CN114573468A

CN114573468A - Protein inhibitor and application thereof

Info

Publication number: CN114573468A
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: 2022-06-03
Anticipated expiration: 2042-02-21
Also published as: CN114573468B

Abstract

Legumain protein inhibitor and application thereof, wherein the structural formula of Legumain protein inhibitor is shown in the specification

The inhibitor can specifically inhibit Legumain protein highly expressed in tumor cells, inhibits the activity of the Legumain protein and reduces the protein expression by combining with a binding domain of the Legumain protein, thereby inhibiting the migration and invasion of the tumor cells and inducing and reducing the tolerance of the tumor cells to chemotherapy, 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 a clear action mechanism, and can effectively inhibit the metastasis and invasion of the tumor.

Description

Protein inhibitor and application thereof

Technical Field

The invention relates to a technology in the field of medicines, in particular to a micromolecular asparagines endopeptidase (Legumain) protein inhibitor and application thereof

Background

The asparaginyl endopeptidase (also called AEP) is a lysosome cysteine protease, has a quite conserved structure, is the only asparaginyl endopeptidase in the body of the known mammal, can specifically hydrolyze aspartic peptide bonds, plays roles of shearing protein and signal transduction in cells, is closely related to the pathophysiological processes of antigen presentation, tumor, Alzheimer disease, liver injury, kidney injury and the like, and the mechanistic research of Legumain in the tumor shows that the asparaginyl endopeptidase has a regulating effect on an important pathway PI 3K-AKT-mTOR in the tumor, and the expression of the pathway is up-regulated when the Leggumain is highly expressed, so that the cell adhesion is reduced, the apoptosis is inhibited, the anti-chemotherapy capacity of the tumor cells is enhanced, the tumor growth is promoted, and the tumor angiogenesis and the epithelial-mesenchymal transition (EMT) are regulated to promote the invasion and metastasis of the breast cancer. Besides this pathway, Legumain also exerts a tumor growth promoting effect through the regulatory effect on NF- κ B pathway, Erk pathway, etc. Immunohistochemical experiments showed that Legumain is highly expressed in a variety of cancer species including breast cancer, and its high expression inside tumors is considered to be highly correlated with poor prognosis, clinical staging, etc. Therefore, Legumain is expected to become a new tumor target therapeutic target.

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

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Legumain protein inhibitor and application thereof, which can specifically inhibit Legumain protein 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 and inducing and reducing the tolerance capacity of the tumor cells to chemotherapy, have 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, have clear action mechanism and can effectively inhibit the metastasis and invasion of the tumor.

The invention is realized by the following technical scheme:

the invention relates to a synthetic method of a Legumain protein inhibitor, which uses CH₂CH(NH₂) COOH is taken as raw material, added with CH₃CHO, NaBH3CN and as H₂O and CH₃OH is used as a solvent to be mixed and then reacts at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]COOH; by addition of tert-butyl carbazate (NH)₂NHBoc), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), 1-hydroxybenzotriazole (HOBt), N-Diisopropylethylamine (DIEA) and CH₂Cl₂Reacting at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CONHNHBoc; trifluoroacetic acid (TFA), Dichloromethane (DCM) and CH are further added₂Cl₂Is a solvent and reacts at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CONHNF₂TFA, addition of NH₂COCH₂Br、 K₂CO₃And reacting at room temperature by taking Tetrahydrofuran (THF) as a solvent to obtain CH₂CH[N(CH₂CH₃)₂]CONHCH₂CONH₂And adding ClCOCH₂R，Na₂CO₃And THF is used as a solvent to react at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CON(COCH₂R)CH₂CONH₂。

The synthetic route of the method is specifically as follows:

wherein: the group R is H, C_1-6Alkyl radical, C_2-6Unsaturated fatty alkanyl radical, C_2-6Unsaturated aliphatic chain hydroxyl, C_3-6Cycloalkyl, halo C_1-6Alkyl, hydroxy C_1-6Alkyl, amino C_1-6Alkyl, halo C_2-6Unsaturated fatty alkanyl radical, C_1-6alkyl-C_3-6Cycloalkyl or halo C_3-6A cycloalkyl group.

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 deuterates thereof:

①

②

③

④

⑤

⑥

⑦

and (b)

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

The tumor is specifically human breast cancer cell MDA-MB-231 or mouse highly metastatic breast cancer cell 4T 1.2.

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

Technical effects

Compared with the prior art, the preparation 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 inhibits the metastasis and invasion of the tumor cells on the basis of no cytotoxicity, and has an inhibiting effect on the bone metastasis of the breast cancer cells by combining the inhibiting effect on the osteoclasts.

Drawings

FIG. 1 shows example S6 (CH)₂CH[N(CH₂CH₃)₂]CON(COCH₂Cl)CH₂CONH₂) Nuclear magnetic resonance spectrum of¹H-NMR(400MHz，DMSO-d₆)；

FIGS. 2A-D are schematic diagrams of activation energy changes and binding sites of a compound of formula I after docking of a drug and an AEP enzyme molecule, respectively;

FIG. 2E, F is a graph showing the time-varying pattern and fold-difference of AEP enzyme activity after administration of different concentrations of compound (i) 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 compound (i) at different concentrations in the examples, wherein FIG. 2G is a graph showing the expression level of AEP after compound (i) in the examples has been treated for 96 hours on 4T1.2 cells;

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

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

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

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

FIG. 4F is a schematic representation of the different concentrations of compound (i) in the examples and the cells invaded after stimulation by AEP KO;

FIG. 4G is a corresponding statistical chart;

FIG. 5 is a graph showing survival curves of 4 groups of mice in example;

FIGS. 6A-D are schematic illustrations of bioluminescence results for four groups of mice in example PBS, 0.44mg/ml epirubicin +0.7mg/ml compound (r), and 0.44mg/ml epirubicin +10mg/ml compound (r), respectively;

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

FIG. 7E is a schematic view showing the bone erosion volumes of 4 groups of samples in example;

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

FIG. 7G, H is a schematic diagram showing the PBS group and the 0.44mg/ml epirubicin group in the example respectively;

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

FIGS. 7J-M are schematic diagrams showing TRAP staining results of bone sections of four groups of mice in examples;

fig. 7N is a graph showing the number of osteoclasts in four groups of slices in the example.

Detailed Description

Example 1

The synthesis route of the compound (I) related in the embodiment is as follows, and the compounds (II) to (III) can be prepared by adopting a similar mode:

the embodiment specifically comprises the following steps:

step 1) starting from S1, i.e. CH₂CH(NH₂) COOH was dissolved in a mixed solvent of ice water and methanol, and then NaBH was added₃CN was added in portions, and after stirring for a few minutes acetaldehyde (5 equivalents first) was added dropwise. After the completion of the dropwise addition, the reaction was carried out at room temperature for 6 hours, and then the remaining 5 equivalents of acetaldehyde were added thereto and the reaction was carried out for two hours. Then concentrated hydrochloric acid was slowly added to adjust the pH to 1.5 and reacted at 40 ℃ for 1.5 hours.

And (3) post-treatment process: the reaction was checked by TLC (DCM: MeOH ═ 7: 1). After the reaction is finished, firstly, the methanol is removed by spinning, then dichloromethane is added to extract impurities, the product is in a water phase, and after the water phase is dried by spinning, the methanol is added to dissolve silica gel and mix with the sample. Purification by column chromatography (DCM: MeOH: water 1000:100:5) gave S2 as a yellow oil, CH₂CH[N(CH₂CH₃)₂]COOH totaled 9.6 g.

Step 2) converting S2, i.e. CH₂CH[N(CH₂CH₃)₂]COOH was dissolved in dichloromethane, NH was added separately₂NHBoc, EDC, HOBT, and finally DIEA was added and reacted at room temperature for 2 hours.

And (3) post-treatment process: the reaction was checked by TLC. After the reaction is finished, water is added for washing, and dichloromethane is used for extraction to obtain S3, namely CH₂CH[N(CH₂CH₃)₂]The crude product of CONHNHBoc is directly put into the next step.

Step 3) converting S3, i.e. CH₂CH[N(CH₂CH₃)₂]CONHNHBoc was dissolved in dichloromethane, TFA was added and the reaction was allowed to proceed overnight at room temperature.

And (3) post-treatment process: and detecting the reaction by TLC. After the reaction is finished, the solvent is dried by spinning, and column chromatography purification is carried out (DCM: MeOH ═ 20:1) to obtain yellow oily matterS4, i.e. CH₂CH[N(CH₂CH₃)₂]CONHNF₂TFA totaled 3.6 g.

Step 4) converting S4, i.e. CH₂CH[N(CH₂CH₃)₂]CONHNF₂TFA was dissolved in THF, NH was added separately₂COCH₂Br and potassium carbonate, at room temperature for 24 hours.

And (3) post-treatment process: the reaction was checked by TLC. After the reaction is finished, silica gel is directly added, the mixture is stirred and dried, and then column chromatography purification is carried out (DCM: MeOH: ammonia water: 200:20:1) to obtain a yellow oily substance S5, namely CH₂CH[N(CH₂CH₃)₂]CONHCH₂CONH₂The total amount is 2 g.

Step 5) converting S5, i.e. CH₂CH[N(CH₂CH₃)₂]CONHCH₂CONH₂Dissolving in THF, adding sodium carbonate, and slowly adding ClCOCH at low temperature₂R, after the addition was complete, the reaction was carried out at room temperature for 2 hours.

And (3) post-treatment process: the reaction was checked by TLC. After the reaction is finished, water is added for washing, ethyl acetate is used for extraction, spin drying and column chromatography purification (PE: EA is 1:1) are carried out, and white solid S6, namely CH is obtained₂CH[N(CH₂CH₃)₂]CON(COCH₂Cl)CH₂CONH₂I.e. compound (i), in a total amount of 50 mg.

As shown in fig. 1, is S6, CH₂CH[N(CH₂CH₃)₂]CON(COCH₂Cl)CH₂CONH₂Nuclear magnetic resonance spectrum of¹H-NMR(400MHz，DMSO-d₆)。

Example 2

The embodiment relates to a compound (I) for inhibiting the expression and activity of human Legumain protein, which specifically comprises the following steps:

step 1) AEP activity detection experiment: to determine the inhibitory effect of the inhibitors on the activity of AEP enzyme, recombinant human legumain protein (rhLegumain, catalog #2199-CY) was diluted to 100. mu.g/mL, pH4.0, in activation buffer containing 50mM sodium acetate and 100mM NaCl for 2h at 37 ℃. 30 ng/. mu.l of activated rhLegumain was diluted in assay buffer (50mM MES, 250mM NaCl), combined with 30. mu.l of different doses of inhibitors (0nM, 10nM, 100nM and 1. mu.M diluted in assay buffer) at pH5.0, and then added to black well plates for 30 min. Thereafter, 40. mu.l of 200. mu.M substrate Z-AAN-AMC diluted in assay buffer, including a control containing 60. mu.l of assay buffer (50mM MES, 250mM NaCl, pH5.0) and 40. mu.l of 200. mu.M substrate, was added. 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. At the same time legumain was incubated with AMC to exclude the possibility that this compound directly quenches AMC.

As shown in FIG. 2, FIGS. 2A-D show the activation energy changes, binding sites, etc. of the inhibitor drug after docking with the AEP enzyme molecule, respectively. As is clear from the figure, the AEP enzyme molecule is increased in activation energy after docking with the inhibitor drug, and is less likely to be activated for enzymatic activity. FIG. 2E is a graph showing the time course of AEP enzyme activity after administration of different concentrations of inhibitor, showing that AEP enzyme activity was significantly inhibited at 1 μ M (p <0.01) and 100nM inhibitor (p <0.05) concentration in C.C.with time, compared to the control (0 nM).

To further verify whether the inhibitor was effective in inhibiting the endogenous enzymatic activity of AEP, total cell lysates and medium from 293L cells cultured with different doses of inhibitor were collected and analyzed for AEP activity. FIG. 2G shows the enzymatic activity of AEP in cells and supernatants after administration of different concentrations of inhibitor, which indicates that the inhibitor inhibits the enzymatic activity of AEP in cell culture in a dose-dependent manner.

Step 2) immunoblot experiment of AEP expression: breast cancer 4T1.2 cells are stimulated with inhibitors of 100nM, 1. mu.M and 0nM, respectively, after 96h of culture the medium is discarded, washed 1 time with PBS and the cells are lysed by addition of RIPA lysate (containing phosphatase inhibitor and protease inhibitor). Centrifuging, quantifying and boiling the cell lysate for denaturation, separating a protein sample by polyacrylamide gel SDS-PAGE electrophoresis, then electrically transferring the protein sample to a nitrocellulose membrane, sealing the nitrocellulose membrane at room temperature by 5% BSA for 1h, incubating the nitrocellulose membrane at 4 ℃ with AEP primary antibody for overnight, incubating the nitrocellulose membrane at room temperature for 1h with corresponding secondary antibody, incubating the ECL chemiluminescence solution for 2min in a dark place, and finally developing the chemiluminescence imaging system to detect the expression level of the protein.

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

Example 3

The embodiment relates to the fact that the inhibitor improves the metastatic invasive capacity of tumor cells by influencing the expression of key proteins in epithelial cell-mesenchymal transition (EMT) processes such as Snail, E-cadherin, 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 of the tumor cells and the capacity of degrading extracellular matrix are improved, and the malignant tumor cells from epithelial cells obtain important biological processes of migration and invasion capacity. The EMT change process can be detected by detecting E-cadherin, Snail and other key proteins, and the migration and invasion capacity of the tumor cells can be judged.

The embodiment specifically comprises the following steps: the breast cancer 4T1.2 cells are stimulated by inhibitors of 100nM, 1. mu.M and 0nM respectively, the medium is discarded after 96h of culture, washed 1 time with PBS, and the cells are lysed by adding RIPA lysate (containing phosphatase inhibitor and protease inhibitor). Centrifuging, quantifying and boiling the cell lysate for denaturation, separating a protein sample by polyacrylamide gel SDS-PAGE electrophoresis, then electrically transferring the protein sample to a nitrocellulose membrane, sealing the nitrocellulose membrane at room temperature by 5% BSA for 1h, incubating the nitrocellulose membrane at 4 ℃ with AEP primary antibody for overnight, incubating the nitrocellulose membrane at room temperature for 1h with corresponding secondary antibody, incubating the ECL chemiluminescence solution for 2min in a dark place, and finally developing the chemiluminescence imaging system to detect the expression level of the protein.

As shown in FIG. 3, FIGS. 3A-F are the expression of E-cadherin, Snail, MMP-9 protein, respectively, following administration of the inhibitor. As can be seen, the expression of E-cadherin is up-regulated and the expression of Snail, MMP-9 is down-regulated after administration of the inhibitor. This result can indicate that the EMT process of the tumor cells is inhibited and the metastatic invasive ability of the tumor cells is decreased after administration of the inhibitor.

Example 4

This example relates to inhibitors that are non-cytotoxic to cells and do not affect cell proliferation, comprising the steps of:

the cells were collected, and 100. mu.l (approximately 5000-10000 cells) of human breast cancer cell MDA-MB-231 and mouse high metastatic breast cancer cell 4T1.2 suspension was added to a 96-well plate (the marginal well was filled with sterile water or PBS). Controls (100. mu.l medium added) were set for each plate; standing at 37 deg.C and 5% CO₂Incubate overnight and observe under inverted microscope. Mu.l of inhibitor drug solutions of different concentrations were added to each well and incubated at 37 ℃. Mu.l of MTS solution was added to each well and incubated at 37 ℃ for 1-4 hours. Absorbance was measured at 490nm for each well. Blank wells (medium and MTS solution, no cells) and control wells (medium and MTS solution without inhibitor, with cells) were also set, and 3-5 duplicate wells were set for each group.

And (3) calculating the cell viability: the OD value of the zeroing well or the control well was subtracted from the OD value of each test well. The OD values of each replicate well were averaged. Percent cell viability ═ 100 (dosed cells OD-blank OD/control cells OD-blank OD) ×

As shown in FIG. 4A, FIG. 4A shows the results of the cell viability experiment of breast cancer cells MDA-MB-23 and 4T1.2 cells in the presence of inhibitor. As can be seen, the growth and proliferation progress of both cells after administration of the inhibitor differed very little from the control group. 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 the migration and invasion of tumor cells, including in particular:

step 1) cell scratch test: the cell scratching method is a method for detecting cell migration movement and repair capacity, is similar to an in vitro wound healing model, and includes the steps of marking lines on the central region of cell growth by using a microspur gun head or other hard objects on single-layer adherent cells cultured in an in vitro culture dish or a flat plate, removing cells in the central part, continuously culturing the cells, and setting a normal control group and an experimental group. After the experiment, the cell culture plate is taken out, whether the peripheral cells grow to the central scratch area or not is observed, and the growth migration capacity of the cells is judged again.

Collecting MDA-MB-231 cells and 4T1.2 cells of breast cancer in logarithmic growth phase, digesting, counting at 5 × 10⁴The density of each well was seeded in a petri dish. When the cell fusion rate reaches 95%, scratching the cell in a culture dish, washing the cell for 2 times by PBS, removing the scratched cell, and taking a picture under a microscope. Setting a control group and an administration group, adding corresponding culture media into the control group, adding culture media containing inhibitors with different concentrations into the administration group, and repeating 3 wells in each group. When the cells in the periphery of the control group grow to the scratched area, the culture medium is discarded, the floating cells are removed by 1-2 times of PBS washing, and 3 random visual fields are selected for photographing in each hole under a microscope.

As shown in FIG. 4, FIGS. 4B-E show the results of the scratch test on 4T1.2 cells and MDA-MB-231 cells, respectively, and it can be seen that AEPKO or inhibitor treatment reduces the migration ability of 4T1.2 cells and MDA-MB-231 cells, and the effect of inhibiting the migration ability is concentration-dependent.

Step 2) Transwell cell invasion assay: the principle of the Transwell invasion experiment is that a culture hole is divided into an upper chamber and a lower chamber by a Transwell nest, tumor cells are planted in the upper chamber, FBS or certain specific chemotactic factors are added into the lower chamber, and the tumor cells can run to the lower chamber with high nutrient content. A layer of matrigel is laid on the upper chamber side of the polycarbonate membrane (porous membrane) and is used for simulating an in vivo extracellular matrix, and if cells enter the lower chamber, Matrix Metalloproteases (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 reflects the invasive capacity of the tumor cells.

Matrigel was spread in the Transwell upper chamber, after which the logarithmic growth phase breast cancer 4T1.2 cells were digested and counted, the cells were resuspended in medium containing 10% fetal bovine serum, and 5 x 10⁴One/well (300uL) was inoculated into the upper chamber of a Transwell chamber and the lower chamber was filled with medium containing 10% fetal bovine serum. After the cells adhere to the wall, the culture medium in the upper and lower chambers is discarded, PBS is washed once, a control group and an administration group are set, equivalent DMSO is added into the upper chamber of the control group, inhibitors with different concentrations are added into the upper chamber of the administration group, and 3 compound holes are formed in each group. Serum-free culture medium is used in the upper chamber, 500uL of culture medium containing 20% fetal calf serum is added in the lower chamber, and then the chambers are placed at constant temperatureThe incubator continues to culture for 12 h. After 12h, the chamber was removed, the medium was discarded, the PBS was washed once, the upper chamber cells were gently scraped off with a cotton swab, and the cells scraped off the upper chamber were removed by washing with PBS 3 times. The lower chamber cells were then 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 away with running water until the background was clear. After drying at room temperature, pictures were taken under a microscope, and the number of invading cells was counted and counted.

As shown in fig. 4, fig. 4F is a graph of the number of invading cells after stimulation with different concentrations of inhibitor and AEP KO, and fig. 4G is a corresponding statistical graph. As can be seen from the figure, the inhibitor can inhibit each cell from invading the lower compartment by degrading matrigel, reduce the number of invading cells of each cell, and has concentration-dependent invasion inhibition effect. The above results indicate that the inhibitor can significantly inhibit the invasion of breast cancer cells.

Example 6

This example relates to the prolongation of survival of breast cancer mice by inhibitors.

The embodiment specifically includes: 40 BALB/C mice were injected subcutaneously with 5X 10⁴4T1.2 cells of breast cancer, which are divided into 4 groups, are respectively injected into the abdominal cavity with PBS, 0.44mg/ml epirubicin +0.7mg/ml inhibitor, 0.44mg/ml epirubicin +10mg/kg inhibitor after the injection of the tumor cells for 3 days, the dosage is 5ml/kg, and the administration interval is 3 days. The death time of each group of mice was recorded, and the survival data of 4 groups of mice was counted.

The Epirubicin (Epirubicin) belongs to antibiotic antitumor drugs. Is an isomer of adriamycin, is a traditional tumor chemotherapy medicament, and is effective to various transplantation tumors.

Fig. 5 is a graph of survival curves for 4 groups of mice, as in fig. 5. As can be seen, the survival of the two groups of mice administered with 0.44mg/ml epirubicin +0.7mg/ml inhibitor and 0.44mg/ml epirubicin +10mg/ml inhibitor was significantly different (p <0.01) from the two groups of mice administered with PBS and 0.44mg/ml epirubicin, but not significantly different between the two groups of mice administered with different concentrations of inhibitor. This result demonstrates that inhibitors significantly extend the survival of tumor mice.

Example 7

This example relates to inhibitors that inhibit 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 breast cancer patients are seriously influenced, the bones of the patients are irreversibly damaged, and the life quality is seriously influenced.

The embodiment specifically includes: 2 x 10 of⁴A total of 24 mice were injected with 4T1.2-luc cells for breast cancer into their left ventricles, and then directly entered into the bone via the arterial system, to create a mouse model of breast cancer bone metastasis. These mice were divided into 4 groups, and 3 days after the injection of tumor cells, PBS, 0.44mg/ml epirubicin +0.7mg/ml inhibitor, 0.44mg/ml epirubicin +10mg/ml inhibitor were administered intraperitoneally at a dose of 5ml/kg with 3 days intervals. In the mouse bone metastasis model, the most common metastatic sites are the proximal tibia, femur, and dorsal spine.

Bioluminescence imaging experiments: luciferase (English name: Luciferase) is a generic name of enzymes capable of generating bioluminescence in nature, and can emit emission light of a specific wavelength after excitation by excitation light of a specific wavelength. By means of the principle, luciferase gene can be transfected into tumor cells, the tumor cells can express luciferase, and after potassium luciferin is given as a substrate of the luciferase, the tracking and quantitative detection of the tumor cells in vivo of a living mouse can be realized on a bioluminescence instrument.

As shown in FIG. 6, FIGS. 6A-D show the bioluminescence results of four groups of mice, PBS, 0.44mg/ml epirubicin +0.7mg/ml inhibitor, and 0.44mg/ml epirubicin +10mg/ml inhibitor, respectively. As can be seen, all mice in the control and epirubicin monotherapy groups had tumor growth on 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 mass in the bone and none of the epirubicin + inhibitor high groups had tumor metastasis (fig. 6D). This result indicates that the bone metastasis of tumor cells in mice is inhibited after administration of the inhibitor, and that the inhibitory effect of the inhibitor is concentration-dependent.

X-ray CT experiment: x-ray (X-ray), also known as roentgen ray or X-ray, is an electromagnetic wave with a wavelength in the range of 0.01 nm to 10 nm. The characteristic of extremely short wavelength makes it able to penetrate through the material, but will attenuate with the density of the material, and after receiving the information of the material tissue density can be obtained by the receiving instrument, the internal structure of the material can be known. CT refers to high-speed, high-precision, and high-sensitivity cross-sectional scanning around a material using X-rays together with a detector having extremely high sensitivity, and computer simulation is performed to obtain the internal structural condition of the material on the 3D plane, which is of great importance in materials science, medicine, and the like.

After 4 groups of mice naturally died due to tumors, hind limb bones of the dead mice were taken and muscles were isolated. Fixed in formalin for 3 days and decalcified in EDTA decalcification solution for 14 days. Hind limb bone samples from 4 groups of mice were X-ray CT examined and 3D reconstructed to explore intra-bone tumor erosion.

As shown in FIGS. 7A-D, X-ray CT results of four groups of mouse bone samples, i.e., PBS, 0.44mg/ml epirubicin +0.7mg/ml inhibitor, and 0.44mg/ml epirubicin +10mg/ml inhibitor, respectively, are shown, FIG. 7E is the bone erosion volume of the 4 groups of samples, and FIG. 7F is the ratio of the bone surface area to the bone volume of the 4 groups of samples, representing the bone integrity. As can be seen from the figure, the bone structures of the bone samples of the PBS group and the 0.44mg/ml epirubicin group are incomplete, and bone erosion exists. The two groups given the inhibitor had complete bone structure and less bone erosion. And the ratios of the bone erosion volume to the bone surface area and the bone volume of the four groups of bone samples have obvious trend (p is less than 0.05) and have concentration dependence. The results show that the inhibitor can effectively inhibit the distal bone metastasis and bone erosion of mice.

Tissue morphology experiments: during tumor bone metastasis, the number of osteoclasts in bone tissue will increase significantly, causing bone tissue erosion and tumor cells will grow in the bone sample. Therefore, the distribution and the number of osteoclasts and tumor cells in a mouse bone sample can be researched through HE staining aiming at the tumor cells and TRAP staining aiming at the osteoclasts, so that the bone erosion condition can be verified.

The HE staining refers to hematoxylin-eosin staining, and hematoxylin staining solution is alkaline, so that chromatin in cell nucleus and nucleic acid in cytoplasm are bluish; eosin stain is acidic, primarily coloring cytoplasmic and extracellular matrix components red. Different species of cells can be differentiated in HE staining due to different cell characteristics and cytoplasm.

TRAP staining refers to anti-tartrate acid phosphatase staining, which is a staining method for specifically detecting osteoclasts, and can make osteoclasts red and make background green or blue. Tartrate-resistant acid phosphatase (TRAP) is a marker enzyme for osteoclasts, specifically distributed in osteoclasts, specific to osteoclasts, and 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, an insoluble red dye is formed at the enzyme activity site, the activity of acid phosphatase can be indirectly known by observing the formation of the red dye, and the state of osteoclast is further identified and analyzed.

HE staining, which is to embed four groups of mouse bone samples in paraffin and cut them into 5 μm sections. Placing the slices in xylene I10 min-xylene II 10 min-absolute ethanol I5 min-absolute ethanol II 5 min-95% ethanol 5 min-90% ethanol 5 min-80% ethanol 5 min-70% ethanol 5 min-distilled water washing. The slices are stained with hematoxylin for 3-8min, washed with tap water, differentiated by 1% hydrochloric acid-alcohol for several seconds, washed with tap water, rewetted with 0.6% ammonia water, and washed with running water. The sections were stained in eosin stain for 1-3 min. Placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, air drying, and sealing with neutral gum. And finally, microscopic examination and image acquisition and analysis are carried out.

TRAP staining: and dewaxing the paraffin sections for 5-10 min, and repeating the process once. Anhydrous ethanol for 5min, 90% ethanol and 70% ethanol each for 2 min. Washing with water for 2 min. And (4) naturally drying, and fixing the TRAP stationary liquid at 4 ℃ for 30-3 min, wherein the TRAP stationary liquid is used for 30-60 s in most cases. Washed with water and dried slightly. And (3) putting the slices into TRAP incubation liquid, putting the slices into a 37' C incubator, dip-dyeing for 45-60 min, and washing with water. And dyeing with hematoxylin staining solution for 5-8 min or dyeing with methyl green staining solution for 2-3 min. Washing with water, air drying, and performing microscopic examination.

The results of histomorphometric examinations are shown in fig. 7G-N, wherein fig. 7G, H shows that the PBS group and the 0.44mg/ml epirubicin group, respectively, the tumor cells stained in deep blue color break through the bone tissue and show invasion, while in fig. 7I, the 0.44mg/ml epirubicin + inhibitor group, the tumor cells do not show invasion and the interface of bone metastasis and tumor is reduced.

As shown in fig. 7J-M, which are TRAP staining results of bone sections of four groups of mice, fig. 7N is the number of osteoclasts in the four groups of sections, and it can be seen from the graph that the number of osteoclasts in bone lesions of the control group or the epirubicin-treated group was increased 4-fold and 3.6-fold, respectively, compared to 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 and invasion of the tumor cells. The invention specifically inhibits the bone metastasis of breast cancer by inhibiting LGMN and inhibits bone erosion in the bone metastasis process of a breast cancer patient.

The small molecular compound obtained by screening can obviously inhibit Legumain from playing a catalytic role and inhibit Legumain expression by inhibiting the binding domain of Legumain protein in a breast cancer cell, so that the phosphorylation of downstream proteins AKT and mTOR is further reduced, the epithelial-mesenchymal transition (EMT) level of a tumor cell is reduced, and the metastasis and invasion capacity of the breast cancer cell is finally inhibited. The small molecular compound obtained by screening has the characteristics of good specificity, high bioavailability and the like, does not directly inhibit the proliferation of tumor cells, almost has no toxic or side effect, only inhibits the metastasis and invasion of the tumor cells, and can inhibit the growth and metastasis of breast cancer tumors when being used together with chemotherapeutic drugs, thereby greatly prolonging the life cycle.

The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A synthetic method of Legumain protein inhibitor is characterized in that CH is used₂CH(NH₂) COOH is taken as raw material, added with CH₃CHO, NaBH3CN and as H₂O and CH₃OH is used as a solvent to be mixed and then reacts at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]COOH; by adding NH₂NHBoc, EDC, HOBt, DIEA and CH₂Cl₂Reacting at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CONHNHBoc; further add TFA, DCM and CH₂Cl₂Reacting at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CONHNF₂TFA, addition of NH₂COCH₂Br、K₂CO₃And THF is used as a solvent to react at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CONHCH₂CONH₂Finally adding ClCOCH₂R，Na₂CO₃And THF is used as a solvent to react at room temperature to obtain CH₂CH[N(CH₂CH₃)₂]CON(COCH₂R)CH₂CONH₂；

The synthetic route of the method is specifically as follows:

2. The method for synthesizing a Legumain protein inhibitor according to claim 1, wherein said method comprises:

step 1) raw material CH₂CH(NH₂) COOH was dissolved in a mixed solvent of ice water and methanol, and then NaBH was added₃CN is added in batches, acetaldehyde is dropwise added after stirring for a few minutes and then reacts at room temperature, concentrated hydrochloric acid is slowly added to adjust the pH value to 1.5, TLC detection reaction is carried out after further reaction, methanol is firstly screwed off after the reaction is finished, dichloromethane is added to extract impurities, methanol is added to dissolve silica gel after water phase is screwed off, the sample is stirred, and then column chromatography purification is carried out to obtain yellow oily CH₂CH[N(CH₂CH₃)₂]COOH；

Step 2) converting CH₂CH[N(CH₂CH₃)₂]COOH was dissolved in dichloromethane, NH was added separately₂NHBoc, EDC and HOBT, and then DIEA is added, TLC detection is carried out after reaction, water is added for washing and dichloromethane extraction, CH is obtained₂CH[N(CH₂CH₃)₂]CONHNHBoc；

Step 3) converting CH₂CH[N(CH₂CH₃)₂]Dissolving CONHNHBoc in dichloromethane, adding TFA, reacting at room temperature overnight, detecting by TLC, spin-drying solvent, and purifying by column chromatography to obtain yellow oily substance CH₂CH[N(CH₂CH₃)₂]CONHNF₂TFA；

Step 4) converting CH₂CH[N(CH₂CH₃)₂]CONHNF₂TFA was dissolved in THF, NH was added separately₂COCH₂Br and potassium carbonate, reacting at room temperature, detecting by TLC, directly adding silica gel, stirring, spin-drying, and purifying by column chromatography to obtain yellow oily CH₂CH[N(CH₂CH₃)₂]CONHCH₂CONH₂；

Step 5) converting CH₂CH[N(CH₂CH₃)₂]CONHCH₂CONH₂Dissolving in THF, adding sodium carbonate, and slowly adding ClCOCH at low temperature₂R, after the dropwise addition, reacting at room temperature, detecting by TLC, adding water for washing, extracting by ethyl acetate, spin-drying, and purifying by column chromatography to obtain white solid CH₂CH[N(CH₂CH₃)₂]CON(COCH₂Cl)CH₂CONH₂I.e. a compound.

3. A Legumain protein inhibitor characterized by the chemical structural formula:

4. The Legumain protein inhibitor according to claim 3, characterized by any one of the following structures and pharmaceutically acceptable salts or deuterides thereof: firstly

②

③

④

⑤

⑥

⑦

And (b)

5. Use of the Legumain protein inhibitor according to any one of claims 1 to 4 for the preparation of an anti-tumor drug or an anti-alzheimer drug;

the tumor is specifically human breast cancer cell MDA-MB-231 and/or mouse highly metastatic breast cancer cell 4T 1.2.

6. The use of claim 5, wherein the effective amount of the medicament is 1mg to 800mg per day.