Derivatives having a repaglinide tetravalent platinum structure, and methods of making and using the same

Abstract

The present application provides a derivative with a repaglinide tetravalent platinum structure and a preparation method and use thereof, the compound with the repaglinide tetravalent platinum structure is shown as general formula (I), wherein, selected from cisplatin or oxaliplatin; L is hydroxyl or a series of repaglinide platinum (IV) compounds targeting p53 pathway described in the present application, which shows good anti-proliferation and anti-metastasis activity in vitro and in vivo. The severe DNA damage caused by platinum core can up-regulate the expression of p53, and repaglinide (RPG) can also effectively promote the secretion of p53 through the lumican / p53 / p21 pathway. The present application designs and synthesizes a series of repaglinide tetravalent platinum derivatives, which can start mitochondrial-mediated apoptosis through the Bcl-2 / Bax / caspase-3 pathway. Subsequently, with the up-regulation of p53, the compound can effectively activate pro-apoptotic autophagy, inhibit the epithelial-mesenchymal transition (EMT) process, and then inhibit tumor angiogenesis by inhibiting COX-2, MMP9 and VEGFA. The compound can also increase the number of CD3 + And CD8 + T cells in tumors by blocking immune checkpoint PD-L1, and stimulate anti-tumor immunity. The above multiple anti-tumor mechanisms synergistically inhibit the proliferation and metastasis of tumor cells.

Description

Technical Field

[0001] The present 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 application thereof in anti-tumor drugs. Background Art

[0002] Cancer remains a major obstacle to human health, with metastatic tumors, in particular, responsible for over 90% of cancer deaths. Platinum-based drugs, represented by cisplatin (CDDP), oxaliplatin (OXP), and carboplatin (CBP), are widely used in clinical cancer treatment. They possess potent and broad-spectrum antitumor activity and are the cornerstone of cancer chemotherapy. Despite their tremendous success, poor stability, severe dose-limiting toxicity, and drug resistance limit their clinical application. Notably, classic platinum (II) drugs exhibit limited efficacy against metastatic tumors. Therefore, the development of new platinum candidate compounds with the potential to overcome the inherent limitations of platinum (II) drugs is a pressing priority for pharmaceutical researchers. Platinum (IV) compounds with bioactive axial ligands offer an effective approach for developing novel anti-tumor proliferation and anti-metastatic drugs and are expected to become a new generation of platinum-based drugs.

[0003] The tumor suppressor p53 is a crucial barrier preventing the development and progression of cancer and is considered a powerful "guardian" of the human genome. Accumulating evidence indicates that p53 gene inactivation is the most common single gene mutation in human cancers. This inactivation not only results in loss of p53's anti-cancer function but also promotes cancer cell motility, invasion, and metastasis through multiple pathways. Therefore, targeting p53 to develop novel anti-cancer drugs is an attractive strategy for chemotherapy drug development.

[0004] Protective autophagy is a key cellular process involved in maintaining tumor cell homeostasis and shaping the tumor microenvironment (TME). It is crucial for tumor proliferation, survival, and differentiation, and also plays a significant role in promoting tumor cell resistance to platinum-based drugs. Studies have demonstrated that p53 and autophagy interact in tumors, jointly contributing to tumor development and progression. Protective autophagy is a key factor in inactivating the anti-tumor function of p53 and has great potential in promoting tumor progression. On the other hand, p53 plays a double-edged sword role in regulating autophagy. Notably, increasing nuclear p53 expression with chemotherapeutic drugs can effectively inhibit the function of Bcl-2 family anti-apoptotic proteins. This, in turn, upregulates Beclin1 activity by blocking the interaction between Bcl-2 and Beclin1, promoting autophagic flux, disrupting cellular homeostasis, and ultimately leading to autophagic cell death. Therefore, targeting p53 to induce pro-apoptotic autophagy and further enhance tumor cell sensitivity to platinum-based drugs holds great potential.

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

[0006] Immune suppression is one of the significant features of malignant tumors, which largely promotes the development of cancer. Recent studies have shown that p53 can regulate innate and adaptive immunity by regulating immune checkpoint (IC) PD-L1. 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 dawn to 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 platinum (IV) system and designed a series of repaglinide platinum (IV) compounds. These compounds can damage DNA through platinum-based parent nucleus; activate lumican / p53 / p21 signaling axis to increase the expression of p53 through repaglinide functional fragments; and then reverse EMT and immune suppression, synergistically exerting anti-tumor proliferation and anti-metastasis effects. SUMMARY

[0009] In view of the problems in the prior art, the present application provides a compound with a repaglinide tetravalent platinum structure, by introducing a repaglinide molecule into a tetravalent platinum nucleus, a series of novel repaglinide compound modified tetravalent platinum compounds are synthesized, the repaglinide tetravalent platinum compounds have significant anti-tumor proliferation ability, good treatment effect on metastatic malignant tumors, improve 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. The drug opens up a new direction for the development of anti-tumor drugs, especially the research and development of anti-metastatic malignant tumor drugs.

[0010] To achieve the above purpose, the compound with the repaglinide tetravalent platinum structure provided by the present application,

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

[0012]

[0013] Among them, selected from cisplatin or oxaliplatin; L is hydroxyl or

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

[0015]

[0016] The compound provided by the present application is selected from:

[0017]

[0018] Another object of the present application provides a preparation method of the compound as shown in general formula (I), and the synthesis route of the compound is as follows.

[0019] The synthesis route one is as follows:

[0020]

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

[0022] The synthesis route two is as follows:

[0023]

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

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

[0026] Under an inert gas atmosphere, compound III is dissolved in an anhydrous organic solvent for reaction, compound II is added, and the reaction is carried out in the dark. After post-treatment, compound Ia is isolated;

[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 the reaction is carried out in the dark. After post-treatment, compound Ib is isolated;

[0030] The 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 between compound II and organic solvent is that 10-100 mL of organic solvent is added for every 1 g 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 a reaction vessel, 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 column chromatography is performed to obtain an asymmetric monosubstituted repaglinide-modified tetravalent platinum compound Ia.

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

[0035] Further, the compound II is composed of After being oxidized with 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 a therapeutically effective amount of a compound represented by formula (I) and pharmaceutically acceptable excipients thereof.

[0039] 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 can 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; and 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, and Ringer's solution.

[0040] The tetravalent platinum compound or pharmaceutical composition of the repaglinide of 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 nanoformulations prepared using conventional methods based on currently recognized pharmaceutical knowledge.

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

[0042] The administration route of the repaglinide tetravalent platinum compound of the present application can be intravenous administration. Injection includes intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection, and acupoint injection, etc.

[0043] The method for preparing the active ingredient into a drug in the present application can be prepared by the method known to those skilled in the art, for example: the active ingredient can be diluted with a carrier or encapsulated in a carrier, so as to release the active ingredient quickly, slowly or delayed after administration to the subject.

[0044] Another object of the present application provides the use of the compound or pharmaceutical composition as shown in the general formula (I) in the preparation of an anti-tumor drug, specifically in the preparation of an anti-tumor proliferation and anti-tumor metastasis drug.

[0045] The repaglinide tetravalent platinum compound of the present application can have 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 good therapeutic effect on metastatic malignant tumors.

[0046] Further, the anti-tumor is anti-lung cancer, anti-drug-resistant lung cancer, anti-liver cancer, or anti-breast cancer, etc.; wherein the anti-tumor proliferation is specifically anti-human lung adenocarcinoma, anti-drug-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 present application also provides a combined preparation comprising the compound or pharmaceutical composition as shown in the general formula (I), and the combination of paclitaxel, fluorouracil, gemcitabine, vinblastine, antibody, and other anti-tumor drugs.

[0048] The repaglinide tetravalent platinum compound of the present application is expected to be used alone or in combination with marketed platinum, paclitaxel, fluorouracil, gemcitabine, vinblastine, antibody, etc., to prepare a combined preparation with anti-tumor activity. The combined preparation can be tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, oral preparations, granules, decoctions, pills, powders, pastes, suspensions, solutions, injections, powder injections, lyophilized powder injections, suppositories, rubs, ointments, plasters, creams, sprays, aerosols, drops, patches, etc.

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

[0050] (1) The present invention introduces the repaglinide molecule into the tetravalent platinum system to synthesize a series of novel repaglinide compound-modified tetravalent platinum compounds, which are proven to have good anti-tumor and anti-cancer abilities through anti-tumor activity tests; this type of repaglinide tetravalent platinum compound has a good therapeutic effect on metastatic malignant tumors and has 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 p53 expression 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 innovative structure of the compound of formula I described in the present invention is expected to produce a variety of lead molecules that are effective against tumors, providing new candidate drug molecules to address the defects of traditional divalent platinum drugs, and also opening up new avenues for the modification of tetravalent platinum compounds. In terms of pharmacological activity, the tetravalent platinum compound 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 large amounts of capital investment, promote the development of related industries such as biotechnology, chemical pharmaceuticals, and pharmaceutical R&D services, create more employment opportunities, and contribute to national economic growth. 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 the experimental design. (b) Relative body weights of mice during treatment. (c) Tumor volume changes over time. (d) Tumor weights of mice in each group at the end of the experiment. The tumor growth inhibition rate (TGI) of the test drug is indicated [TGI = (1 - tumor weight of the drug-treated group / tumor weight of the control group) × 100%). (e) Tumor images after mouse sacrifice.

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

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

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

[0059] Figure 4 In vitro Transwell assays were used to evaluate the inhibitory effects of compound 3 (5 μM), CDDP (5 μM), and CDDP + RPG (5 μM / 5 μM) on 4T1 cell migration. Tumor cells were treated with the compounds for 24 hours, while untreated cells served as blank controls. (a) Representative images. (b) Relative migration rate analysis. ***P < 0.001.

[0060] Figure 5 An in vitro scratch-healing assay was used to evaluate the migration inhibitory 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 used as blanks. (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 photographs of the front and back of lung tissue from each group at the end of the experiment. (c) Pulmonary nodule counts for each group. (d) H&E staining of pulmonary metastatic nodules. Nodules are indicated by arrows.

[0062] Figure 7 Stability of Compound 3 in biological culture medium. A 0.5 mM solution of Compound 3 was prepared in RPMI 1640 and incubated at 37°C for 24 hours. Chromatograms were obtained using an Agilent C18 column (250 mm × 4.6 mm, 5 μm) on a Thermo Ultimate 3000RS. A fixed eluent (75% methanol / 25% water) was used, with a flow rate of 1.0 mL / min, λ = 250 nm, and an injection volume of 10 μL.

[0063] Figure 8 HPLC chromatogram 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] Figure 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] The chromatogram was obtained on a Thermo Ultimate 3000RS (250 mm × 4.6 mm, 5 μm). A fixed eluent (75% methanol / 25% water) was used, with a flow rate of 1.0 mL / min, λ = 250 nm, and an injection volume of 10 μL. A decrease in the peak of compound 3 was observed in the HPLC chromatogram, while a peak of RPG appeared and gradually increased.

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

[0068] Figure 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] Figure 12 In vitro drug uptake and distribution in 4T1 cells: 4T1 cells were treated with CDDP (5 μM), CDDP+RPG (5 μM / 5 μM), and compound 3 (5 μM) for 24 hours, and then the drug uptake and distribution were determined by atomic absorption spectrometry.

[0070] Figure 13Analysis of the 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: water, phase B: 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 and 24 hours. (b) Reduction reaction in reducing medium and the process of CDDP-GMP adduct formation.

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

[0072] Figure 15 The p53 signaling regulation properties of RPG platinum (IV) compound 3 were examined by Western blotting and immunohistochemistry. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM), and a CDDP + RPG mixture (5 μM / 5 μM) for 24 hours: (a) Blots; (b) Relative grayscale intensity analysis. Immunohistochemical staining analysis of p53 in tumor tissues obtained from in vivo antitumor experiments: (c) Representative images; (d) Quantitative data. **p < 0.01, ***p < 0.001

[0073] Figure 16Western blot and immunohistochemistry were used to examine the properties of RPG platinum (IV) compound 3 in inducing pro-death autophagy. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM), and a mixture of CDDP and RPG (5 μM / 5 μM) for 24 hours: (a) Western blot; (b) Analysis of LC3I, LC3II, p62, and Beclin1 expression data. In vivo antitumor experiments were performed using immunohistochemical staining of p62 in tumor tissues: (c) Representative images; (d) Quantification data. (e) MTT assay was used to assess the viability of 4T1 cells treated with compound 3 (5 μM), CDDP (5 μM), 3 + 3MA (5 μM / 70 μM), and CDDP + 3MA (5 μM / 70 μM) for 24 hours. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM), a mixture of 3 + 3MA (5 μM / 70 μM), and CDDP + 3MA (5 μM / 70 μM) for 24 hours: (a) Western Blot; (b) Data analysis of LC3I, LC3II, p62, and Beclin1 expression. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical difference

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

[0075] Figure 18 Western blot immunohistochemistry was used to examine the inhibitory effects of RPG platinum (IV) compound 3 on EMT in tumor cells. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM), and a CDDP + RPG mixture (5 μM / 5 μM) for 24 hours. (a) Western blot analysis; (b) E-cadherin, N-cadherin, Vimentin, and Snail1 expression data analysis. Immunohistochemical staining of E-cadherin and N-cadherin in tumor tissues obtained from in vivo antitumor experiments: (c) Representative images; (d) Quantitative data. **p < 0.01, ***p < 0.001.

[0076] Figure 19Western blot and immunohistochemistry were used to examine the inhibitory effects of RPG platinum (IV) compound 3 on tumor inflammation and angiogenesis. 4T1 cells were incubated with compound 3 (5 μM), CDDP (5 μM), and a CDDP + RPG mixture (5 μM / 5 μM) for 24 hours: (a) Western blot; (b) COX-2, MMP9, and VEGFA expression data analysis. Immunohistochemical staining of VEGFA and CD34 in tumor tissues obtained from in vivo antitumor experiments: (c) Representative images; (d) Quantitative data. **p < 0.01, ***p < 0.001.

[0077] Figure 20 Western Blot and immunohistochemistry were used to detect the anti-tumor immune activation 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 expression in tumor tissues obtained from in vivo anti-tumor experiments were detected. + 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 solutions of the present invention are further described with reference to specific examples. However, these examples are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the experiments were performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used, unless otherwise specified, are conventional biochemical reagents. The experimental methods described, 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 represented by compound II

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

[0084]

[0085] To a 250 mL round-bottom flask, 1.0 g of cisplatin and 30 mL of distilled water were added, and the mixture was stirred at room temperature. 50 mL of 30% hydrogen peroxide was slowly added dropwise to the reaction system, and the temperature was raised to 60°C and stirred for 4 h. The reaction was stopped, and the mixture was placed in a 4°C refrigerator for crystallization for 12 h. The yellow solid was separated by filtration. An appropriate amount of distilled water was added, and the mixture was heated to 80°C to dissolve it. The mixture was placed at 4°C for crystallization for 12 h, and compound IIa was filtered to obtain yellow crystals (0.74 g, 67%).

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

[0087]

[0088] To a 250 mL round-bottom flask, 1.0 g of oxaliplatin and 30 mL of distilled water were added, and the mixture was stirred at room temperature. 50 mL of 30% hydrogen peroxide was slowly added dropwise to the reaction system, and the temperature was raised to 60 ° C. and stirred for 4 h. The reaction was stopped and the mixture was placed in a 4 ° C refrigerator for crystallization for 12 h. The yellow solid was separated by filtration. An appropriate amount of distilled water was added, and the mixture was heated to 80 ° C to dissolve it. The mixture was placed at 4 ° C for crystallization for 12 h, and filtered to obtain compound IIb as a white crystal (0.80 g, 72%).

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

[0090]

[0091] To a solution of RPG (500 mg, 1.14 mmol) in anhydrous DMF (10 mL) was added EDC·HCl (364 mg, 1.89 mmol) and stirred at room temperature overnight. After completion of the reaction, the reaction system was concentrated and extracted with aqueous citric acid and aqueous NaHCO₃. The organic phases were combined, dried over anhydrous Na₂SO₄, and dried to afford III as a white solid (315 mg, 32%).

[0092] 3. 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 in a dark environment at 55 ° C for 48 h. 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%). The purity was determined by HPLC to be 99.8% (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 bis-repaglinide 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 at 55 ° C in the dark for 48 h. After the reaction was completed, the mixture was concentrated and further purified by silica gel column chromatography to obtain white solid compound 2 (75.2 mg, 25%), which was determined to be 98.6% pure 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 under nitrogen for 24 h. After the reaction, the mixture was concentrated and further purified by silica gel column chromatography to obtain white solid compound 3 (46.3 mg, 20%). The purity 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 under nitrogen for 24 h. After the reaction, the mixture was concentrated and further purified by silica gel column chromatography to obtain compound 4 (37.2 mg, 19%) as a white solid 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] To better understand the essence of the present invention, the following pharmacological experimental results demonstrating the tumor inhibition effects of the compounds in vivo and in vitro are used to illustrate the potential uses of these compounds in the pharmaceutical field. The pharmacological experiments provide partial activity data for some of the compounds. It should be noted that the pharmacological experiments of the present invention are intended to illustrate the present invention and not to limit it. Simple modifications to the present invention based on its essence fall within the scope of protection of the present invention.

[0111] 1. In vitro antitumor activity assay

[0112] Experimental method: This experiment uses the MTT method to determine the cell viability, and 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, remove it, remove the culture medium, add 150 μL of DMSO, and shake in a 37°C shaker in the dark for 20 minutes. Measure the absorbance OD value of each well at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader 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 was tested by MTT assay using platinum (II) drugs CDDP and OXP, ligand RPG, and CDDP+RPG mixture as reference drugs, 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). Table 1 lists the maximum 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 anti-proliferative activity against all tested tumor cell lines. 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 anti-proliferative 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 anti-tumor efficacy against all tumor cell lines, with an IC 50 The value was lower than 3.57 μM, 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 similar effects to CDDP, indicating that the addition of RPG to the platinum system to construct the RPG platinum (IV) compound is the key to improving its antitumor efficacy and 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 comparing 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 value to a lower level (1.06-1.93) compared with CDDP and CDDP+RPG (RF=5.06, 3.08), indicating that the RPG platinum (IV) skeleton has 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 with 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, particularly compound 3, derived from CDDP and bearing a single RPG ligand, exhibited promising antiproliferative activity in vitro and demonstrated 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 using CDDP, OXP, RPG, and CDDP+RPG as references. After 48 hours of drug treatment, the half-maximal 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 experiments

[0122] Experimental Methods: The anti-tumor activity was evaluated using a 4T1 tumor-bearing female BALB / c mouse model. 4T1 cells were injected into the left axilla of the mice. Mice with palpable tumor nodules on day 3 after inoculation 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 via 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 volume of matrix solution. Tumor volume (V = W2 × L / 2, where W represents tumor width and L represents tumor length) and body weight of the mice were monitored throughout the experiment. On day 11, the mice were sacrificed, and blood samples were collected for ELISA analysis. Organ samples, including tumor, heart, liver, spleen, lung, and kidney, were also collected for immunohistochemistry and H&E staining. TGI was determined based on tumor weight using the following formula: TGI (%) = (1 - tumor weight in the drug-treated group / tumor weight in the 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. 3 , TGI = 43.5%) and CDDP + RPG compound (425mm 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 image of the tumor in Figure f further demonstrates its strong anti-tumor 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 anti-tumor 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), thereby 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 showed that compared with the blank group, compound 3 did not significantly reduce body weight after three doses (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 anti-tumor 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. To test the potential of RPG platinum (IV) compounds as anti-tumor metastasis drugs, the anti-metastatic activity of compound 3 was evaluated using in vitro Transwell and wound healing assays.

[0127] Transwell assay: serum-deprived 4T1 cells (5 × 10 4 The cells were resuspended in 200 μL RPMI 1640 medium (600 μL / well) and inoculated into the upper chamber of a 24-well Transwell plate (Costar 3422). In the lower chamber, a mixture of culture 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. Cells migrating from the bottom side of the membrane were observed using an inverted microscope, and images of five fields of view were randomly collected per well for analysis.

[0128] Wound-Healing Assay: Serum-deprived 4T1 cells were resuspended in RPMI 1640 medium containing 1% FBS and plated at 4 × 10 cells per well. 5 Cells were seeded at a density of 1 μg / ml in 6-well culture dishes. After overnight preincubation, when cell confluence reached approximately 90%, a scratch wound was created. Subsequently, cells were treated with platinum compound 3 (5 μM), CDDP (5 μM), or CDDP + RPG (5 μM / 5 μM) for 24 hours. The degree of cell healing was measured.

[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 wound healing assay shown, the wound closure rate induced by compound 3 was lower than that of the blank group, CDDP group, and CDDP+RPG group, further confirming its anti-metastatic effect. Therefore, RPG platinum (IV) compound 3 can effectively inhibit tumor cell metastasis in vitro.

[0130] 4. In vivo metastasis inhibition experiment

[0131] Experimental method: The lung is the main site of tumor cell metastasis in the body, so this study used the lung metastasis model to study the anti-tumor metastasis ability of the drug 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 the 4th day, 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 the 4th, 7th and 10th days, 2 mg Pt / kg was intravenously injected ( Figure 6 a) On day 12, mice were sacrificed and lung tissue was collected for analysis. Nodules on the lung surface were counted, and nodules within the lung tissue were observed using H&E staining.

[0132] Experimental results: Statistical analysis of pulmonary nodules ( Figure 6 bc) showed that 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, compared with the blank group, CDDP group and CDDP + RPG group, fewer and smaller tumor nodules were observed in the H&E staining images of the lung tissue of the compound 3 treatment 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 acting as prodrugs of platinum(II) drugs are expected to be stable in biological media and readily reduced in the reducing TME. Therefore, we evaluated the stability of RPG platinum(IV) compound 3 in the biological culture medium RPMI1640 by high-performance liquid chromatography, as well as its reduction potential in RPMI1640, a reducing medium 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 for improving antitumor activity and reducing toxicity.

[0136] 6. Induce mitochondria-mediated apoptosis.

[0137] Apoptosis, a key form of programmed cell death, plays a crucial role in maintaining cellular homeostasis and clearing damaged cells. Mitochondria are key organelles that regulate tumor cell apoptosis through the Bcl-2 / Bax / caspase-3 signaling pathway. Platinum-based drugs can effectively exert antiproliferative effects by inducing apoptosis. Therefore, we evaluated the apoptosis-inducing properties of compound 3 using the classic Annexin V-FITC / PI staining method.

[0138] Figure 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, JC-1 staining was used to assess the changes in mitochondrial membrane potential (ΔΨm). Figure 11 The results showed that compared with the blank group, the compound 3-treated group induced a more severe collapse of the mitochondrial membrane potential (ΔΨm) (34.9%), even higher than that of CDDP (25.5%) and CDDP+RPG (20.5%). Mitochondrial membrane potential collapse is a hallmark of apoptosis, indicating that RPG platinum (IV) compound 3 significantly affects mitochondrial integrity and function.

[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, Bax, caspase-3, and c-caspase-3 proteins related to the Bcl-2 cascade was evaluated by Western Blot. Figure 10 c, d). The results showed that compared with the blank group, compound 3 significantly downregulated the expression of the anti-apoptotic protein Bcl-2 (P < 0.001) and upregulated the expression of the pro-apoptotic protein Bax (P < 0.001). Furthermore, an increase in the ratio of c-caspase-3 / caspase-3 (P < 0.001) was observed in the compound 3-treated group. c-caspase-3 / caspase-3 is a key executioner of apoptosis. Therefore, compound 3 effectively induces tumor cell apoptosis through the mitochondrial-mediated Bcl-2 / Bax / caspase-3 pathway.

[0140] 7. Inducing DNA damage in tumor cells

[0141] DNA damage is the primary mechanism by which platinum-based drugs induce apoptosis in tumor cells. Furthermore, nuclear accumulation of platinum-based drugs is a key factor influencing their DNA-damaging capacity. We used high-performance liquid chromatography and Western blot to examine whether the reduced RPG platinum(IV) compound 3 causes DNA damage. AAS was used to assess drug accumulation in DNA by measuring platinum content.

[0142] Figure 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 (1mM) and 5'-GMP (3mM) was monitored ( Figure 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 damaging ability of RPG platinum (IV) compounds.

[0143] Phosphorylation of H2AX (γ-H2AX) is one of the hallmarks of DNA damage. In this study, CDDP and CDDP+RPG mixture were used as positive reference drugs. Western Blot was used to determine the expression of γ-H2AX in 4T1 tumor cells treated with compound 3 to further confirm the role of RPG platinum (IV) in DNA damage ( Figure 14 As expected, the 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 various anti-tumor processes. It has been reported that RPG can increase 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 signaling. Therefore, we detected the effect of RPG platinum (IV) compound 3 on the lumican / p53 / p21 pathway by Western Blot and immunohistochemistry.

[0147] Figure 15 a-b shows that compared with the blank group (P<0.001) and the CDDP group (P<0.001), compound 3 significantly inhibited the 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 up-regulated (P<0.001). In addition, Figure 15 c-d shows that in vivo tumor tissues, high expression of p53 was also observed in tumor tissues treated with compound 3 compared with the blank group and the CDDP treatment group (P<0.01), which further verified that RPG platinum (IV) compound 3 has an advantage in activating the p53 cascade. Notably, compared with CDDP alone, the effect of CDDP+RPG combination on lumican, p53 and p21 protein expression is relatively more obvious, which is mainly due to RPG. These research results show that compound 3 has an advantage 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, known as the second type of programmed cell death, is another mechanism of cell death. Increasing autophagic flux through drugs can disrupt the balance of tumor cells, leading to tumor cell apoptosis. In addition, it has been reported that RPG can effectively enhance autophagic flux. Next, we studied the effect of drugs on autophagic flux by Western Blot and MDC staining.

[0150] It has been proven that protein p53 has a double-edged sword function in regulating the autophagy process. In this study, we determined whether the up-regulation of p53 caused by RPG platinum (IV) compound 3 would promote autophagy in tumor cells by measuring the proteins LC3II / I and P62 related to autophagic flux by Western Blot and immunohistochemistry. Figure 16 The results in a, b show that compound 3 effectively promotes autophagy, and the ratio of LC3II / I in tumor cells is significantly increased (P<0.01), while the expression of autophagy substrate p62 in tumor cells is reduced compared with the blank group (P<0.01). In addition, the immunohistochemical results of tumor tissues Figure 16 c, d) further demonstrated this trend, with compound 3 significantly reducing the expression of p62 in tumors in vivo (P<0.001). MDC staining is widely used for autophagosome staining in tumor cells. Figure 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 with 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 decrease 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 considered a double-edged sword in tumor cells. Therefore, evaluating whether autophagy induced by the RPG platinum(IV) compound 3 can enhance tumor cell apoptosis is a key question. 3-Methyladenine (3MA), a potent autophagy inhibitor, is frequently used in the study of autophagy-modulating drugs. In this study, 3MA was used to examine the effects of autophagy on 4T1 cell viability. Figure 16 Western blot results in f, g showed that compared with the compound 3-treated group, the expression of LC3II / I (P<0.01) and Beclin1 in the tumor cells of the 3+3MA-treated 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. Figure 17 ) further verified this trend, that is, the number of autophagosomes in the 3+3MA treatment group was reduced compared with the compound 3 treatment group. Subsequently, the cell viability in the 3+3MA treatment group was significantly improved compared with the compound 3 group (P<0.01) ( Figure 16 e). This shows 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] Growing 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, including E-cadherin, N-cadherin, Vimentin, and Snail1.

[0154] Figure 18 Western blot results in a and b show that, compared with the blank control group, RPG platinum (IV) compound 3 significantly inhibited N-cadherin expression in 4T1 cells (P < 0.001), while increasing E-cadherin secretion (P < 0.001). Subsequently, Vimentin and Snail1 proteins were suppressed to lower levels (P < 0.001). These results demonstrate the effectiveness of RPG platinum (IV) compound 3 in reversing the EMT process in tumors, which was also confirmed by immunohistochemical staining of tumor tissues in vivo. Figure 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. Notably, platinum (II) drugs had minimal effects on E-adherin and N-adherin both in vitro and in vivo, indicating 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 pathway for tumor cell metastasis. Tumor cell EMT and protective autophagy have great potential in maintaining the chronic inflammatory TME. COX-2 and MMP9 proteins are key enzymes involved in the inflammatory TME and play a synergistic role in promoting angiogenesis by upregulating VEGFA. In this study, we evaluated the expression of COX-2, MMP9, and VEGFA using Western blotting and immunohistochemistry. We also examined the expression of the vascularization marker CD34 in tumors using immunohistochemistry.

[0157] As shown in Western Blot ( Figure 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 reducing 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, Figure 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). This shows that RPG platinum (IV) compound 3 inhibits tumor angiogenesis by inhibiting COX-2, MMP9 and VEGFA, thereby further inhibiting tumor cell metastasis.

[0158] 12. Activate immunity and inhibit tumor metastasis

[0159] Immunosuppression is a major characteristic 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 be inactivated. 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 studied CD3 + and CD8 + T cells were immunohistochemically stained to determine their effect on activating anti-tumor immunity in vivo. In addition, their effect on PD-L1 expression was also examined by Western blotting and immunohistochemistry.

[0160] Figure 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 ( Figure 20 CD3 + and CD8 + T cell counts increased to 3.0-fold and 3.7-fold compared to the blank group, respectively (P<0.01), significantly superior to those in the CDDP and CDDP+RPG groups. This suggests that RPG platinum (IV) compound 3 can effectively stimulate T cell anti-tumor immunity in tumor tissue by blocking the immune checkpoint PD-L1, further enhancing anti-proliferative and anti-metastatic activity in vivo.

[0161] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A compound having a tetravalent platinum structure of repaglinide as shown in 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. The 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: The molar ratio of compound II to compound III is 1:1.0-1.5, and compound II and compound III undergo a coupling reaction to obtain an asymmetric monosubstituted repaglinide-modified tetravalent platinum compound Ia, where 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, wherein: 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 Ia 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 Ib is isolated after post-treatment. 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, thereby obtaining an asymmetric monosubstituted repaglinide-modified tetravalent platinum compound Ia; The 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 to obtain a symmetrically disubstituted repaglinide-modified tetravalent platinum compound Ib.

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: The invention comprises the compound represented by the general formula (I) as claimed in claim 1 or 2, and pharmaceutically acceptable excipients thereof.

7. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to claim 6 in the preparation of drugs for treating lung adenocarcinoma, cisplatin-resistant lung adenocarcinoma, liver cancer, breast cancer proliferation or breast cancer cell metastasis.

8. A combined preparation comprising the compound of formula (I) according to claim 1 or 2 or the pharmaceutical composition according to claim 6, and an anti-tumor drug of the platinum type, paclitaxel type, fluorouracil type, gemcitabine type, vinca alkaloid type or antibody type.

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