

Compared with the prior art, the method has the advantages of fewer steps of the synthetic route, simple reaction conditions, lower industrial synthesis threshold and better industrial practicability, and the prepared inhibitor can inhibit tumor cell metastasis invasion on the basis of no cytotoxicity and has an inhibition effect on breast cancer cell bone metastasis by combining the inhibition effect on osteoclast.

Drawings

FIG. 1 shows the S6 (CH) ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CON(COCH ₂ Cl)CH ₂ CONH ₂ ) Nuclear magnetic resonance spectrum of (2) ¹ H-NMR(400MHz，DMSO-d ₆ )；

FIGS. 2A-D are schematic diagrams of the change in activation energy and binding site of compound (1) drug after docking with AEP enzyme molecule, respectively, in the examples;

FIG. 2E, F is a graph showing the time course and fold difference in AEP enzyme activity after administration of different concentrations of compound (1) in the examples;

FIG. 2G, H is a graph showing the enzymatic activity and fold difference of AEP in cells and supernatant after administration of different concentrations of Compound (1) in the examples, wherein FIG. 2G is a graph showing the expression level of AEP after treatment of 4T1.2 cells for 96 hours with Compound (1) in the examples;

FIG. 2I, J is a graph showing the expression level and fold difference of AEP after 96h of compound (1) treatment of 4T1.2 cells and 4T1.2 AEP KO cells;

FIGS. 3A-F are schematic diagrams showing the expression of E-cadherin, snail, MMP-9 protein, respectively, after administration of Compound (1) in examples;

FIG. 4A is a schematic diagram showing the results of cell viability experiments of breast cancer cells MDA-MB-23 and 4T1.2 in the case of administration of the compound (1) in the example;

FIGS. 4B-E are schematic diagrams showing the results of the scratch experiments on 4T1.2 cells and MDA-MB-231 cells, respectively, in the examples;

FIG. 4F is a schematic representation of invading cells after stimulation with different concentrations of Compound (1) and AEP KO in the examples;

FIG. 4G is a corresponding statistical graph;

FIG. 5 is a schematic diagram of survival curves of 4 mice in the example;

FIGS. 6A-D are graphs showing the bioluminescence results of four groups of mice of PBS, 0.44mg/ml epirubicin, 0.44mg/ml epirubicin+0.7 mg/ml compound (1), 0.44mg/ml epirubicin+10 mg/ml compound (1), respectively, in the examples;

FIGS. 7A-D are graphs showing X-ray CT results of four groups of mouse bone samples of PBS, 0.44mg/ml epirubicin, 0.44mg/ml epirubicin+0.7 mg/ml compound (1), 0.44mg/ml epirubicin+10 mg/ml compound (1), respectively, in examples;

FIG. 7E is a schematic representation of the bone erosion volume of the 4-group sample of the example;

FIG. 7F is a graph showing the ratio of the bone surface area to the bone volume of the 4 groups of samples according to the example;

FIG. 7G, H is a schematic representation of the PBS group and the 0.44mg/ml epirubicin group, respectively, of the examples;

FIG. 7I is a schematic representation of a group of 0.44mg/ml epirubicin+ compound (1) in the example;

FIG. 7J-M is a graph showing TRAP staining results of bone sections of four groups of mice in the examples;

FIG. 7N is a schematic representation of osteoclast numbers in four sections of the example.

Detailed Description

Example 1

The synthetic route of the compound (1) is as follows, and the compounds (2) to (8) can be prepared in a similar manner:

the embodiment specifically comprises the following steps:

step 1) starting material S1, i.e. CH ₂ CH(NH ₂ ) COOH was dissolved in a mixed solvent of ice water and methanol, and NaBH was then added ₃ CN was added in portions and after stirring for a few minutes acetaldehyde was added dropwise (5 eq. First). After the completion of the dropwise addition, the reaction was carried out at room temperature for 6 hours, and then 5 equivalents of acetaldehyde were added and reacted for two hours. Concentrated hydrochloric acid was then slowly added to adjust the pH to 1.5 and reacted at 40 ℃ for 1.5 hours.

Post-treatment process: TLC detection reaction (DCM: meoh=7:1). After the reaction is finished, methanol is firstly removed, then dichloromethane is added to extract impurities, the product is placed in a water phase, and methanol is added to dissolve silica gel after the water phase is dried in a spinning mode to mix the sample. Purification by column chromatography (DCM: meOH: water=1000:100:5) then afforded a yellow oil S2, CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]COOH was 9.6g in total.

Step 2) is performed ₂ CH[N(CH ₂ CH ₃ ) ₂ ]COOH was dissolved in dichloromethane and NH was added separately ₂ NHBoc, EDC, HOBT, DIEA was added last and reacted at room temperature for 2 hours.

Post-treatment process: the reaction was checked by TLC. After the reaction is finished, adding water for washing, extracting with dichloromethane to obtain S3, namely CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]The crude product of CONHNHBoc is directly fed to the next step.

Step 3) mixing S3, namely CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHBoc was dissolved in dichloromethane, TFA was added and reacted overnight at room temperature.

Post-treatment process: the reaction was checked by TLC. After completion of the reaction, column chromatography purification after spin-drying the solvent (DCM: meoh=20:1) gave S4 as a yellow oil, CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNF ₂ TFA amounted to 3.6g.

Step 4) is performed ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNF ₂ TFA was dissolved in THF and NH was added separately ₂ COCH ₂ Br and potassium carbonate, at room temperature for 24 hours.

Post-treatment process: the reaction was checked by TLC. After the reaction, silica gel is directly added, the mixture is dried by spin-drying, and then column chromatography is carried out to obtain yellow oily substance S5, namely CH, after the purification (DCM: meOH: ammonia water=200:20:1) ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHCH ₂ CONH ₂ Together with 2g.

Step 5) step 5 (A), CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CONHCH ₂ CONH ₂ Dissolving in THF, adding sodium carbonate, and slowly adding ClCOCH at low temperature ₂ R, after completion of the dropwise addition, was reacted at room temperature for 2 hours.

Post-treatment process: the reaction was checked by TLC. After the reaction, water is added for washing, and after ethyl acetate extraction and spin drying, column chromatography is performed for purification (PE: EA=1:1), white solid S6, namely CH is obtained ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CON(COCH ₂ Cl)CH ₂ CONH ₂ Namely compound (1), in total 50mg.

As shown in FIG. 1, S6, CH ₂ CH[N(CH ₂ CH ₃ ) ₂ ]CON(COCH ₂ Cl)CH ₂ CONH ₂ Nuclear magnetic resonance spectrum of (2) ¹ H-NMR(400MHz，DMSO-d ₆ )。

Example 2

The present example relates to the demonstration of the inhibition of expression and activity of human Legumain proteins by compound (1), comprising in particular:

step 1) AEP activity assay: to determine the inhibition of AEP enzyme activity by inhibitors, recombinant human legumain protein (rhLegumain, catalog # 2199-CY) was diluted to 100 μg/mL in an activation buffer containing 50mM sodium acetate and 100mM NaCl, pH4.0, for 2h at 37 ℃.30 ng/. Mu.l of activated rhLegum was diluted in assay buffer (50 mM MES, 250mM NaCl), combined with 30. Mu.l of different doses of inhibitors (0 nM, 10nM, 100nM and 1. Mu.M diluted in assay buffer) at pH5.0 and then added to a black well plate for 30 min. Thereafter, 40. Mu.l of 200. Mu.M substrate Z-AAN-AMC diluted in assay buffer was added, including a control containing 60. Mu.l of assay buffer (50 mMMES, 250mMNaCl,pH5.0) and 40. Mu.l of 200. Mu.M substrate. Enzyme activity was measured using a microplate reader (Bio-tek) at 380 and 460nm (top reading), respectively, and read in kinetic mode for 5 minutes for 13 cycles. Meanwhile, legumain was incubated with AMC to exclude the possibility that the compound directly quenched AMC.

As shown in fig. 2, fig. 2A-D are the activation energy change, binding site, etc. after docking of inhibitor drug with AEP enzyme molecule, respectively. From the figure, it is clear that the activation energy increases after the AEP enzyme molecule is docked with the inhibitor drug, and the AEP enzyme molecule is difficult to activate the enzyme activity. Fig. 2E shows the time course of AEP enzyme activity after administration of different concentrations of inhibitor, and shows that AEP enzyme activity was significantly inhibited over time at 1 μm (p < 0.01) and 100nM of inhibitor (p < 0.05) concentration compared to control (0 nM).

To further verify whether inhibitors were effective in inhibiting the endogenous enzymatic activity of AEP, total cell lysates and media of 293L cells cultured with different doses of inhibitors were collected and analyzed for AEP activity. Fig. 2G shows the enzymatic activity of AEP in cells and supernatant following administration of different concentrations of inhibitor, which can inhibit the enzymatic activity of AEP in cell culture in a dose-dependent manner.

Step 2) immunoblotting experiments of AEP expression: breast cancer 4t1.2 cells were stimulated with 100nM, 1 μm and 0nM inhibitors, respectively, the medium was discarded after 96h incubation, washed 1 time with PBS, and the cells were lysed by adding RIPA lysate (containing phosphatase inhibitor and protease inhibitor). Centrifuging, quantifying, boiling and denaturing a cell lysate, separating a protein sample by polyacrylamide gel SDS-PAGE electrophoresis, then electrotransferring to a nitrocellulose membrane, blocking for 1h by 5% BSA at room temperature, incubating overnight by AEP primary antibody at 4 ℃, incubating for 1h by corresponding secondary antibody at room temperature, incubating ECL chemiluminescent liquid for 2min in a dark place, and finally developing and detecting the expression level of the protein by a chemiluminescent imaging system.

As shown in FIG. 2, FIG. 2G shows the AEP expression levels after inhibitor treatment of 4T1.2 cells for 96 hours, and FIG. 2I shows the AEP expression levels after inhibitor treatment of 4T1.2 cells and 4T1.2 AEP KO cells for 96 hours, showing that the AEP expression in 1. Mu.M inhibitor-cultured cells is reduced by 82.31% (0 nM) (p < 0.05) compared to the control group. In the AEPKO cell line, AEP expression was reduced by more than 90% compared to the control and scrambling groups (scramble group). This result indicates that the inhibitor is capable of inhibiting the level of AEP protein expression.

Example 3

This example relates to inhibitors to enhance tumor cell metastasis invasion by affecting the expression of key proteins during epithelial-mesenchymal transition (EMT) of Snail, E-cadherein, MMP-9, and the like.

The EMT refers to a biological process in which epithelial cells are transformed into cells having a mesenchymal phenotype by a specific procedure. In the EMT process of tumor cells, the migration and invasion capacity and the extracellular matrix degradation capacity of the tumor cells are improved, and the malignant tumor cells from epithelial cells acquire important biological processes of migration and invasion capacity. The tumor cell migration invasion capacity can be judged by detecting the EMT change process through detecting key proteins such as E-cadherin, snail and the like.

The embodiment specifically comprises the following steps: breast cancer 4t1.2 cells were stimulated with 100nM, 1 μm and 0nM inhibitors, respectively, the medium was discarded after 96h incubation, washed 1 time with PBS, and the cells were lysed by adding RIPA lysate (containing phosphatase inhibitor and protease inhibitor). Centrifuging, quantifying, boiling and denaturing a cell lysate, separating a protein sample by polyacrylamide gel SDS-PAGE electrophoresis, then electrotransferring to a nitrocellulose membrane, blocking for 1h by 5% BSA at room temperature, incubating overnight by AEP primary antibody at 4 ℃, incubating for 1h by corresponding secondary antibody at room temperature, incubating ECL chemiluminescent liquid for 2min in a dark place, and finally developing and detecting the expression level of the protein by a chemiluminescent imaging system.

As shown in FIG. 3, FIGS. 3A-F show the expression of E-cadherin, snail, MMP-9 protein, respectively, after administration of the inhibitor. As can be seen, E-cadherein expression was up-regulated, snail, MMP-9 expression was down-regulated following inhibitor administration. This result may indicate that the EMT process of tumor cells is inhibited and the metastatic invasion capacity of tumor cells is decreased after administration of the inhibitor.

Example 4

This example relates to inhibitors that have no cytotoxic effect on cells and do not affect cell proliferation, comprising the specific steps of:

cells were collected and 100 μl (about 5000-10000 cells) of a suspension of human breast cancer cells MDA-MB-231 and mouse highly metastatic breast cancer cells 4T1.2 was added to a 96-well plate (the marginal well was filled with sterile water or PBS). Control (100. Mu.l medium) was placed per plate; placing at 37deg.C, 5% CO ₂ Incubate overnight and observe under inverted microscope. Mu.l of inhibitor drug solution of different concentrations was added to each well and incubated at 37 ℃. Mu.l MTS solution was added to each well and incubated at 37℃for 1-4 hours. The absorbance of each well at 490nm was measured. Blank wells (medium and MTS solution, no cells) and control wells (medium without inhibitor and MTS solution, with cells) were simultaneously set, each set having 3-5 multiplex wells.

Cell viability calculation: the OD value of each test well was subtracted by either the zeroed well OD value or the control well OD value. OD values for each replicate well were averaged. Cell viability% = (dosing cell OD-blank OD/control cell OD-blank OD) ×100

As shown in fig. 4A, fig. 4A shows the results of cell viability experiments of breast cancer cells MDA-MB-23 and 4t1.2 cells administered with the inhibitor. It can be seen that the growth and propagation rates of the two cells were slightly different from that of the control group after the inhibitor was administered. The results show that the inhibitor has no cytotoxic effect on cells and does not affect cell proliferation.

Example 5

This example relates to inhibitors that inhibit migration and invasion of tumor cells, and specifically includes:

step 1) cell scratch experiment: the cell scratch method is a method for detecting migration movement and repair capability of cells, and is similar to an in-vitro wound healing model, in-vitro culture dishes or flat-plate culture of single-layer adherent cells, a trace gun head or other hard substances are used for scribing in a central area of cell growth, cells in the central part are removed, then the cells are continuously cultured, and a normal control group and an experimental group are arranged. After the experiment is finished, the cell culture plate is taken out, and whether peripheral cells grow to a central scratch area is observed, so that the growth and migration capacity of the cells is judged again.

Taking logarithmic growth phase breast cancer MDA-MB-231 cells and 4T1.2 cells, counting after digestion, and taking 5×10 ⁴ The density of the spots/wells was inoculated into a petri dish. When the cell fusion rate reaches 95%, scratches are formed in a culture dish, PBS is used for washing for 2 times, the scratched cells are removed, and the microscope is used for photographing. A control group and a dosing group were set, the control group was added with the corresponding medium, the dosing group was added with medium containing different concentrations of inhibitor, 3 duplicate wells per group. When the peripheral cells of the control group grew to the scratch area, the medium was discarded, the floating cells were removed by washing with PBS 1-2 times, and 3 visual fields were randomly selected for each well under a microscope to photograph.

As shown in FIG. 4, FIGS. 4B-E show the results of scratch experiments on 4T1.2 cells and MDA-MB-231 cells, respectively, and it is understood that AEPKO or inhibitor treatment reduces the migration capacity of 4T1.2 cells and MDA-MB-231 cells, and the effect of inhibiting the migration capacity has concentration dependence.

Step 2) Transwell chamber invasion assay: the principle of the Transwell invasion experiment is that a Transwell nest divides a culture hole into an upper chamber and a lower chamber, tumor cells are planted in the upper chamber, FBS or certain specific chemokines are added into the lower chamber, and the tumor cells can run to the lower chamber with high nutrition. A layer of matrigel is spread on the side of the upper chamber of the polycarbonate membrane (porous membrane) to simulate extracellular matrix in vivo, and if cells enter the lower chamber, matrix Metalloproteinases (MMPs) are secreted to degrade the matrigel, so that the matrigel can pass through the polycarbonate. Counting the number of cells entering the lower chamber may reflect the invasive capacity of the tumor cells.

Matrigel was spread in Transwell upper chamber, after which log phase breast cancer 4t1.2 cells were digested and counted, cells were resuspended in medium containing 10% fetal bovine serum at 5 x 10 ⁴ Each well (300 uL) was inoculated into the upper chamber of a Transwell chamber, and the lower chamber was fed with medium containing 10% fetal bovine serum. After cells were attached, the medium in the upper and lower chambers was discarded, washed once with PBS, and a control group and a dosing group were set, with equal amounts of DMSO added to the upper chamber of the control group, and different concentrations of inhibitors were added to the upper chamber of the dosing group, with 3 duplicate wells per group. The upper chamber was used with serum-free medium, and the lower chamber was used with 500uL of medium containing 20% fetal bovine serum, and then placed in a constant temperature incubator for continuous culture for 12h. After 12h, the cells were removed, medium was discarded, washed once with PBS, upper cells were gently scraped with a cotton swab, washed 3 times with PBS, and the upper scraped cells were removed. Then, the lower chamber cells were fixed with 4% paraformaldehyde for 15min, washed 3 times with PBS, stained with crystal violet for 30min, and then the excess staining solution was slowly washed with running water until the background was clean. After drying at room temperature, photographs were taken under a microscope, and the number of invading cells was counted and counted.

As shown in fig. 4, fig. 4F shows the affected cells after different concentrations of inhibitor and AEP KO stimulation, and fig. 4G shows the corresponding statistical graph. From the figure, the inhibitor can inhibit invasion of each cell degradation matrigel to the lower chamber, reduce the number of invasion cells of each cell, and has concentration dependence of invasion inhibition effect. The above results demonstrate that inhibitors can significantly inhibit invasion of breast cancer cells.

Example 6

This example relates to inhibitors to extend survival of breast cancer mice.

The embodiment specifically comprises the following steps: subcutaneous injections of 5X 10 were performed on 40 BALB/C mice ⁴ The breast cancer 4T1.2 cells are divided into 4 groups, and PBS, 0.44mg/ml epirubicin, 0.44mg/ml epirubicin+0.7 mg/ml inhibitor are respectively injected intraperitoneally 3 days after tumor cell injectionThe preparation, 0.44mg/ml epirubicin+10 mg/kg inhibitor, was 5ml/kg at 3 days intervals. The death time of each group of mice was recorded, and the survival data of 4 groups of mice were counted.

The Epirubicin (Epirubicin) belongs to antibiotic antitumor drugs. Is an isomer of doxorubicin, is a traditional tumor chemotherapeutic drug, and is effective on various transplanted tumors.

As shown in fig. 5, fig. 5 is a survival curve of 4 mice. As can be seen, the mice given 0.44mg/ml epirubicin plus 0.7mg/ml inhibitor and 0.44mg/ml epirubicin plus 10mg/ml inhibitor had significantly different survival (p < 0.01) than the mice given PBS, 0.44mg/ml epirubicin, but no significant difference was seen between the mice given different concentrations of inhibitor. This result demonstrates that the inhibitor significantly prolongs the survival of tumor mice.

Example 7

This example relates to inhibitors of distal bone erosion and metastasis of breast cancer 4t1.2 cells in mice.

In the occurrence and development process of breast cancer, bone metastasis is often accompanied, so that the treatment and prognosis of a breast cancer patient are seriously affected, irreversible damage is caused to bones of the patient, and the quality of life is seriously affected.

The embodiment specifically comprises the following steps: will be 2 x 10 ⁴ The individual breast cancer 4t1.2-luc cells were injected into the left ventricle of 24 mice in total, so as to directly enter the bone through the arterial system, creating a model of breast cancer bone metastasis in the mice. The mice were divided into 4 groups, and PBS, 0.44mg/ml epirubicin, 0.44mg/ml epirubicin+0.7 mg/ml inhibitor, 0.44mg/ml epirubicin+10 mg/ml inhibitor were administered by intraperitoneal injection, respectively, 3 days after tumor cell injection, at a dose of 5ml/kg, at an interval of 3 days. In the mouse bone metastasis model, the most common metastasis locations are the proximal tibia, femur, and spine.

Bioluminescence imaging experiments: luciferases (english name: luciferases) are a general term for enzymes capable of generating bioluminescence in nature, and emit light of a specific wavelength after excitation by excitation light of a specific wavelength. By means of the principle, the luciferase gene can be transfected into tumor cells, so that the tumor cells express the luciferase, and after potassium luciferin is given as a substrate of the luciferase, the tumor cells can be tracked and quantitatively detected in vivo in a living mouse on a bioluminescence instrument.

As shown in FIG. 6, FIGS. 6A-D are bioluminescence results of four groups of mice, PBS, 0.44mg/ml epirubicin +0.7mg/ml inhibitor, 0.44mg/ml epirubicin +10mg/ml inhibitor, respectively. As can be seen, in the control and epirubicin single drug treatment groups, all mice had tumor growth in the proximal tibia, femur or dorsal spine (fig. 6A, B), whereas in the epirubicin + inhibitor low group (fig. 6C), only half of the mice had tumor masses in the bone, while the epirubicin + inhibitor high group had no tumor metastasis (fig. 6D). This result demonstrates that tumor cell bone metastasis is inhibited in mice after administration of the inhibitor, and that the inhibition effect of the inhibitor is concentration-dependent.

X-ray CT experiment: x-rays (X-rays), also known as ethion rays or X-rays, are an electromagnetic wave having a wavelength in the range of 0.01 nm to 10 nm. The characteristic of extremely short wavelength makes it can pass through the material, but can decay with the density of material, can obtain the information of material tissue density after receiving instrument receives, knows material internal structure. CT refers to the use of X-rays, and the X-rays and a detector with extremely high sensitivity are used for carrying out high-speed, high-precision and high-sensitivity section scanning around a material, and the internal structure condition of the material on the 3D layer is obtained through computer simulation, so that the CT has important application in the aspects of materialism, medicine and the like.

After the 4 groups of mice die naturally due to tumors, the hind bones of the dead mice are taken, and muscles are separated. Formalin was fixed for 3 days and decalcified in EDTA decalcification solution for 14 days. Hind limb bone samples from 4 groups of mice were subjected to X-ray CT testing and 3D reconstruction to investigate the tumor erosion conditions inside the bone.

As shown in fig. 7A-D, X-ray CT results for four groups of mouse bone samples, PBS, 0.44mg/ml epirubicin +0.7mg/ml inhibitor, 0.44mg/ml epirubicin +10mg/ml inhibitor, respectively, fig. 7E for bone erosion volume for the 4 groups of samples, fig. 7F for the ratio of bone surface area to bone volume for the 4 groups of samples, representing bone integrity. From the figure, it can be seen that the bone sample bone structure of PBS group and 0.44mg/ml epirubicin group is incomplete and bone erosion exists. While the two groups given the inhibitor had complete bone structure and a lesser degree of bone erosion. The ratio of the bone erosion volume to the bone surface area to the bone volume of the four groups of bone samples all had a clear trend (p < 0.05) and had a concentration dependence. The results show that the inhibitor can effectively inhibit distal bone metastasis and bone erosion of mice.

Histomorphology experiments: during tumor bone metastasis, the number of osteoclasts in bone tissue will rise significantly, causing bone tissue erosion, and tumor cells will grow in the bone sample. Therefore, the distribution and the quantity of the osteoclast and the tumor cells in the mouse bone sample can be explored through HE staining aiming at the tumor cells and TRAP staining aiming at the osteoclast, and the bone erosion condition can be verified.

HE staining refers to hematoxylin-eosin staining, wherein hematoxylin staining solution is alkaline, and is mainly used for coloring chromatin in cell nuclei and nucleic acid in cytoplasm into purple blue; eosin stain is acidic and primarily reds the cytoplasmic and extracellular matrix components. Different cell types can be distinguished in HE staining due to different cell properties and cytoplasms.

TRAP staining refers to staining with tartaric acid-resistant acid phosphatase, and is a staining method for specifically detecting osteoclasts, which can make the osteoclasts red and the background green or blue. Tartrate-resistant acid phosphatase (TRAP) is a marker enzyme of osteoclasts, is specifically distributed in osteoclasts, is unique to osteoclasts, and is generally used as an important marker for identifying osteoclasts. Under the acidic condition containing tartaric acid, TRAP can hydrolyze naphthol AS-BI phosphate, the generated naphthol AS-BI is immediately combined with hematoxylin, insoluble red dye is formed at the enzyme active site, the activity of acid phosphatase can be indirectly known by observing the formation of the red dye, and the state of osteoclasts can be further identified and analyzed.

HE staining four groups of mouse bone samples were paraffin embedded and cut into 5 μm sections. Sequentially placing the slices into xylene I10 min-xylene II 10 min-absolute ethanol I5 min-absolute ethanol II 5min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5 min-distilled water for washing. The slices are stained with hematoxylin for 3-8min, washed with tap water, differentiated with 1% hydrochloric acid-alcohol for several seconds, washed with tap water, and flushed with 0.6% ammonia water. The slices are dyed in eosin dye solution for 1-3min. Sequentially placing the slices into 95% alcohol I5 min-95% alcohol II 5 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 5min for dehydration and transparency, taking out the slices from the xylene, slightly airing, and sealing the slices with neutral resin. And finally, microscopic examination and image acquisition and analysis are carried out.

TRAP staining: dewaxing paraffin sections for 5-10 min, repeating once. Absolute ethanol 5min,90% ethanol and 70% ethanol each for 2min. Washing with water for 2min. Naturally airing, and fixing the TRAP fixing solution for 30-3 min at 4 ℃ and 30-60 s in most cases. Washing with water and slightly airing. Slicing into TRAP incubation liquid, placing in a 37' C incubator, dip-dyeing for 45-60 min, and washing with water. Hematoxylin staining solution is stained for 5-8 min or methyl green staining solution is stained for 2-3 min. Washing with water, airing and microscopic examination.

The results of the histomorphometric assay are shown in FIGS. 7G-N, wherein FIG. 7G, H shows PBS and 0.44mg/ml epirubicin, respectively, and it was seen that tumor cells stained in dark blue broken through bone tissue and appeared invasive, whereas in FIG. 7I,0.44mg/ml epirubicin + inhibitor, tumor cells did not appear invasive bone tissue and bone metastasis tumor interface was reduced.

As shown in fig. 7J-M, which shows TRAP staining results of bone sections of four groups of mice, fig. 7N shows the number of osteoclasts in the four groups of sections, and it is understood that the number of osteoclasts in the control group or the epirubicin-treated group was increased by 4-fold and 3.6-fold, respectively, compared to the number of osteoclasts in the 2 groups to which the inhibitor was administered (fig. 7N).

Compared with the prior art, the invention can inhibit the expression and activity of LGMN under the condition of no cytotoxicity to tumor cells, thereby inhibiting the metastasis invasion of tumor cells. The invention specifically inhibits bone metastasis of breast cancer by inhibiting LGMN, and inhibits bone erosion in the bone metastasis process of breast cancer patients.

The small molecular compound obtained by screening can obviously inhibit the Legumain from playing a catalytic role and inhibit the Legumain from expressing by inhibiting the binding domain of Legumain protein in breast cancer cells, so that the phosphorylation of downstream proteins AKT and mTOR is further down-regulated, the epithelial-mesenchymal transition (EMT) level of tumor cells is down-regulated, and finally the metastasis and invasion capacity of the breast cancer cells are inhibited. The small molecular compound obtained by screening in the invention has the characteristics of good specificity, high bioavailability and the like, does not directly inhibit proliferation of tumor cells, has little toxic or side effect, only inhibits metastasis and invasion of tumor cells, can inhibit growth and metastasis of breast cancer tumors when being combined with chemotherapeutics, and greatly prolongs the survival period.

The foregoing embodiments may be partially modified in numerous ways by those skilled in the art without departing from the principles and spirit of the invention, the scope of which is defined in the claims and not by the foregoing embodiments, and all such implementations are within the scope of the invention.

Claims

1. A Legumain protein inhibitor, which is characterized by being any one of the following structures and pharmaceutically acceptable salts thereof:

①

②/>

③

④/>

⑤

⑥/>

⑦

(8)/(8)>

2. A method for synthesizing the Legumain protein inhibitor (1), (2), (3), (5), (6) or (8) according to claim 1, wherein the Legumain protein inhibitor is represented by CH ₃ CH(NH ₂ ) COOH as raw material, adding CH ₃ CHO, naBH3CN and with H ₂ O and CH ₃ Mixing OH as solvent and then reacting at room temperature to obtain CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]COOH; by addition of NH ₂ NHBoc, EDC, HOBt, DIEA by CH ₂ Cl ₂ The solvent is reacted at room temperature to obtain CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHBoc; further TFA, DCM and CH ₂ Cl ₂ The solvent is reacted at room temperature to obtain CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNH ₂ TFA, further addition of NH ₂ COCH ₂ Br、K ₂ CO ₃ And reacting at room temperature by using THF as a solvent to obtain CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHCH ₂ CONH ₂ Finally add ClCOCH ₂ R，Na ₂ CO ₃ And reacting at room temperature by using THF as a solvent to obtain CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHN(COCH ₂ R)CH ₂ CONH ₂ ；

The synthetic route of the method specifically comprises the following steps:

wherein: the R group is shown as a structural formula of a compound (1), (2), (3), (5), (6) or (8) in the claim 1.

3. The method for synthesizing the Legumain protein inhibitor according to claim 2, which is characterized by comprising the following steps:

step 1) raw material CH ₃ CH(NH ₂ ) COOH was dissolved in a mixed solvent of ice water and methanol, and NaBH was then added ₃ Adding CN in batches, stirring for several minutes, dropwise adding acetaldehyde dropwise, reacting at room temperature, slowly adding concentrated hydrochloric acid to adjust pH to 1.5, further reacting, performing TLC detection reaction, removing methanol after reaction, adding dichloromethane to extract impurities, drying water phase, adding methanol to dissolve silica gel, stirring, and purifying by column chromatography to obtain yellow oily substance CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]COOH；

Step 2) CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]COOH was dissolved in dichloromethane and NH was added separately ₂ NHBoc, EDC, HOBT DIEA is added last, and after reaction, TLC detection and water washing and dichloromethane extraction are carried out to obtain CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHBoc；

Step 3) CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHBoc was dissolved in dichloromethane, TFA was added, TLC was used after overnight reaction at room temperature and column chromatography was performed after spin-drying the solvent to give yellow oil CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNH ₂ TFA；

Step 4) CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNH ₂ TFA was dissolved in THF and NH was added separately ₂ COCH ₂ Br and potassium carbonate, TLC detection after room temperature reaction, direct silica gel sample addition, spin drying and column chromatography purification to obtain yellow oily matter CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHCH ₂ CONH ₂ ；

Step 5) CH ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHNHCH ₂ CONH ₂ Dissolving in THF, adding sodium carbonate, and slowly adding ClCOCH at low temperature ₂ R, after completion of the dropwise addition, TLC detection after reaction at room temperature and washing with water, extraction with ethyl acetateThe white solid CH is obtained after column chromatography purification after drying ₃ CH[N(CH ₂ CH ₃ ) ₂ ]CONHN(COCH ₂ R)CH ₂ CONH ₂ I.e. the compound.

4. Use of a Legumain protein inhibitor according to claim 1 for the preparation of an anti-tumour agent;

the tumor is human breast cancer cells MDA-MB-231 and/or mouse highly metastatic breast cancer cells 4T1.2.

5. The use according to claim 4, wherein the effective dose of the medicament is 1mg-800 mg/day.