Derivative with repaglinide tetravalent platinum structure and preparation method and application thereof

Abstract

The invention provides a derivative with a repaglinide tetravalent platinum structure and a preparation method and application of the derivative, the compound with the repaglinide tetravalent platinum structure is shown in the general formula (I), # imgabs0 #, # imgabs1 # is selected from cis-platinum or oxaliplatin, # imgabs2 # is selected from iso-platinum or oxaliplatin, and # imgabs2 # is selected from iso-platinum or oxaliplatin. L is hydroxyl or # imgabs2. A series of repaglinide platinum (IV) compounds taking the p53 pathway as the target spot disclosed by the invention show good anti-proliferation and anti-metastasis activity in vitro and in vivo. The expression of p53 can be up-regulated due to serious DNA damage caused by platinum nucleus, and repaglinide (RPG) can also effectively promote the secretion of p53 through a lumican / p53 / p21 pathway. According to the invention, a series of repaglinide tetravalent platinum derivatives are designed and synthesized, and the compounds start mitochondrial mediated apoptosis through a Bcl-2 / Bax / caspase-3 pathway. Afterwards, along with up-regulation of p53, the compound can effectively activate apoptosis-promoting autophagy and inhibit an epithelial-mesenchymal transition (EMT) process, and further inhibit angiogenesis of tumors by inhibiting COX-2, MMP9 and VEGFA (vascular endothelial growth factor A). The compound can also increase the number of CD3 + and CD8 + T cells in tumors by blocking an immune checkpoint PD-L1 and stimulate anti-tumor immunity. The multiple anti-tumor mechanisms synergistically inhibit proliferation and metastasis of tumor cells.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to a derivative having a tetravalent platinum structure of repaglinide, a preparation method thereof, and an application thereof in anti-tumor drugs. Background Art

[0002] Cancer remains a major obstacle to human health, especially metastatic tumors, which cause more than 90% of cancer patients to die. Platinum drugs represented by cisplatin (CDDP), oxaliplatin (OXP) and carboplatin (CBP) are widely used in clinical tumor treatment. They have potent and broad-spectrum anti-tumor activity and are the main cornerstone of tumor chemotherapy. Despite their great success, problems such as poor stability, severe dose-limiting toxicity and drug resistance limit their clinical application. It is worth noting that classic platinum (II) drugs have weak efficacy against metastatic tumors. Therefore, the development of new candidate platinum compounds with the potential to overcome the inherent defects of platinum (II) drugs is a top priority for researchers in the medical field. Platinum (IV) compounds with bioactive axial ligands provide an effective way to develop new anti-tumor proliferation and anti-metastatic drugs, and are expected to develop into a new generation of platinum drugs.

[0003] Tumor suppressor p53 is an important barrier to prevent the occurrence and development of cancer and is regarded as a powerful "guardian" of the human genome. Increasing evidence shows that p53 gene mutation and inactivation is the most common single gene mutation in human cancer, which not only leads to the loss of p53 anti-cancer function, but also promotes the movement, invasion and metastasis of cancer cells through multiple pathways. Therefore, the development of new anti-tumor drugs targeting p53 is an attractive strategy for the development of chemotherapy drugs.

[0004] Protective autophagy is an important cellular process involved in maintaining the balance of tumor cells and the formation of the tumor microenvironment (TME). It is essential for tumor proliferation, survival and differentiation, and also plays an important role in promoting the resistance of tumor cells to platinum drugs. Studies have shown that p53 and autophagy interact with each other in tumors and jointly participate in the occurrence and development of tumors. Protective autophagy is an important factor causing the inactivation of p53's anti-tumor function and has great potential in promoting tumor development. On the other hand, p53 plays a double-edged sword role in regulating autophagy. It is worth noting that the use of chemotherapeutic drugs to increase the expression of nuclear p53 can effectively inhibit the function of Bcl-2 family anti-apoptotic proteins, and then upregulate the activity of Beclin1 by blocking the interaction between Bcl-2 and Beclin1, promote autophagic flux, destroy cell balance, and ultimately lead to autophagic death of tumor cells. Therefore, it is of great potential to target p53 to induce pro-apoptotic autophagy and further improve the sensitivity of tumor cells to platinum drugs.

[0005] Epithelial-mesenchymal transition (EMT) causes tumor cells to transform from an epithelial state to a mesenchymal state, plays a crucial role in the process of tumor metastasis, and is closely related to chemotherapy failure and poor prognosis in cancer. Mutated p53 and protective autophagy can promote EMT by regulating key transcription factors associated with EMT (such as Snail1 and Vimentin). In addition, p53 and EMT work together to participate in the regulation of extracellular matrix and angiogenesis by regulating key proteins MMPs and VEGAs, and further promote tumor metastasis.

[0006] Immunosuppression is one of the prominent features of malignant tumors and contributes to the development of cancer to a great extent. Recent studies have shown that p53 can regulate innate and adaptive immunity by regulating the immune checkpoint (ICs) PD-L1. The upregulation of PD-L1 can inhibit T cell immunity and weaken the anti-tumor effect of chemotherapy drugs. Therefore, targeting the key protein p53, further reversing the EMT process, and activating the immune response of tumor cells will bring new hope for the treatment of cancer, especially metastatic tumors.

[0007] Lumican protein plays an important role in the structural organization of collagen fibers in the extracellular matrix (ECM) and can affect tumor development through multiple signaling pathways. High expression of Lumican can inhibit the p53 / p21 signaling pathway, thereby promoting the occurrence of EMT, invasion and metastasis of tumor cells. Repaglinide (RPG) is a widely used insulin secretagogue for the treatment of type II diabetes. Recent studies have found that RPG can effectively inhibit the invasion and metastasis of tumor cells by targeting the Lumican / p53 axis, and it also plays an important role in increasing autophagy and inhibiting inflammation and tumor angiogenesis. Therefore, RPG has the potential for further research in the field of anti-tumor.

[0008] Based on this, we introduced repaglinide as a functional group into the platinum (IV) system and designed a series of repaglinide platinum (IV) compounds, which destroy DNA through the platinum nucleus; activate the lumican / p53 / p21 signaling axis through the repaglinide functional fragment to increase the expression of p53; and then reverse EMT and immunosuppression, and synergistically exert anti-tumor proliferation and anti-metastasis effects. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention proposes a compound having a tetravalent platinum structure of repaglinide, and synthesizes a series of novel tetravalent platinum compounds modified by repaglinide compounds by introducing repaglinide molecules into a tetravalent platinum mother nucleus. Such tetravalent platinum compounds of repaglinide have significant anti-tumor proliferation ability and good therapeutic effects on metastatic malignant tumors, and increase the expression of p53 protein through the Lumican / p53 / p21 signal axis, thereby reversing the EMT process and immunosuppression, and further exerting anti-proliferation and anti-metastasis effects. Such drugs have opened up a new direction for the development of anti-tumor drugs, especially the research and development of anti-metastatic malignant tumor drugs.

[0010] In order to achieve the above object, the present invention provides a compound having a tetravalent platinum structure of repaglinide,

[0011] The general formula is shown in (Ⅰ):

[0012]

[0013] in, Selected from cisplatin or oxaliplatin; L is hydroxyl or

[0014] Further, the compound is any one of the following:

[0015]

[0016] The compound of the present invention is selected from:

[0017]

[0018] Another object of the present invention is to provide a method for preparing the compound represented by general formula (I), and the synthesis route of the compound is shown below.

[0019] Described synthetic route 1 is as follows:

[0020]

[0021] Compound II and compound III (repaglinide anhydride) undergo coupling reaction to obtain an asymmetric monosubstituted repaglinide-modified tetravalent platinum compound Ia; wherein the molar ratio of compound II to compound III is 1:1.0-1.5;

[0022] Described synthetic route 2 is as follows:

[0023]

[0024] Compound II and compound RPG undergo coupling reaction to obtain a symmetrical disubstituted repaglinide-modified tetravalent platinum compound Ib; wherein the feed molar ratio of compound II to compound RPG is 1:2.0-5.0.

[0025] Furthermore, in the synthetic route 1, the preparation steps of the asymmetric monorepaglinide tetravalent platinum compound are:

[0026] Under an inert gas atmosphere, compound III is dissolved in an anhydrous organic solvent for reaction, compound II is added, and after the reaction is protected from light, compound Ia is isolated through post-treatment;

[0027] The molar ratio of compound II to compound III is 1:1.0-1.5; the feeding relationship between compound II and organic solvent is that 10-100 mL of organic solvent is added for every 1 g of compound II;

[0028] In the second synthetic route, the preparation steps of the symmetrical tetravalent platinum compound of bisrepaglinide are as follows:

[0029] Under an inert gas atmosphere, compound RPG, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, and after the reaction is protected from light, compound Ib is isolated through post-treatment;

[0030] Among them, the feeding molar ratio of compound II, compound RPG, condensation agent and organic base is 1:2.0~5.0:2.0~5.0:2.0~5.0; the feeding relationship between compound II and organic solvent is that 10~100mL of organic solvent is added for every 1g of compound II.

[0031] Furthermore, the inert gas is nitrogen, helium or argon; the condensing agent is TBTU, HATU or EDCI; the organic base is triethylamine, N,N-diisopropylethylamine or 4-dimethylaminopyridine; and the organic solvent is DMF or DMSO.

[0032] The preparation process can be specifically as follows:

[0033] II and compound III (repaglinide anhydride) are added to the reaction container, the air in the system is replaced with nitrogen, anhydrous DMSO is added, and the reaction system is placed at 25-120° C. in the dark for 24-72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and an asymmetric monosubstituted repaglinide modified tetravalent platinum compound Ia is obtained by column chromatography.

[0034] TBTU and compound RPG (repaglinide) are added to a reaction container, the air in the system is replaced with nitrogen, anhydrous DMF is added, the reaction is stirred at room temperature, anhydrous triethylamine is added to the reaction system, the reaction is stirred at room temperature, a tetravalent platinum compound II is added to the reaction system, the air in the system is replaced with nitrogen again, and the reaction system is placed at 25-120° C. in the dark for reaction for 24-72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and a symmetrical disubstituted repaglinide-modified tetravalent platinum compound Ib is obtained by column chromatography.

[0035] Further, the compound II is composed of After being oxidized by hydrogen peroxide, the product is prepared. The specific preparation process is as follows:

[0036]

[0037] Divalent platinum compounds The dihydroxy tetravalent platinum compound II is prepared by oxidizing with hydrogen peroxide at 60-70° C. for 1-8 hours.

[0038] In another aspect, the present invention provides a pharmaceutical composition comprising an effective therapeutic amount of a compound represented by the general formula (I) and a pharmaceutically acceptable excipient thereof.

[0039] The pharmaceutically acceptable excipients in the present invention include one or more of carriers, excipients, and diluents; such as binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, preservatives, solubilizers, and bases. Pharmaceutically acceptable excipients may be aqueous or non-aqueous. Conventional excipients include colloids, such as gelatin; starches, such as corn starch and potato starch; sugars, such as lactose, glucose, and sucrose; cellulosic materials and mixtures thereof, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate. Pharmaceutically acceptable excipients include, but are not limited to, tragacanth powder, malt, talc, oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.), alcohols (such as propylene glycol, ethanol, glycerol, sorbitol, mannitol, polyethylene glycol, etc.), esters (such as ethyl oleate, ethyl laurate, agar), buffers (such as magnesium hydroxide, aluminum hydroxide, boric acid and sodium borate and phosphate buffer), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution.

[0040] The tetravalent platinum compound or pharmaceutical composition of repaglinide described in the present invention is in the form of tablets, capsules, aerosols, dispersible tablets, oral liquids, suppositories, pills, large infusions, small injections, freeze-dried powder injections, ointments or liniments, including various sustained-release, controlled-release dosage forms or nano-preparations prepared by conventional methods using currently recognized pharmaceutical common sense.

[0041] The tetravalent platinum compound of repaglinide of the present invention can be administered in the form of a unit dose, and the administration route can be enteral and parenteral, such as oral, intramuscular, subcutaneous, nasal, etc.

[0042] The administration route of the tetravalent platinum compound of repaglinide of the present invention can be intravenous administration, including intravenous injection, intramuscular injection, intratumor injection, subcutaneous injection and acupoint injection.

[0043] The method of preparing the active ingredient into a drug in the present invention can be prepared by methods known to ordinary technicians in the field, for example: the active ingredient can be diluted with a carrier or encapsulated in a carrier so that it can be quickly released, slowly released or delayed released after administration to a subject.

[0044] Another object of the present invention is to provide the use of the compound or pharmaceutical composition represented by the general formula (I) in the preparation of anti-tumor drugs, specifically in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

[0045] The tetravalent platinum compound of repaglinide described in the present invention can have a good therapeutic effect on metastatic malignant tumors, can effectively inhibit the tumor epithelial-mesenchymal transition process, and effectively activate the tumor immune response, and has a good therapeutic effect on metastatic malignant tumors.

[0046] Furthermore, the anti-tumor is anti-lung cancer, anti-resistant lung cancer, anti-liver cancer or anti-breast cancer, etc.; wherein, the anti-tumor proliferation is specifically anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer; the anti-tumor metastasis is anti-mouse breast cancer cells.

[0047] The invention also provides a combined preparation, comprising a compound or pharmaceutical composition as shown in general formula (I) and a combination of paclitaxel, fluorouracil, gemcitabine, vinca alkaloids, and antibodies as anti-tumor drugs.

[0048] The tetravalent platinum compound of the repaglinide compound of the present invention is expected to be used alone or in combination with marketed platinum drugs, paclitaxel drugs, fluorouracil drugs, gemcitabine drugs, vinca alkaloid drugs, antibodies and the like to prepare a combined preparation with anti-tumor activity. The combined preparation can be in the form of tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, lozenges, granules, electuary preparations, pills, powders, pastes, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, liniments, ointments, plasters, creams, sprays, aerosols, drops, patches and the like.

[0049] Compared with the prior art, the compound having the tetravalent platinum structure of repaglinide described in the present invention has the following advantages:

[0050] (1) The present invention introduces the repaglinide molecule into the tetravalent platinum system, and synthesizes a series of novel repaglinide compounds modified with tetravalent platinum compounds, which are proved to have good anti-tumor and anti-cancer abilities through anti-tumor activity tests; such repaglinide tetravalent platinum compounds have good therapeutic effects on metastatic malignant tumors and have excellent anti-tumor metastasis activity;

[0051] (2) This type of repaglinide tetravalent platinum compound can induce severe DNA damage in tumor cells and induce mitochondrial-mediated apoptosis, thereby exerting an anti-tumor effect;

[0052] (3) This type of repaglinide tetravalent platinum compound increases the expression of p53 through the lumican / P53 / p21 signaling axis, thereby reversing tumor EMT and immunosuppression, and further exerting anti-proliferative and anti-metastatic effects;

[0053] (4) The compound structure of the general formula I described in the present invention is innovative, and it is expected to obtain a variety of lead molecules that are effective against tumors, provide new candidate drug molecules to solve the defects of traditional divalent platinum drugs, and open up new ways for the modification of tetravalent platinum compounds; in terms of pharmacological activity, the tetravalent platinum compound of repaglinide can inhibit tumor proliferation and metastasis by increasing the expression of P53, reversing EMT, activating immunity, and damaging DNA. This mechanism of action is significantly different from that of traditional platinum drugs, and can effectively overcome the defects of traditional platinum drugs such as severe drug resistance and strong toxic side effects, and effectively expand the application of platinum drugs in the treatment of malignant metastatic tumors.

[0054] Innovative drug research and development can drive the upgrading and development of the pharmaceutical industry, attract a large amount of capital investment, promote the development of related industries such as biotechnology, chemical pharmaceuticals, and pharmaceutical research and development services, create more employment opportunities, and contribute to the growth of the national economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 In vivo antitumor activity of compound 3, CDDP and CDDP+RPG against 4T1 tumors in female BALB / c mice (n=5). (a) Schematic diagram of experimental design. (b) Relative body weight of mice during treatment. (c) Curve of tumor volume change over time. (d) Tumor weight of mice in each group at the end of the experiment. The tumor growth inhibition rate TGI of the tested drug is indicated [TGI=(1-tumor weight of drug treatment group / tumor weight of blank group)×100%]. (e) Tumor image after mice were sacrificed.

[0056] (f) H&E staining images of tumors. *p<0.05, ***p<0.001.

[0057] Figure 2. H&E staining images of liver, spleen and kidney of mice in the blank group, CDDP group, CDDP+RPG group and compound 3 treatment group. Tissues were obtained from in vivo anti-tumor experiments.

[0058] Figure 3 . Drug accumulation in tumor tissues in vivo. Tumor tissues were from the CDDP, CDDP+RPG and compound 3 treatment groups in the in vivo anti-tumor experiment.

[0059] Figure 4 .In vitro Transwell assay was used to evaluate the migration inhibition properties of compound 3 (5 μM), CDDP (5 μM) and CDDP+RPG (5 μM / 5 μM) on 4T1 cells. Tumor cells were treated with compounds for 24 hours, and untreated cells were set as blank. (a) Representative images. (b) Relative mobility analysis. ***P<0.001.

[0060] Figure 5 .In vitro scratch healing assay was used to evaluate the migration inhibition properties of compound 3 (5 μM), CDDP (5 μM) and CDDP+RPG (5 μM / 5 μM) on 4T1 cells. Tumor cells were treated with platinum compounds for 24 hours, and untreated cells were set as blank. (a) Representative images. (b) Relative scratch healing rate analysis. ***P<0.001.

[0061] Figure 6 .Inhibitory effects of CDDP, CDDP+RPG, and platinum (IV) compound 3 on 4T1 tumor lung metastasis in vivo (n=5). ***P<0.001. (a) Schematic diagram of experimental design. (b) Representative photos of the front and back of lung tissues in each group at the end of the experiment. (c) Statistical data of lung nodules in each group. (d) H&E staining of lung metastatic nodules. Nodules are indicated by arrows.

[0062] Figure 7 .Stability of compound 3 in biological culture medium. A solution of compound 3 (0.5 mM) in RPMI1640 was prepared and incubated at 37°C for 24 hours. The chromatogram was detected on a ThermoUltimate 3000RS using an Agilent C18 column (250 mm×4.6 mm, 5 μm). A fixed eluent (75% methanol / 25% water) was used, flow rate = 1.0 mL / min, λ = 250 nm, and injection volume was 10 μL.

[0063] Figure 8 . HPLC spectrum of RPG (0.5 mM) in RPMI1640. The chromatogram was detected on a Thermo Ultimate 3000RS using an Agilent C18 column (250 mm × 4.6 mm, 5 μm). A fixed eluent (75%

[0064] methanol / 25% water), flow rate = 1.0 mL / min, λ = 250 nm, injection volume 10 μL.

[0065] Fig. 9 .Reduction potential of compound 3. A solution of compound 3 (0.5 mM) and AsA (1 mM, similar to that in TME) was prepared in RPMI1640 and incubated at 37°C for 24 hours. An Agilent C18 column was used

[0066] (250 mm × 4.6 mm, 5 μm) The chromatogram was detected on a Thermo Ultimate 3000RS. A fixed eluent (75% methanol / 25% water) was used, flow rate = 1.0 mL / min, λ = 250 nm, injection volume 10 μL. In the HPLC spectrum, it was observed that the peak of compound 3 decreased, and the peak of RPG appeared and gradually increased.

[0067] Fig.10 .Apoptosis of 4T1 cells treated with CDDP (5μM), CDDP+RPG (5μM / 5μM) and compound 3 (5μM) for 24 hours. Cell apoptosis was measured by flow cytometry using Annexin V-FITC / PI staining. (a) Flow cytometry analysis, and (b) quantitative analysis. (c) Western Blot of Bcl-2, Bax, caspase3 and c-caspase3 expression in 4T1 cells, and (d) data quantitative analysis. ***P<0.001.

[0068] Fig.11 Analysis of mitochondrial membrane potential in 4T1 cells treated with CDDP (5 μM), CDDP+RPG (5 μM / 5 μM) and compound 3 (5 μM) for 24 hours. Cell apoptosis was measured by flow cytometry using JC-1 staining.

[0069] Fig.12 In vitro drug uptake and distribution in 4T1 cells. After 4T1 cells were treated with CDDP (5 μM), CDDP+RPG (5 μM / 5 μM) and compound 3 (5 μM) for 24 hours, the drug uptake and distribution were detected by atomic absorption spectrometry.

[0070] Fig.13.Analysis of DNA binding ability of compound 3. A RPMI1640 solution containing compound 3 (0.5 mM), AsA (1 mM) and 5'-GMP (3 mM) was prepared and incubated at 37°C for 24 hours. The chromatographic conditions were as follows (λ=300 nm): phase A was water, phase B was methanol; 10% phase B (0-5 minutes); 10%-60% phase B (5-25 minutes); 60%-90% phase B (25-26 minutes); 90% phase B (26-34 minutes). The flow rate was 1.0 mL / min. (a) HPLC chromatograms recorded at 0 hours and 24 hours. (b) Reduction reaction in a reducing medium and the adduct formation process of CDDP-GMP.

[0071] Fig.14 .Western Blot analysis of γ-H2AX expression. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM) and CDDP+RPG mixture (5 μM / 5 μM) for 24 hours. (a) Blots. (b) Relative gray intensity analysis. Relative gray = (gray of the indicated protein) / (gray of GAPDH). ***P<0.001

[0072] Fig.15 .The p53 signaling regulation properties of RPG platinum (IV) compound 3 were detected by Western Blot and immunohistochemistry. 4T1 cells were incubated with compound 3 (5μM), CDDP (5μM) and CDDP+RPG mixture (5μM / 5μM) for 24 hours: (a) Blots; (b) relative gray intensity analysis. Analysis of p53 immunohistochemical staining in tumor tissues obtained from in vivo antitumor experiments: (c) representative photos; (d) quantitative data. **p<0.01, ***p<0.001

[0073] Fig.16.The properties of RPG platinum (IV) compound 3 in causing pro-death autophagy were detected by Western Blot and immunohistochemistry. 4T1 cells were incubated with compound 3 (5μM), CDDP (5μM) and CDDP+RPG mixture (5μM / 5μM) for 24 hours: (a) Western Blot; (b) Data analysis of the expression of LC3I, LC3II, p62 and Beclin1. Analysis of p62 immunohistochemical staining in tumor tissues obtained from in vivo anti-tumor experiments: (c) Representative photos; (d) Quantification data. (e) MTT method was used to detect the viability of 4T1 cells treated with compound 3 (5μM), CDDP (5μM) and 3+3MA (5μM / 70μM), CDDP+3MA (5μM / 70μM) for 24 hours. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM), mixture 3+3MA (5 μM / 70 μM), and CDDP+3MA (5 μM / 70 μM) for 24 hours: (a) Western Blot; (b) Data analysis of the expression of LC3I, LC3II, p62, and Beclin1. *p<0.05, **p<0.01, ***p<0.001, ns: no statistical difference

[0074] Fig.17 .MDC staining was used to evaluate the autophagy induced by CDDP (5μM), CDDP+RPG (5μM / 5μM), compound 3 (5μM), compound 3+3MA (5μM / 70μM), CCCP (20μM), and CCCP+3MA (20μM / 70μM) in 4T1 cells. (a) Image. (b) Relative fluorescence intensity analysis. ***P<0.001.

[0075] Fig.18 .The inhibitory effect of RPG platinum (IV) compound 3 on EMT of tumor cells was detected by Western Blot immunohistochemistry. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM) and CDDP+RPG mixture (5 μM / 5 μM) for 24 hours: (a) Western Blot; (b) Data analysis of the expression of E-cadherin, N-cadherin, Vimentin and Snail1. Analysis of immunohistochemical staining of E-cadherin and N-cadherin in tumor tissues obtained from in vivo antitumor experiments: (c) Representative photos; (d) Quantitative data. **p<0.01, ***p<0.001.

[0076] Fig.19.The properties of RPG platinum (IV) compound 3 in inhibiting tumor inflammation and angiogenesis were detected by Western Blot and immunohistochemistry. 4T1 cells were incubated with compound 3 (5μM), CDDP (5μM) and CDDP+RPG mixture (5μM / 5μM) for 24 hours: (a) Western Blot; (b) Data analysis of COX-2, MMP9 and VEGFA expression. Analysis of immunohistochemical staining of VEGFA and CD34 in tumor tissues obtained from in vivo antitumor experiments: (c) Representative photos; (d) Quantitative data. **p<0.01, ***p<0.001.

[0077] Fig. 20 Western Blot and immunohistochemistry were used to detect the anti-tumor immunity activating properties of RPG platinum (IV) compound 3. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM) and CDDP + RPG mixture (5 μM / 5 μM) for 24 hours: (a) Western Blot; (b) PD-L1 expression data analysis. PD-L1 and CD3 in tumor tissues obtained from in vivo anti-tumor experiments + and CD8 + T cell immunohistochemical staining analysis: (c) representative photos;

[0078] (d) Quantitative data. **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0079] The technical scheme of the present invention is further described in conjunction with specific embodiments, but the implementation is intended to explain the present invention and cannot be understood as limiting the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in the field or the product instructions shall be followed. Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as those generally understood by technicians in the field to which the present invention belongs; the test reagents used, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

[0080] In order to make the objectives, technical solutions and advantages of the present invention more clear, representative embodiments of the present invention are described in detail below, but are not limited thereto.

[0081] Example 1.

[0082] 1. Preparation of tetravalent platinum shown in compound II

[0083] 1. Synthesis of dihydroxycisplatin (IV) IIa

[0084]

[0085] Add 1.0 g of cisplatin to a 250 mL round-bottom flask, add 30 mL of distilled water, stir at room temperature, slowly drop 50 mL of 30% hydrogen peroxide into the reaction system, raise the temperature to 60 ° C and stir for 4 h; stop the reaction, place in a 4 ° C refrigerator for crystallization for 12 h, filter and separate to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 ° C to dissolve it, place at 4 ° C for crystallization for 12 h, and filter to obtain compound IIa as yellow crystals (0.74 g, 67%).

[0086] 2. Synthesis of dihydroxyoxaliplatin (IV) IIb

[0087]

[0088] Add 1.0 g of oxaliplatin to a 250 mL round-bottom flask, add 30 mL of distilled water, stir at room temperature, slowly drop 50 mL of 30% hydrogen peroxide into the reaction system, raise the temperature to 60 ° C and stir for 4 h; stop the reaction, place in a 4 ° C refrigerator for crystallization for 12 h, filter and separate to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 ° C to dissolve it, place at 4 ° C for crystallization for 12 h, and filter to obtain compound IIb as a white crystal (0.80 g, 72%).

[0089] 2. Preparation of Repaglinide Anhydride as Compound III

[0090]

[0091] EDC·HCl (364 mg, 1.89 mmol) was added to a solution of RPG (500 mg, 1.14 mmol) in anhydrous DMF (10 mL), and stirred at room temperature overnight. After the reaction was completed, the reaction system was concentrated, and citric acid aqueous solution and NaHCO3 aqueous solution were added for extraction. The organic phases were combined, and anhydrous Na2SO4 was added to dry the organic phase, and III was obtained as a white solid (315 mg, 32%) by spin drying.

[0092] III. Preparation of the Repaglinide Compound Shown as Compound I

[0093] 1. Preparation of symmetrical bisrepaglinide tetravalent platinum compound 1.

[0094]

[0095] To a solution of RPG (339 mg, 0.75 mmol) in anhydrous DMF (5 mL) was added TBTU (241 mg, 0.75 mmol) and triethylamine (104 μL, 0.75 mmol). The mixture was stirred for 15 min under nitrogen at room temperature. Subsequently, IIa (100 mg, 0.30 mmol) was added, and the mixture was stirred for 48 h in a dark environment at 55 ° C. After the reaction was completed, the mixture was concentrated and further purified by silica gel column chromatography to obtain white solid compound 1 (79.4 mg, 22%), with a purity of 99.8% as determined by HPLC (eluent: MeOH / H2O=80:20, T R =7.456min).

[0096] 1 H NMR (500MHz, DMSO-d6) δ8.41(d,J=8.6Hz,2H),7.50(d,J=7.8Hz,2H),7.32(s,2H),7. 18–7.13(m,2H),7.11–7.02(m,4H),6.90(s,2H),6.80(d,J=7.9Hz,2H),6.66(br,6H,N H3),5.42–5.35(m,2H),3.98(q,J=7.0Hz,4H),3.52–3.41(m,4H),3.08(s,4H),2.54( s,4H),1.73–1.59(s,4H),1.62–1.44(m,12H),1.42–1.25(m,8H),0.95–0.85(m,12H). 13 CNMR(126MHz,DMSO-d6)δ173.6,168.8,156.5,151.5,140.6,140.4,131.0,127.1,126.0,123.9,12 2.1,120.5,120.3,114.2,64.2,46.5,45.8,42.6,26.3,24.8,23.8,23.1,21.7,14.7.MS-ESI:calcd for[M+H] + :1204(M=C 54 H 76 Cl2N6O8Pt),found:1204.HRMS:calcd for[M+H] + :1202.4828(M=C 54 H 76 Cl2N6O8Pt), found:1202.4832.

[0097] 2. Preparation of symmetrical bisrepaglinide tetravalent platinum compound 2.

[0098]

[0099] To a solution of RPG (260 mg, 0.56 mmol) in anhydrous DMF (5 mL) was added TBTU (185 mg, 0.56 mmol) and triethylamine (80 μL, 0.56 mmol). The mixture was stirred for 15 min under nitrogen at room temperature. Subsequently, IIb (100 mg, 0.23 mmol) was added, and the mixture was stirred for 48 h in a dark environment at 55 ° C. After the reaction was completed, the mixture was concentrated and further purified by silica gel column chromatography to obtain a white solid compound 2 (75.2 mg, 25%), with a purity of 98.6% as determined by HPLC (eluent: MeOH / H2O=80:20, T R =8.097min).

[0100] 1 H NMR (500MHz, DMSO-d6) δ8.49(br,4H,NH2),8.40(d,J=8.5Hz,2H),7.41(d,J=7.8Hz,2H),7.30(d,J=7.4Hz,2H),7.16( t,J=7.3Hz,2H),7.08(d,J=7.7Hz,2H),7.03(t,J=7.3Hz,2H),6.90(s,2H),6.79(d,J=7.9Hz,2H),5.41–5.34(m,2H), 3.97(q,J=6.9Hz,4H),3.47–3.41(m,4H),3.15–2.96(m,4H),2.72–2.51(m,6H),2.18(d,J=11.0Hz,2H),1.74–1.66(m ,4H),1.62–1.41(m,15H),1.35–1.29(m,2H),1.25(t,J=7.0Hz,6H),1.24–1.10(t,J=7.3Hz,3H),1.02–0.80(m,12H). 13 C NMR(126MHz,MeOD)δ180.3,174.4,174.4,158.9,153.0,145.8,137.4,134.0,131.4,129.5,128.3,127.4,12 6.7,120.7,111.8,60.1,46.6,41.9,41.5,41.1,38.9,38.1,37.4,31.3,29.0,23.9,16.0,7.9.MS-ESI:calcd for[M] + :1300(M=C 62 H 84 N6O12 Pt),found:1300.HRMS:calcd for[M+H] + :1300.5873(M=C 62 H 84 N6O 12 Pt),found:1300.5881.

[0101] 3. Preparation of asymmetric monorepaglinide tetravalent platinum compound 3.

[0102]

[0103] RPG anhydride III (319 mg, 0.36 mmol) was dissolved in anhydrous DMSO (5 mL). IIa (100 mg, 0.30 mmol) was added, and the mixture was stirred at 75 ° C for 24 h under nitrogen. After the reaction was completed, the mixture was concentrated and further purified by silica gel column chromatography to obtain a white solid compound 3 (46.3 mg, 20%), the purity of which was 96.9% as determined by HPLC (eluent: MeOH / H2O=80:20, T R =5.803min).

[0104] 1 H NMR (500MHz, DMSO-d6) δ8.39(d,J=8.6Hz,1H),7.44(d,J=7.8Hz,1H),7.30(d,J=6.4Hz,1H),7.18–7 .13(m,1H),7.08(d,J=6.9Hz,1H),7.03(t,J=7.4Hz,1H),6.86(s,1H),6.77(d,J=7.9Hz,1H),6.23– 5.90(br,6H,NH3),5.37(d,J=9.0Hz,1H),3.95(q,J=7.0Hz,2H),3.47–3.40(m,2H),3.11–3.03(m,2 H),2.57–2.51(m,2H),1.70–1.63(m,2H),1.58–1.46(m,6H),1.33–1.27(m,4H),0.93–0.85(m,6H). 13C NMR (126MHz, DMSO-d6) δ174.0,168.9,156.3,151.4,140.4,139.7,130.7,127.1,126.0,124.4,123.9,120.4,120 .2,114.3,64.2,46.5,45.8,42.6,39.8,39.7,39.5,39.3,39.2,26.3,24.8,23.8,23.2,21.7,14.8.MS-ESI:calcd for[M] + :769(M=C 27 H 42 Cl2N4O5Pt),found:769.HRMS:calcd for[M+H] + :768.2258(M=C 27 H 42 Cl2N4O5Pt), found:768.2263.

[0105] 4. Preparation of asymmetric monorepaglinide tetravalent platinum compound 4.

[0106]

[0107] RPG anhydride III (245 mg, 0.27 mmol) was dissolved in anhydrous DMSO (5 mL). IIb (100 mg, 0.23 mmol) was added, and the mixture was stirred at 75 °C for 24 h under nitrogen. After the reaction was completed, the mixture was concentrated and further purified by silica gel column chromatography to obtain white solid compound 4 (37.2 mg, 19%), with a purity of 99.7% as determined by HPLC (eluent: MeOH / H2O=80:20, T R =6.356min).

[0108] 1H NMR(500MHz,DMSO-d6)δ8.64(s,1H,NH2),8.39(d,J=8.6Hz,1H),8.27–7.70(m,3H,NH2),7.33(d,J=7.7Hz,1H),7.29(d, J=7.6Hz,1H),7.17–7.13(m,1H),7.08(d,J=8.9Hz,1H),7.03(t,J=7.9Hz,1H),6.86(s,1H),6.76(d,J=8.8Hz,1H),5.41 –5.35(m,1H),3.91(d,J=7.0Hz,2H),3.42(s,2H),3.07(s,2H),2.54(s,5H),2.10(dd,J=36.2,10.2Hz,2H),1.67(s,2H) ,1.53(d,J=25.5Hz,8H),1.33(d,J=24.0Hz,2H),1.23(t,J=7.0Hz,3H),1.17–1.07(m,2H),0.89(dd,J=6.5,3.7Hz,6H). 13 C NMR(126MHz,DMSO-d6)δ175.4,168.9,163.8,156.0,151.5,140.4,140.1,129.6,127.1,126.0,124.0,123.9,1 20.5,120.2,113.5,63.6,61.6,60.1,46.5,45.8,42.6,30.9,26.3,24.8,23.8,23.2,21.8,14.4.MS-ESI:calcd for[M] + :866(M=C 35 H 50 N4O9Pt),found:866.HRMS:calcd for[M+H] + :866.3304(M=C 35 H 50 N4O9Pt), found:866.3298.

[0109] 4. Experimental Test

[0110] In order to better understand the essence of the present invention, the pharmacological test results of the inhibitory effect of the compounds on tumors in vivo and in vitro experiments are used below to illustrate the potential use of these compounds in the pharmaceutical field. The pharmacological experiments provide partial activity data of some compounds. It must be noted that the pharmacological experiments of the present invention are used to illustrate the present invention rather than to limit the present invention. Simple improvements made to the present invention according to the essence of the present invention all belong to the protection scope of the present invention.

[0111] 1. In vitro antitumor activity assay

[0112] Experimental method: This experiment uses the MTT method to determine the cell survival rate. The half-inhibitory concentration (IC 50 ) value to measure the in vitro anticancer activity of the compound. 100 μL of tumor cells in the logarithmic growth phase were inoculated into a 96-well plate with a cell density of 3000-5000 / well, and the last column was reserved as a zero well. Place in a 37°C cell culture incubator for 12 hours, then add 100 μL of compound culture medium solution with gradient concentrations to the 96-well plate, and continue to culture in a 37°C cell culture incubator for 48 hours. Add 20 μL of 5 mg / mL MTT solution to each well of the 96-well plate, culture in a 37°C cell culture incubator for 4 hours, take out, remove the culture medium, add 150 μL of DMSO, and shake in a 37°C shaker in the dark for 20 minutes. Use an enzyme-linked immunosorbent assay reader to measure the absorbance OD value of each well at 490 nm to calculate its IC 50 Each set of experiments was repeated at least three times.

[0113] The cytotoxicity of RPG platinum (IV) compounds 1-4 against four tumor cell lines and one normal human cell line, including mouse breast cancer (4T1), human lung cancer (A549), human liver cancer (HepG2), CDDP-resistant human lung cancer (A549R) and normal human liver cell line (LO2), was tested by MTT assay using platinum (II) drugs CDDP and OXP, ligand RPG and CDDP+RPG mixture as reference drugs. Table 1 lists the maximum half inhibitory concentrations (IC50) based on three parallel experiments. 50 )result.

[0114] RPG platinum (IV) compounds (compounds 1, 2, 3 and 4 synthesized in the examples) showed moderate to strong antiproliferative activity against all tumor cell lines tested. The platinum core has a great influence on the anti-tumor ability. The IC values ​​of compounds 1 and 3 with CDDP as the core are 50 The values ​​were relatively lower than those of compounds 2 and 4 with OXP as the parent core. In addition, the ligand RPG also had a significant effect on the antiproliferative efficacy, and the activity of the single RPG platinum (IV) compound was significantly better than that of the dual RPG platinum (IV) compound. In particular, the single RPG platinum (IV) compound 3 with CDDP as the parent core had the most effective antitumor effect against all tumor cell lines, with an IC 50 The value was lower than 3.57 μM, which was even stronger than the platinum (II) drug CDDP. It is worth noting that the antitumor activity of the ligand RPG is very weak, and the mixture of CDDP and RPG (CDDP+RPG) shows a similar effect to CDDP, which indicates that adding RPG to the platinum system to construct RPG platinum (IV) compounds is the key to improving its antitumor efficacy, and it exhibits an antitumor mechanism different from the mixture CDDP+RPG.

[0115] Subsequently, the resistance factor (RF) (i.e., IC 50 The potential of these compounds to overcome CDDP resistance was evaluated by the ratio of the IC50 value to the IC50 value of A549. All prepared RPG platinum (IV) compounds were able to effectively reduce the RF values ​​to lower levels (1.06-1.93) compared to CDDP and CDDP+RPG (RF=5.06, 3.08), indicating that the RPG platinum (IV) skeleton is potential in overcoming platinum drug resistance. Then, the tumor selectivity index (SI) was calculated to mainly determine the toxicity of the test compounds in vitro. The results showed that the SI=3.41 of the potent compound 3 was significantly improved compared to the platinum (II) drugs CDDP and OXP and the CDDP+RPG mixture (SI=0.50, 1.19, 0.81), which demonstrated its efficacy in reducing toxicity.

[0116] Therefore, RPG platinum (IV) compounds, especially CDDP-derived compound 3 with a single RPG ligand, showed promising antiproliferative activity in vitro and showed great potential in overcoming CDDP-induced drug resistance and reducing toxicity. Considering these results, compound 3 was subsequently evaluated for its antiproliferative and anti-metastatic activities in vivo.

[0117] Discussion of Antitumor Activity:

[0118] Table 1 Antitumor activity of RPG platinum (IV) compounds 1-4 with CDDP, OXP, RPG and CDDP+RPG as references. After cells were treated with drugs for 48 hours, the half maximum inhibitory concentration (IC 50 , μM).

[0119]

[0120] a RF: resistance factor, RF = IC 50 (A549R) / IC 50 (A549); b SI: Selectivity index, SI = IC 50 (LO2) / IC 50 (HepG2); c ND: not tested or not calculated; d CDDP+RPG: A mixture of CDDP and RPG with a molar ratio of 1:1.

[0121] 2. In vivo antitumor activity experiment

[0122] Experimental methods: The evaluation of antitumor activity was performed using a 4T1 tumor female BALB / c mouse model. 4T1 cells were injected into the left axilla of the mice. The mice had palpable tumor nodules on the third day after inoculation and were randomly divided into four groups (blank group, CDDP group, CDDP+RPG group, and compound 3 group), with 5 mice in each group. The drug was dissolved in saline containing 5% DMSO and administered by tail vein injection at a dose of 2 mg Pt / kg on days 3, 6, and 9 ( Figure 1 a), while the blank group received an equal amount of matrix solution. The tumor volume (V = W2 × L / 2, where W represents the width of the tumor and L represents the length of the tumor) and body weight changes of the mice were monitored during the experiment. On day 11, the mice were sacrificed and blood samples were collected for ELISA detection. Organ samples including tumor, heart, liver, spleen, lung and kidney were collected for immunohistochemical analysis and H&E staining. TGI was determined based on tumor weight using the following formula: TGI (%) = (1-tumor weight of drug-treated group / tumor weight of blank group) × 100%.

[0123] Experimental results: Figure 1 The results shown in ce indicate that RPG platinum (IV) compound 3 can effectively inhibit tumor growth in vivo. Compared with the platinum (II) drug CDDP (486 mm 3 , TGI = 43.5%) and CDDP + RPG compound (425 mm 3 , TGI = 40.2%), its effect of inhibiting tumor growth is more significant. This trend is basically consistent with the in vitro anti-tumor results. In addition, Figure 1 The hematoxylin and eosin (H&E) staining images of the tumor in f further demonstrate its strong antitumor effect. Severe degeneration, necrosis and nuclear dispersion were observed in the compound 3-treated group, which was completely different from the blank group. The absorption of drugs in tumor tissue is a key factor affecting the antitumor effect. The present invention uses the AAS method to determine the accumulation of drugs in tumor tissue ( Figure 3 The results showed that compound 3 had a higher absorption level in tumors than CDDP and CDDP+RPG (P<0.001), thus further improving the anti-tumor efficacy.

[0124] Changes in body weight during the experiment can reflect the toxicity of the drug in the body. Figure 1 The results in b show that compared with the blank group, compound 3 did not significantly reduce body weight after three administrations (P = ns), while the reference drug CDDP significantly reduced the body weight of mice during the experiment (P < 0.01). In addition, compared with the blank group, there were no significant histological differences in the liver, spleen, and kidney tissues treated with compound 3 ( Figure 2). Therefore, RPG platinum (IV) compound 3 has effective antitumor properties and no obvious toxicity in vivo, and is more effective and less toxic than CDDP.

[0125] 3. In vitro metastasis inhibition assay

[0126] Tumor cell metastasis is a major obstacle to cancer treatment. In order to test the potential of RPG platinum (IV) compounds as anti-tumor metastasis drugs, the anti-metastatic activity of compound 3 was evaluated by in vitro Transwell and scratch healing assays.

[0127] Transwell assay: Serum-deprived 4T1 cells (5×10 4 / well) were resuspended in 200 μL RPMI 1640 medium and inoculated into the upper chamber of a 24-well Transwell plate (Costar 3422). In the lower chamber, a mixed solution of medium containing 10% FBS (600 μL) and platinum compound 3 (5 μM), CDDP (5 μM) or CDDP+RPG (5 μM / 5 μM) was added. After 24 hours of cell culture, the cells were fixed with 4% paraformaldehyde for 20 minutes and then stained with 0.1% crystal violet for 20 minutes. The cells migrating on the bottom side of the membrane were observed with an inverted microscope, and images of five fields of view were randomly collected for analysis in each well.

[0128] Wound-Healing Assay: Serum-deprived 4T1 cells were resuspended in RPMI 1640 medium containing 1% FBS and plated at 4 × 10 5 The cells were seeded at a density of 100 μM into 6-well culture dishes. After pre-incubation overnight, when the cell confluence reached about 90%, scratches were made. Subsequently, the cells were treated with platinum compound 3 (5 μM), CDDP (5 μM), or CDDP+RPG (5 μM / 5 μM) for 24 hours. The healing degree of the cells was detected.

[0129] Figure 4 The results of the Transwell assay shown in the figure indicate that RPG platinum (IV) compound 3 can effectively inhibit the migration of tumor cells, which is 43% of the blank group (P<0.001), higher than CDDP (85%, P<0.001) and CDDP+RPG mixture (69%, P<0.001). Figure 5 In the scratch healing test shown, the wound closure rate induced by the compound 3 treatment group was lower than that of the blank group, CDDP group and CDDP+RPG group, further verifying its anti-metastatic effect. Therefore, RPG platinum (IV) compound 3 can effectively inhibit the metastasis of tumor cells in vitro.

[0130] 4. In vivo metastasis inhibition assay

[0131] Experimental method: The lung is the main site of tumor cell metastasis in vivo, so this study used the lung metastasis model to study the anti-tumor metastasis ability of drugs in vivo. Female BALB / c mice were injected via the tail vein with 1×10 5 4T1 cells were used to establish an in vivo 4T1 lung metastasis model. On day 4, the mice were randomly divided into four groups (blank group, CDDP group, CDDP+RPG group and compound 3 group), with 5 mice in each group. On days 4, 7 and 10, 2 mg Pt / kg was intravenously injected ( Figure 6 a). On day 12, mice were sacrificed and lung tissues were collected for analysis. Nodules on the lung surface were counted, and nodules in the lung tissue were observed using H&E staining.

[0132] Experimental results: Statistical analysis of pulmonary nodules ( Figure 6 bc) showed that the RPG platinum (IV) compound 3 also had stronger anti-metastatic activity in vivo than CDDP and the mixture CDDP + RPG, reducing lung nodules to 25% of the blank group (P < 0.001), which was also significantly lower than CDDP (70%, P < 0.001) and CDDP + RPG (62%, P < 0.001). In addition, fewer and smaller tumor nodules were observed in the H&E staining images of the lung tissues of the compound 3-treated group compared with the blank group, CDDP group, and CDDP + RPG group. This trend is mainly consistent with the anti-metastatic activity in vitro ( Figure 6 d). Therefore, candidate RPG platinum (IV) compound 3 can effectively inhibit the metastasis of tumor cells both in vitro and in vivo, showing great potential for further development as an anti-metastatic agent.

[0133] 5. Stability in biological media and reducibility in the tumor microenvironment

[0134] Platinum (IV) complexes as prodrugs of platinum (II) drugs are expected to be stable in biological media and to be easily reduced in the reducing TME. Therefore, we evaluated the stability of RPG platinum (IV) compound 3 in biological culture medium RPMI1640 by HPLC, as well as its reduction potential in the reducing medium RPMI1640 containing ascorbic acid (AsA, 1 mM, similar to that in the TME).

[0135] Figure 7 The spectrum in Figure 2 shows that compound 3 remains stable in RPMI1640 for at least 24 hours, indicating that RPG platinum (IV) compound 3 is stable in biological culture medium. In reducing medium (RPMI1640, containing 1mM AsA), platinum (IV) is easily reduced to platinum (II) and RPG fragments, which is manifested by the reduction of the peak of compound 3 and the appearance and gradual increase of the RPG peak ( Figure 8,9). Therefore, the candidate compound RPG platinum (IV) compound 3 remains stable during transport in biological media and is easily reduced in the reducing media of TME, which is beneficial to improve antitumor activity and reduce toxicity.

[0136] 6. Induce mitochondria-mediated apoptosis.

[0137] Apoptosis is an important form of programmed cell death and plays a vital role in maintaining cell balance and clearing damaged cells. Mitochondria are key organelles that regulate tumor cell apoptosis through the Bcl-2 / Bax / caspase-3 signaling pathway. Platinum drugs can effectively exert anti-proliferative effects by inducing cell apoptosis. Therefore, we used the classic Annexin V-FITC / PI staining method to evaluate the apoptosis-inducing properties of compound 3.

[0138] Fig.10 The results in a and b showed that RPG platinum (IV) compound 3 could significantly induce apoptosis in 4T1 cells. Then, to further evaluate the effect of compound 3 on mitochondria, the changes in mitochondrial membrane potential (ΔΨm) were evaluated by JC-1 staining. Fig.11 The results showed that compared with the blank group, the mitochondrial membrane potential ΔΨm induced by compound 3 was severely collapsed (34.9%), even higher than CDDP (25.5%) and CDDP+RPG (20.5%). Mitochondrial membrane potential collapse is a sign of apoptosis, indicating that RPG platinum (IV) compound 3 has a significant effect on the integrity and function of mitochondria.

[0139] To further determine the involvement of the Bcl-2 / Bax / caspase-3 pathway in the process of cell apoptosis, the expression of Bcl-2 cascade-related proteins Bcl-2, Bax, caspase-3, and c-caspase-3 ( Fig.10 c, d). The results showed that compared with the blank group, compound 3 significantly downregulated the expression of anti-apoptotic protein Bcl-2 (P<0.001) and upregulated the expression of pro-apoptotic protein Bax (P<0.001). In addition, an increase in the ratio of c-caspase-3 / caspase-3 (P<0.001) was observed in the compound 3 treatment group, and c-caspase-3 / caspase-3 is a key execution protein of apoptosis. Therefore, compound 3 effectively induces tumor cell apoptosis through the mitochondria-mediated Bcl-2 / Bax / caspase-3 pathway.

[0140] 7. Inducing DNA damage in tumor cells

[0141] DNA damage is the main mechanism by which platinum drugs induce apoptosis of tumor cells. In addition, the accumulation of platinum drugs in the cell nucleus is a key factor affecting their ability to damage DNA. We used high-performance liquid chromatography and Western Blot to detect whether RPG platinum (IV) compound 3 causes DNA damage after reduction, and used AAS detection to evaluate the accumulation of drugs in DNA by measuring the platinum content.

[0142] Fig.12 The results showed that the accumulation level of RPG platinum (IV) compound 3 in tumor cells was significantly higher than that in the CDDP group and the CDDP+RPG mixed group (P < 0.01), which further led to a higher DNA accumulation level (P < 0.001). Subsequently, guanosine-5'-monophosphate (5'-GMP) was used as a DNA base model to determine the DNA binding ability of compound 3, and the HPLC profile of the RPMI1640 solution containing AsA (1 mM) and 5'-GMP (3 mM) was monitored ( Fig.13 After 24 hours of culture, a peak of platinum and GMP (platinate GMP) appeared, indicating that 5'-GMP can effectively bind to the platinum (II) group released from compound 3, which verifies the DNA damage ability of RPG platinum (IV) compounds.

[0143] Phosphorylation of H2AX (γ-H2AX) is one of the hallmarks of DNA damage. In this paper, CDDP and CDDP+RPG mixture were used as positive reference drugs, and the expression of γ-H2AX in 4T1 tumor cells treated with compound 3 was determined by Western Blot to further confirm the DNA damage caused by RPG platinum (IV) ( Fig.14 As expected, compound 3-treated group induced a significant upregulation of γ-H2AX compared with the blank group (P<0.001), which was similar to the results of CDDP and CDDP+RPG.

[0144] In summary, RPG platinum (IV) compound 3 can accumulate at high levels in the nuclei of tumor cells, and the platinum (II) groups released after reduction in the reductive TME can effectively induce DNA damage and cause high expression of γ-H2AX.

[0145] 8. Promote P53 signaling through the Lumican / P53 / p21 pathway

[0146] P53 is a key protein involved in a variety of anti-tumor processes. It has been reported that RPG increases the expression of p53 by inhibiting the expression of lumican, further affecting the expression of its downstream target protein p21. At the same time, platinum drugs can also effectively stimulate p53 signal transduction. Therefore, we detected the effect of RPG platinum (IV) compound 3 on the lumican / p53 / p21 pathway by Western Blot and immunohistochemistry.

[0147] Fig.15 Ab showed that compared with the blank group (P<0.001) and the CDDP group (P<0.001), compound 3 significantly inhibited the expression of lumican protein in tumor cells. Subsequently, after treatment with compound 3, the expression of key protein p53 in tumor cells was significantly increased (P<0.001), and the downstream protein p21 was also significantly upregulated (P<0.001). In addition, Fig.15 CD showed that in tumor tissues in vivo, high expression of p53 was also observed in tumor tissues treated with compound 3 compared with the blank group and CDDP-treated group (P<0.01), which further verified that RPG platinum (IV) compound 3 has advantages in activating the p53 cascade reaction. It is worth noting that compared with CDDP alone, the effect of CDDP+RPG combination on lumican, p53 and p21 protein expression was relatively more obvious, which was mainly attributed to RPG. These findings indicate that compound 3 has advantages in regulating the lumican / p53 / p21 pathway, which is mainly due to the RPG ligand, which can further enhance the anti-tumor effect in vitro and in vivo.

[0148] 9. Activation of pro-apoptotic autophagy

[0149] Autophagy is known as the second type of programmed cell death and is another mechanism of cell death. Increasing autophagic flux by drugs and disrupting the balance of tumor cells can lead to tumor cell apoptosis. In addition, RPG has been reported to effectively enhance autophagic flux. Next, we studied the effect of drugs on autophagic flux by Western Blot and MDC staining.

[0150] It has been shown that protein p53 has a double-edged sword function in regulating autophagy. In this study, we determined the autophagic flux-related proteins LC3II / I and P62 by Western Blot and immunohistochemistry to determine whether upregulation of p53 caused by RPG platinum (IV) compound 3 would promote autophagy in tumor cells. Fig.16 The results in a and b showed that compound 3 effectively promoted autophagy, and the ratio of LC3II / I in tumor cells increased significantly (P<0.01). At the same time, compared with the blank group, the expression of autophagy substrate p62 in tumor cells decreased (P<0.01). In addition, the immunohistochemical results of tumor tissue ( Fig.16 c, d) further demonstrated this trend, compound 3 significantly reduced the expression of p62 in tumors in vivo (P<0.001). MDC staining is widely used for autophagosome staining in tumor cells. Fig.17 It was observed that after treatment with compound 3, the accumulation of autophagosomes in tumor cells was significantly increased compared with the blank group (P<0.001), which was similar to the situation of the positive reference drug CCCP. It is worth noting that the level of autophagy was also increased in the CDDP+RPG mixed group compared with the CDDP group, which was mainly due to the effect of RPG. Subsequently, the protein Beclin1 was regulated to a higher level (P<0.01). Considering the reduction in Bcl-2 expression caused by compound 3, it is reasonable to conclude that the upregulation of p53 by RPG platinum (IV) compounds effectively promotes autophagy by reducing the Beclin1-Bcl-2 interaction, which is mainly attributed to the introduction of RPG ligands in the platinum (IV) system.

[0151] Autophagy is regarded as a double-edged sword in tumor cells. Therefore, it is a key issue to evaluate whether autophagy induced by RPG platinum (IV) compound 3 can enhance apoptosis of tumor cells. 3-Methyladenine (3MA), as an effective autophagy inhibitor, is often used to study autophagy-regulating drugs. In this paper, 3MA was used to detect the effect of autophagy on the viability of 4T1 cells. Fig.16 Western blot results in f and g showed that compared with the compound 3 treatment group, the LC3II / I (P<0.01) and Beclin1 in the tumor cells of the 3+3MA treatment group decreased (P<0.05), and p62 increased (P<0.05), and the trend was similar to that of the CCCP group and CCCP+3MA group. Then, MDC staining was performed to detect ( Fig.17 ) further verified this trend, that is, the number of autophagosomes in the 3+3MA treatment group decreased compared with the compound 3 treatment group. Subsequently, the cell viability of the 3+3MA treatment group was significantly improved compared with the compound 3 group (P<0.01)( Fig.16 e). It can be seen that RPG platinum (IV) compounds can effectively induce autophagy and have a positive effect on promoting tumor cell apoptosis.

[0152] 10. Inhibit EMT to inhibit tumor metastasis

[0153] Increasing evidence suggests that the EMT process plays a key role in promoting tumor metastasis. Dysregulated p53 function and protective autophagy are key drivers of EMT in tumor tissues. Regarding the efficacy of RPG platinum (IV) compound 3 in stimulating p53 function and promoting autophagy, the researchers evaluated its effect on the EMT process by monitoring the expression of key enzymes associated with the EMT process, E-cadherin, N-cadherin, Vimentin, and Snail1.

[0154] Fig.18 Western Blot results in a and b showed that RPG platinum (IV) compound 3 significantly inhibited the expression of N-cadherin in 4T1 cells compared with the blank group (P<0.001), while the secretion of E-cadherin increased (P<0.001). Subsequently, Vimentin and Snail1 proteins were inhibited to a lower level (P<0.001). These facts proved the effectiveness of RPG platinum (IV) compound 3 in reversing the tumor EMT process, which was also verified by immunohistochemical staining of tumor tissues in vivo. Fig.18 c, d Immunohistochemistry results showed that E-cadherin was upregulated (P<0.01) and N-cadherin was downregulated (P<0.001) in tumors treated with compound 3. It is worth noting that platinum (II) drugs have minimal effects on E-adherin and N-adherin both in vitro and in vivo, indicating that they have a weak effect on the EMT process. Therefore, we believe that RPG platinum (IV) compound 3 has good EMT inhibitory properties, which is mainly attributed to the RPG ligand.

[0155] 11. Inhibit angiogenesis and prevent tumor metastasis

[0156] Angiogenesis is a key event in promoting tumor metastasis, as it not only provides nutrients for tumor proliferation, but also serves as a channel for tumor cell metastasis. EMT and protective autophagy of tumor cells have great potential in maintaining chronic inflammatory TME. COX-2 and MMP9 proteins are key enzymes involved in inflammatory TME, and they play a synergistic role in promoting angiogenesis by upregulating VEGFA. In this paper, the expression of COX-2, MMP9 and VEGFA was evaluated by Western Blot and immunohistochemistry, and the expression of vascularization marker CD34 in tumors was further detected by immunohistochemistry.

[0157] As shown in Western Blot ( Fig.19a, b) Compared with the blank group, RPG platinum (IV) compound 3 can effectively inhibit the expression of COX-2 and MMP9 in tumor cells (P<0.001), indicating that it has the effect of alleviating chronic inflammatory TME. Subsequently, compound 3 significantly downregulated the expression of VEGA (P<0.001), which was also confirmed by immunohistochemical staining of tumor tissue in vivo (P<0.001, Fig.19 c,d). Compared with the blank group, the expression of CD34 in tumor tissues + The labeled microvessels were reduced to a lower level (P<0.001). It can be seen that RPG platinum (IV) compound 3 inhibits tumor angiogenesis by inhibiting COX-2, MMP9 and VEGFA, thereby further inhibiting the metastasis of tumor cells.

[0158] 12. Activate immunity and inhibit tumor metastasis

[0159] Immunosuppression is a major feature of malignant tumors, which can greatly promote the proliferation and metastasis of tumor cells, and ultimately lead to chemotherapy failure in cancer patients. The immune checkpoint PD-L1 / PD-1 plays a key role in promoting immunosuppression by causing T cells in tumors to inactivate. Studies have found that p53 can regulate immunity by regulating the expression of PD-L1; reversing EMT can also activate anti-tumor immunity. Given the efficacy of RPG platinum (IV) compound 3 in upregulating p53 and inhibiting EMT, we investigated the effects of CD3 + and CD8 + T cells were immunohistochemically stained to determine its effect on activating anti-tumor immunity in vivo. In addition, its effect on PD-L1 expression was detected by Western Blot and immunohistochemistry.

[0160] Fig. 20 The results of ab showed that compound 3 could effectively inhibit the expression of PD-L1 in 4T1 cells in vitro (P<0.001) and in tumor tissues in vivo (P<0.001). Subsequently, the anti-tumor immunity of the compound 3-treated group was effectively improved ( Fig. 20 CD3 + and CD8 + The T cells increased to 3.0 times and 3.7 times of the blank group, respectively (P<0.01), which was significantly better than the CDDP group and CDDP+RPG group. This shows that RPG platinum (IV) compound 3 can effectively stimulate T cell anti-tumor immunity in tumor tissues by blocking the immune checkpoint PD-L1, and further enhance the anti-proliferation and anti-metastasis activity in vivo.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound having a tetravalent platinum structure of repaglinide as shown in the general formula (I): in, Selected from cisplatin or oxaliplatin; L is hydroxyl or 2. The compound according to claim 1, characterized in that: The compound is selected from:

3. A method for preparing the compound according to any one of claims 1 to 2, characterized in that: The synthetic route of the compound is as follows: in: When the molar ratio of compound II to compound III is 1:1.0-1.5, compound II and compound III undergo a coupling reaction to obtain an asymmetric monosubstituted repaglinide-modified tetravalent platinum compound Ia, that is, L is a hydroxyl group; in: When the molar ratio of compound II to repaglinide (RPG) is 1:2.0-5.0, compound II and compound RPG undergo coupling reaction to obtain a symmetrical disubstituted repaglinide modified tetravalent platinum compound Ib, that is, L is 4. The method for preparing the compound according to claim 3, characterized in that: The specific preparation steps of the synthetic route are: Under an inert gas atmosphere, compound III is dissolved in an anhydrous organic solvent for reaction, compound II is added, the reaction is carried out in the dark, and the target compound is isolated after post-treatment; under an inert gas atmosphere, compound RPG, a condensing agent, and an organic base are dissolved in an anhydrous organic solvent for reaction, compound II is added, the reaction is carried out in the dark, and the target compound is isolated after post-treatment; Wherein, when the molar ratio of compound II to compound III is 1:1.0-1.5; the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II, an asymmetric monosubstituted repaglinide modified tetravalent platinum compound Ia is obtained; When the feeding molar ratio of compound II, compound RPG, condensing agent and organic base is 1:2.0-5.0:2.0-5.0:2.0-5.0; the feeding relationship of compound II and organic solvent is that 10-100 ml of organic solvent is added for every 1 g of compound II, a symmetrical disubstituted repaglinide modified tetravalent platinum compound Ib is obtained.

5. The method for preparing the compound according to claim 4, characterized in that: The inert gas is nitrogen, helium or argon; the condensing agent is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI); the organic base is triethylamine (TEA), N,N-diisopropylethylamine or 4-dimethylaminopyridine, and the organic solvent is N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

6. A pharmaceutical composition, characterized in that: It comprises the compound represented by the general formula (I) as described in claim 1 or 2, and its pharmaceutically acceptable excipients.

7. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: Specifically, it is used in the preparation of anti-tumor proliferation and anti-tumor metastasis drugs.

9. The use according to claim 8, characterized in that: The anti-tumor proliferation is anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer or anti-mouse breast cancer proliferation; the anti-tumor metastasis is anti-mouse breast cancer cell metastasis.

10. A combined preparation comprising the compound of general formula (I) according to claim 1 or 2 or the pharmaceutical composition according to claim 6, Combination with platinum, paclitaxel, fluorouracil, gemcitabine, vinca alkaloids or antibody anti-tumor drugs.

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