Derivative with captopril-valproic acid tetravalent platinum structure and preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a three-component platinum (IV) complex containing captopril (CTP) and valproic acid (VPA), a preparation method of the three-component platinum (IV) complex and application of the three-component platinum (IV) complex in preparation of anti-tumor proliferation and anti-metastasis drugs. The anti-tumor proliferation and anti-metastasis effects of the target compound are tested through in-vivo and in-vitro testing means, and the anti-tumor mechanism of the medicine is researched. Results prove that the compound has a remarkable anti-tumor capability, particularly has an excellent treatment effect on metastatic malignant tumors, is expected to provide a new candidate drug for clinical treatment of tumors, and provides a new direction for research and development of novel platinum drugs and anti-tumor metastasis drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a three-component platinum (IV) hybrid containing captopril (CTP) and valproic acid (VPA), its preparation method, and its application in the preparation of antitumor proliferation and antimetastasis drugs. Background Technology

[0002] Cancer remains one of the most intractable diseases globally. Metastatic cancer, in particular, is a key obstacle to improving patient survival, accounting for approximately 90% of clinical cancer-related deaths. While traditional chemotherapy has been a core treatment for cancer for decades and has proven highly effective in controlling primary tumors, its ability to suppress metastatic cancer cells has been less than satisfactory. Developing novel anti-metastatic drugs has become an urgent task in the field of cancer research.

[0003] Platinum(II) compounds, as the cornerstone of cancer chemotherapy, exert their anti-tumor effects by inducing damage to intracellular DNA through cross-linking. However, their efficacy is significantly limited by severe toxicity and acquired resistance. DNA repair mechanisms are a crucial reason for multidrug resistance (MDR) in tumor cells. Tumor fibrosis, characterized by extracellular matrix (ECM) deposition, remodeling, and cross-linking, is a key factor promoting the formation of the tumor microenvironment (TME) and hinders drug penetration and immune attack by forming a dense ECM, further reducing the efficacy of platinum(II) drugs. Notably, platinum(IV) provides an excellent framework for the development of novel platinum compounds. Therefore, introducing functional groups into the platinum(IV) system to develop multifunctional platinum(IV) compounds with both DNA repair inhibition and fibrosis inhibition functions is an effective strategy to overcome the drawbacks of traditional platinum drugs and can bring new drug candidates to cancer treatment.

[0004] Histone deacetylases (HDACs), key enzymes involved in epigenetic regulation, are highly expressed in various tumors. Aberrant upregulation of HDACs has been shown to be a crucial enzyme promoting tumorigenesis, proliferation, and metastasis. Inhibiting HDAC activity can block the self-repair of tumor cell DNA damage by regulating key proteins such as P-gp, PARP, and PTEN, thereby further overcoming tumor renal degeneration (MDR). More importantly, HDAC inhibitors can effectively suppress tumor fibrosis by inhibiting the TGF-β / Smad cascade and reversing the COX and MMP-mediated inflammatory environment. Tumor epithelial-mesenchymal transition (EMT) is a critical step in tumor invasion and metastasis. Inhibiting tumor fibrosis and inflammatory TME can synergistically inhibit EMT. Therefore, developing platinum-based drugs with HDAC inhibitory activity, which can inhibit tumor fibrosis and inflammatory TME, synergistically inhibit EMT, and thus inhibit tumor metastasis and proliferation, is an effective strategy for developing novel anti-metastatic drugs.

[0005] Valproic acid (VPA), as an HDAC inhibitor, holds great potential in cancer treatment. Its combination with platinum (II) drugs can reduce DNA repair, enhance DNA damage effects, and reverse cancer cell resistance to platinum-based drugs. Several VPA-platinum (IV) compounds have been reported, exhibiting superior antitumor activity compared to traditional platinum (II) drugs; however, their anti-metastatic potential and impact on tumor microenvironment (TME) remain unclear and require further investigation. Captopril (CTP), as an effective clinical ACE inhibitor, can inhibit fibrosis by downregulating factors such as MMPs and TGF-β1. This study uses CTP as a functional linker to couple a platinum (IV) core to a VPA functional group, constructing a novel CTP-VPA platinum (IV) compound. The goal is to develop a novel antitumor drug with combined DNA damage, fibrosis inhibition, and DNA repair inhibition properties.

[0006] Therefore, in this invention, we designed and synthesized novel platinum (IV) compounds with dual or single CTP-VPA ligands. The VPA group is expected to inhibit DNA repair and tumor fibrosis by inhibiting HDAC, while CTP will further modulate immunity by targeting the TGF-β and MMP pathways to inhibit fibrotic TME. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention proposes a compound with a captopril-valproic acid tetravalent platinum structure, its preparation method, and its applications. The antitumor proliferation and antimetastatic effects of the target compound were verified through in vivo and in vitro testing, and the antitumor mechanism of the drug was investigated. The results confirm that this compound possesses significant antitumor activity, particularly showing excellent therapeutic effects against metastatic malignant tumors. It is expected to provide a new candidate drug for clinical cancer treatment and offer new directions for the development of novel platinum-based drugs and antitumor metastasis drugs.

[0008] The compound having a captopril-valproic acid tetravalent platinum structure described in this invention has the general structural formula shown in (Ⅰ):

[0009]

[0010] in, Selected from cisplatin or oxaliplatin; L is a hydroxyl group or

[0011] Furthermore, the compound with the tetravalent platinum structure of captopril-valproic acid is any one of the following:

[0012]

[0013] The captopril-valproate tetravalent platinum derivative described in this invention is selected from:

[0014]

[0015] Another object of the present invention is to provide a method for preparing a compound as shown in general formula (I), the synthetic route of which is shown below.

[0016] The first synthetic route is as follows:

[0017]

[0018] Compound II and compound 6 undergo a coupling reaction to give monosubstituted captopril-valproic acid tetravalent platinum compound Ia; wherein the molar ratio of compound II to compound 6 is 1:1.0 to 1.5.

[0019] The second synthetic route is as follows:

[0020]

[0021] Compound II and compound 6 undergo a coupling reaction to give a symmetrical disubstituted captopril-valproic acid tetravalent platinum compound Ib; wherein the molar ratio of compound II to compound 6 is 1:2.0 to 5.0.

[0022] Furthermore, in the synthetic route, the preparation steps of compound 6 are as follows:

[0023]

[0024] Preparation of compound 6: 1H-benzotriazole (H-Bt, 28 g, 240 mmol) was dissolved in 200 mL of dichloromethane, and thionyl chloride (4 mL, 60 mmol) was added. The mixture was stirred at room temperature for 15 min, and then valproic acid (8.6 g, 60 mmol) was added, and the reaction was continued at room temperature for 2.5 h. After filtration, the organic phase was washed with saturated Na₂CO₃ solution, and 200 mL of hexane was added to precipitate a white solid. The solid was filtered to obtain compound 5 (6 g, 43%). Compound 5 (0.65 g, 2.3 mmol) was dissolved in 20 mL of acetonitrile, and 10 mL of an aqueous solution of CTP (0.5 g, 2.3 mmol) and triethylamine (0.23 g, 2.3 mmol) were added. The mixture was stirred for 20 h. After the reaction was complete, the acetonitrile was removed by vacuum distillation. The residue was extracted with ethyl acetate, evaporated, and concentrated to obtain a white solid, compound 6 (0.31 g, 39.3%).

[0025] Furthermore, in the first synthetic route, the preparation steps of the monocaptopril-valproic acid tetravalent platinum derivative are as follows:

[0026] In an inert gas atmosphere, compound 6, condensing agent, and organic base were dissolved in an anhydrous organic solvent and reacted. Compound II was added, and after reacting in the dark, the monosubstituted captopril-valproic acid tetravalent platinum derivative Ia was obtained by post-treatment.

[0027] The molar ratio of compound II, compound 6, condensing agent, and organic base is 1:1.0-1.5:1.0-1.5:1.0-1.5; the feeding relationship between compound II and organic solvent is 10-100 ml of organic solvent for every 1 g of compound II.

[0028] In the second synthetic route, the preparation steps of the biscaptopril-valproic acid tetravalent platinum derivative are as follows:

[0029] In an inert gas atmosphere, compound 6, condensing agent, and organic base were dissolved in an anhydrous organic solvent and reacted. Compound II was added, and after reacting in the dark, the symmetrical disubstituted captopril-valproic acid tetravalent platinum derivative Ib was obtained by post-treatment.

[0030] The molar ratio of compound II, compound 6, 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 1g of compound II corresponds to 10~100ml of organic solvent.

[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 specific preparation process is as follows: condensing agent and compound 6 are added to the reaction vessel, the air in the system is replaced with an inert gas, anhydrous organic solvent is added, the reaction is stirred at room temperature, then anhydrous organic base is added to the reaction system, the reaction is stirred at room temperature, then tetravalent platinum compound II is added to the reaction system, the air in the system is replaced with an inert gas again, and the reaction system is placed at 25-120℃ in the dark for 24-72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and column chromatography is used to obtain asymmetric monosubstituted captopril-valproic acid tetravalent platinum compound Ia or symmetric disubstituted captopril-valproic acid tetravalent platinum compound Ib.

[0033] Furthermore, compound II is composed of The product is prepared by oxidation with hydrogen peroxide. The specific preparation process is as follows:

[0034]

[0035] The specific preparation process can be as follows: divalent platinum compounds Dihydroxy tetravalent platinum compound II was prepared by oxidation with hydrogen peroxide at 60–70 °C for 1–8 h.

[0036] The present invention provides a pharmaceutical composition comprising an effective therapeutic amount of a compound represented by formula (I) and pharmaceutically acceptable excipients thereof.

[0037] The pharmaceutically acceptable excipients of this invention include one or more carriers, excipients, diluents, etc., and various types of adhesives. Excipients can be aqueous or non-aqueous, and conventional examples include gelatin and other gums, starches such as corn starch, sugars such as lactose, cellulose materials such as sodium carboxymethyl cellulose, and mixtures thereof. Other excipients include tragacanth gum powder, and oils, alcohols, esters, buffers, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, etc., can also be used as excipients. The captopril-valproic acid tetravalent platinum compounds or pharmaceutical compositions of this invention are available in various dosage forms, including tablets, as well as sustained-release, controlled-release, or nano-formulations prepared according to pharmaceutical common sense.

[0038] The captopril-valproic acid tetravalent platinum compound described in this invention can be administered in unit dose form via enteral and non-enteric routes, such as oral, intramuscular, subcutaneous, or nasal administration.

[0039] The captopril-valproic acid tetravalent platinum compound of the present invention can be administered intravenously. Injection includes intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection, and acupoint injection.

[0040] The method for preparing the active ingredient into a drug in this invention can be prepared using methods known to those skilled in the art. For example, the active ingredient can be diluted or encapsulated in a carrier so that it can be released immediately, slowly, or with a delayed release after being administered to a subject.

[0041] Another object of the present invention is to provide the use of compounds or pharmaceutical compositions as shown in general formula (I) in the preparation of antitumor drugs, specifically in antitumor proliferation and antitumor metastasis drugs.

[0042] The captopril-tetravalent platinum derivative described in this invention has a good therapeutic effect on metastatic malignant tumors, can effectively inhibit tumor fibrosis, inhibit DNA repair, and promote DNA damage, thus exhibiting a good therapeutic effect on metastatic malignant tumors.

[0043] Furthermore, the anti-tumor activity is anti-lung cancer, anti-drug-resistant lung cancer, anti-liver cancer, or anti-breast cancer, etc.; wherein, the anti-tumor proliferation activity is specifically anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer, or anti-mouse breast cancer; and the anti-tumor metastasis activity is anti-mouse breast cancer cells.

[0044] The invention also provides a combination formulation comprising a compound or pharmaceutical composition as shown in general formula (I) and an antitumor drug such as paclitaxel, fluorouracil, gemcitabine, vincristine, or an antibody.

[0045] The trifunctional derivative tetravalent platinum compounds described in this invention are expected to be used alone or in combination with marketed platinum-based drugs, paclitaxel-based drugs, fluorouracil-based drugs, gemcitabine-based drugs, vincristine-based drugs, and antibody-based drugs to prepare combination formulations with antitumor activity. These combination formulations can take 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, powders, pills, powders, ointments, suspensions, solutions, injections, powder for injection, lyophilized powder for injection, suppositories, liniments, ointments, hard plasters, creams, sprays, aerosols, drops, patches, etc.

[0046] Compared with existing technologies, the compounds and nanomedicines with a captopril-valproic acid tetravalent platinum structure described in this invention have the following advantages:

[0047] 1. The low sensitivity of metastatic tumors to chemotherapy is a major cause of chemotherapy failure. In this study, captopril, valproic acid and platinum (IV) system were innovatively coupled to synthesize a series of novel captopril-platinum valproate (IV) compounds, which showed significant anti-proliferation and anti-migration activities in in vitro experiments and have the potential to become anti-metastatic targeted anti-tumor drugs.

[0048] 2. Given the key driving role of fibrosis and epithelial-mesenchymal transition (EMT) in cancer progression and metastasis, this study constructed a three-component platinum (IV) compound with captopril (CTP) and valproic acid (VPA) as functional ligands. This compound can effectively reverse EMT and tumor fibrosis, providing a new approach for developing novel drugs with both anti-proliferation and anti-metastatic functions.

[0049] 3. The mechanism of action of this platinum (IV) compound exhibits multi-target synergistic characteristics: the reduced platinum nucleus induces DNA damage and activates the mitochondrial apoptosis pathway; VPA inhibits HDAC3 and regulates P-gp, PARP, and PTEN to block DNA repair and overcome multidrug resistance (MDR); CTP inhibits the TGF-β1 / Smad2 and MMPs cascade reaction, and downregulates COX-2, TNF-α, and IL-6 to inhibit inflammation and fibrosis. These multiple synergistic effects reverse EMT, achieving highly effective anti-tumor metastasis.

[0050] 4. This compound can enhance CD3 levels by blocking PD-L1 activation of T-cell immunity. + and CD8 +The level of T-cell tumor infiltration transforms the "cold" tumor immune microenvironment into a "hot" environment, thereby enhancing the anti-tumor immune response.

[0051] 5. The structural innovation of compounds of general formula I lays the foundation for screening highly efficient platinum-based lead molecules and opens up new pathways for the development of platinum-based drugs. Its original innovative attributes are of great significance to both theoretical research and clinical application in the field of tumor treatment. Attached Figure Description

[0052] Figure 1 Uptake of platinum-based drugs in tumor cells. 4T1 tumor cells were treated with platinum-based drugs CDDP, CDDP+6 (5 μM / 10 μM), CDDP+VPA+CTP (5 μM / 10 μM / 10 μM), and compound 1-2 (5 μM) for 24 h at 37 °C. Uptake levels were determined using the AAS method.

[0053] Figure 2 Antitumor activity of compounds 1, CDDP, and CDDP+6 in female BALB / c mice bearing 4T1 tumors (n=5). (a) Schematic diagram of experimental design. (b) Relative body weight of mice during treatment. (c) Changes in tumor growth over time. (d) Tumor weight of each group at the end of the experiment. TGI of the test drug compared with the control group = (1 - tumor weight of the drug-treated group / tumor weight of the control group) × 100%. (e) Tumor images after mouse sacrifice. (f) H&E staining images of tumors. (g) Detection of platinum accumulation in tumor tissue by atomic absorption spectrometry. (hk) ELISA determination of serum ALT, AST, BUN, and CRE levels. *p<0.05, **p<0.01, ***p<0.001.

[0054] Figure 3 H&E staining images of mouse liver, spleen, and kidney.

[0055] Figure 4 Evaluation of the in vitro and in vivo anti-metastatic properties of platinum(IV) compound 1. (a, b) In vitro transwell assays to evaluate the inhibitory effects of compound 1, CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) on the migration of 4T1 cells. In vivo inhibitory effect of platinum(IV) compound 1 on lung metastases of 4T1 tumors (n=5): (c) Schematic diagram of experimental design. (d) Representative anterior and posterior photographs of the lungs in each group at the end of the experiment. (e) Statistical count of lung nodules in each group. (f) H&E staining of lung metastatic nodules. Nodules are indicated by arrows. **p<0.01, **p<0.001.

[0056] Figure 5In vitro scratch healing assays were used to evaluate the migration-inhibiting properties of compounds 1, CDDP, and CDDP+6 on 4T1 cells. 4T1 cells were treated with 5 μM platinum-based drugs at 37°C for 24 h. (a) Cell scratch images at 0 h, 12 h, and 24 h. (b) Calculation of scratch healing rates at different time points.

[0057] Figure 6 Stability of Compound 1 in RPMI 1640. A solution of Compound 1 (0.25 mM) in RPMI 1640 was prepared and monitored by HPLC for 24 hours.

[0058] Figure 7 Reduction of Compound 1 in the presence of AsA. Solutions of Compound 1 (0.25 mM) and AsA (1 mM, similar to TME) were prepared in RPMI 1640 and monitored by HPLC for 24 h to determine its reduction potential in reducing biological media.

[0059] Figure 8 DNA binding capacity after reduction of compound 1. An RPMI 1640 solution containing compound 1 (0.25 mM) was prepared, and AsA (1 mM) and 5′-GMP (3 mM) were added. The solution was incubated at 37 °C for 24 h, and the results were detected by LC-MS. (a) The process of reduction of compound 1 releasing platinum(II), VPA, and CTP. (b) Mass spectra of platinum-GMP, VPA, and CTP in solution.

[0060] Figure 9 4T1 tumor cells were treated with platinum-based drugs CDDP (5 μM), CDDP+6 (5 μM / 10 μM) and compound 1 (5 μM) at 37 °C for 24 h. The sub-distribution of platinum in cellular DNA, cytoplasm and cell membrane was detected by AAS.

[0061] Figure 10 Western blot analysis of γ-H2AX and p53 expression. 4T1 cells were incubated with compound 1 (5 μM), CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) for 24 h. (a) Blots. (b) Relative grayscale analysis. ***p<0.001.

[0062] Figure 11Western blot and immunohistochemical detection of the mechanism by which CTP-VPA platinum (IV) compounds overcome MDR. Compound 1 (5 μM), CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) were added to 4T1 cells at 37 °C and incubated for 24 h. (a) Western blot images of HDAC3, P-gp, PARP, and PTEN. (b) Western blot data analysis. (c) Immunohistochemical staining of HDAC3 in tumor tissue during in vivo antitumor experiments. (d) Quantitative data analysis of immunohistochemical staining. ***p<0.001.

[0063] Figure 12 Apoptosis of 4T1 cells after co-incubation with the compounds for 24 hours. (a) Apoptosis rates of blank, compound 1 (5 μM), CDDP (5 μM), and CDDP+6 mixture (5 μM / 10 μM) groups were detected by flow cytometry using annexin V-FITC / PI staining; (b) Western blot results of Bcl-2, Bax, caspase 3, and c-caspase 3 in 4T1 cells; (c) Protein expression data analysis. *P<0.001.

[0064] Figure 13 After co-incubating the compound with 4T1 cells for 24 hours, the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. (a) Blank. (b) CDDP (5 μM). (c) CDDP+6 (5 μM / 10 μM) mixture. (d) Compound 1 (5 μM).

[0065] Figure 14 The antitumor fibrosis properties of compound 1. (a) Immunohistochemical staining of α-SMA and FSP1 in in vivo antitumor experimental tissues, and Picrosirius red and Masson staining. (b) Quantitative data. **P<0.01, ***P<0.001.

[0066] Figure 15Western blot and immunohistochemical detection of the regulation of TGF-β1 / Smad2 and MMPs by CTP-VPA platinum (IV) compounds. Compound 1 (5 μM), CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) were added to 4T1 cells at 37 °C and incubated for 24 h. (a) Western blot images of TGF-β1, Smad2, p-Smad2, MMP2, and MMP9. (b) Western blot data analysis. (c) Immunohistochemical staining of TGF-β1 and MMP9 in tumor tissues during in vivo antitumor experiments. (d) Quantitative analysis of immunohistochemical staining data. ***p<0.001.

[0067] Figure 16 Compound 1 (5 μM), CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) were added to 4T1 cells at 37 °C and incubated for 24 h. (a) Western blot images of COX-2, TNF-α, and IL-6. (b) Western blot data analysis. P < 0.001.

[0068] Figure 17 Western blot and immunohistochemical detection of the function of CTP-VPA platinum (IV) compounds in regulating EMT. Compound 1 (5 μM), CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) were added to 4T1 cells at 37 °C and incubated for 24 h. (a) Western blot images of E-cadherin, N-cadherin, Snail1, and Vimentin. (b) Western blot data analysis. (c) Immunohistochemical staining of E-cadherin and N-cadherin in tumor tissue during in vivo antitumor experiments. (d) Quantitative data analysis of immunohistochemical staining. ***p<0.001.

[0069] Figure 18 Immunomodulatory effects of compounds. Compound 1 (5 μM), CDDP (5 μM), and CDDP+6 (5 μM / 10 μM) were added to 4T1 cells at 37 °C and incubated for 24 h. (a) Western blot images of PD-L1. (b) Western blot data analysis. (c) PD-L1 and CD3+ in tumor tissue during in vivo antitumor experiments. + and CD8 + T-cell immunohistochemical staining. (d) Quantitative data analysis of immunohistochemical staining. ***p<0.001. Detailed Implementation

[0070] The technical solutions of the present invention will be further described in conjunction with specific embodiments. However, the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be 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 embodiments have the same meaning as commonly understood by those skilled in the art; the test reagents used, unless otherwise specified, are all conventional biochemical reagents; and the experimental methods, unless otherwise specified, are all conventional methods.

[0071] To make the objectives, technical solutions, and advantages of the present invention clearer, representative embodiments of the present invention will be described in detail below, but are not limited thereto.

[0072] Example 1.

[0073] I. Preparation of tetravalent platinum as shown in structural formula II

[0074] 1. Synthesis of dihydroxycisplatin(IV)IIa

[0075]

[0076] In a 250 mL round-bottom flask, add 1.0 g of cisplatin and 30 mL of distilled water. Under stirring, slowly add 50 mL of 30% hydrogen peroxide dropwise to the reaction system. Raise the temperature to 60 °C and continue stirring for 4 hours. After the reaction is complete, crystallize at 4 °C for 12 hours. Filter to obtain a yellow crude solid, which, upon recrystallization from pure water, yields 0.78 g of yellow crystals of IIa, with a yield of 70%.

[0077] 2. Synthesis of dihydroxyoxaliplatin(IV)IIb

[0078]

[0079] In a 250 mL round-bottom flask, add 1.0 g of oxaliplatin and 30 mL of distilled water. Under stirring, slowly add 50 mL of 30% hydrogen peroxide dropwise to the reaction system. Raise the temperature to 60 °C and continue stirring for 4 hours. After the reaction is complete, crystallize at 4 °C for 12 hours. Filter to obtain a white crude solid, which, upon recrystallization from pure water, yields 0.68 g of white crystals of IIb, with a yield of 63%.

[0080] II. Preparation of valproic acid-captopril tetravalent platinum as shown in structural formula I

[0081] 1. Preparation of symmetrical valproic acid-captopril tetravalent platinum compound 1

[0082]

[0083] Compound 6 (309 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 15 min. Then, triethylamine (TEA, 125 μL, 0.90 mmol) was added, and the mixture was stirred for another 15 min. Platinum(IV)IIa (100 mg, 0.30 mmol) was added, and the mixture was kept in a nitrogen atmosphere at 50 °C for 48 h in the dark. After the reaction was complete, the solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to give compound 1 as a yellow solid (109 mg, 37%). The purity of the final product was determined to be 98.9% by high-performance liquid chromatography (HPLC) using a methanol / water (70 / 30) solvent system.

[0084] 1 H NMR(500MHz,DMSO-d6)δ6.39(br,6H,NH3),4.47–4.23(m,2H),3.54–3.45(m,2H),3.03–2.89(m,4H),2.79–2.69(m,2H),2.59–2.5 3(m,2H),2.22–1.71(m,8H),1.59–1.46(m,4H),1.42–1.33(m,4H),1.31–1.15(m,10H),1.13–0.94(m,6H),0.84(t,J=7.4Hz,12H). 13 C NMR (126MHz, DMSO) δ202.6,179.5,172.2,59.3,53.1,46.3,37.3,34.6,31.1,28.9,23.9,19.8,16.5,13.8.MS-ESI:calcd for[M+H] + :984(M=C 34 H 62 Cl2N4O8PtS2),found:984.HRMS:calcd for[M+Na] + :1006.2932(M=C 34 H 62 Cl2N4O8PtS2), found: 1006.2923.

[0085] 2. Preparation of asymmetric valproic acid-captopril tetravalent platinum compound 2

[0086]

[0087] Compound 6 (103 mg, 0.3 mmol) and TBTU (96 mg, 0.3 mmol) were dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 15 min. Then, TEA (42 μL, 0.3 mmol) was added, and the mixture was stirred for another 15 min. Platinum(IV)IIa (100 mg, 0.30 mmol) was added. The reaction mixture was kept at 50 °C under a nitrogen atmosphere for 48 h, protected from light. After the reaction was complete, the solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to give compound 1 as a yellow solid (61.3 mg, 31%). The purity of the final product was determined to be 97.1% using a methanol / water (70 / 30) solvent system by high-performance liquid chromatography.

[0088] 1 H NMR(500MHz,DMSO-d6)δ6.56(br,1H,OH),6.06–5.66(m,6H,NH3),4.52–4.17(m,1H),3.54-3.47(m,1H),3.11-2.94(m,2H),2.80–2.72(m,1H) ),2.60–2.54(m,1H),2.09–1.71(m,4H),1.57–1.42(m,2H),1.41–1.32(m,2H),1.27–1.19(m,5H),1.13–0.96(m,3H),0.84(t,J=7.2Hz,6H). 13 C NMR (126MHz, DMSO) δ173.9,173.3,172.5,58.5,58.2,46.4,45.9,41.0,30.7,28.6,27.0,24.3,22.0,16.4.MS-ESI:calcd for[MH] - :658(M=C 17 H 35 Cl2N3O5PtS),found:658.HRMS:calcd for[M+Na] + :681.1220(M=C 17 H 35 Cl2N3O5PtS), found: 681.1212.

[0089] III. Experimental Testing

[0090] To better understand the essence of this invention, the following pharmacological experimental results demonstrating the inhibitory effects of the compounds on tumors in in vivo and in vitro experiments illustrate the potential uses of these compounds in the pharmaceutical field. The pharmacological experiments provide partial activity data for some compounds. It must be noted that the pharmacological experiments of this invention are for illustrative purposes only and not for limiting the invention. Simple modifications to this invention based on its essence are all within the scope of protection of this invention.

[0091] 1. In vitro antitumor activity experiment

[0092] This experiment used the MTT assay to determine cell viability, based on the half-inhibitory concentration (IC50) of each test sample on cell growth. 50 The in vitro anticancer activity of a compound is measured by its in vitro anticancer value.

[0093] 100 μL of tumor cells in logarithmic growth phase were seeded into 96-well plates at a density of 5000-8000 cells / well, with the last well reserved for zeroing. The plates were incubated at 37°C for 12 h. Then, 100 μL of a gradient concentration of compound culture medium was added to each well, and the plates were incubated at 37°C for another 48 h. 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated at 37°C for 4 h. The culture medium was then removed, and 150 μL of DMSO was added. The plates were then shaken at 37°C for 20 min in the dark. The absorbance (OD) of each well was measured at 490 nm using an ELISA reader, and the IC50 was calculated. 50 Value. Each experiment should be repeated at least three times.

[0094] The cancer cell lines used in this experiment included: human lung adenocarcinoma cells A549, cisplatin-resistant lung adenocarcinoma cells A549R, mouse breast cancer cells 4T1, human liver cancer cells HepG2, and normal human liver cells LO-2.

[0095] The control compounds included the classic platinum(II) drugs CDDP and oxaliplatin (OXP), and the functional ligands VPA and CTP were tested. Furthermore, the antitumor proliferative effects of CDDP in combination with acid 6 (CDDP+6) and CDDP in a mixture of VPA and CTP (CDDP+VPA+CTP) were evaluated.

[0096] Discussion of antitumor activity:

[0097] Table 1. Antitumor activity of platinum (IV) compounds 1-4. Reference drugs were used: acid 6, VPA, CTP, CDDP, CDDP+6, and CDDP+VPA+CTP. After 48 hours of drug treatment, the half-maximal inhibitory concentration (IC50) was obtained through three parallel experiments. 50 (μM).

[0098]

[0099]

[0100] Table 1 a RF: Drug resistance coefficient, 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-CTZ: A mixture of cisplatin and CTZ in a molar ratio of 1:2.

[0101] Table 1 shows that the CTP-VPA platinum (IV) compounds exhibited effective antitumor activity against all tested tumor cell lines; in particular, the dual-ligand compound 1 showed significantly better antitumor activity than the single-ligand compound 2, with a half-maximal inhibitory concentration (IC50) of 1 / 2. 50 The concentrations reached nanomolar levels of 0.23–0.92 μM. These results confirm that the functional ligands significantly modulate the antitumor activity of platinum (IV) compounds. Specifically, compound 1 exhibited an antitumor effect 5.0–27.3 times greater than that of CDDP. In contrast, the antitumor activity of mixtures of CDDP and functional compound 6 (CDDP+6) and mixtures of CDDP with VPA and CTP (CDDP+VPA+CTP) was similar to that of CDDP alone.

[0102] Cellular uptake is a key factor affecting anti-tumor efficacy. For example... Figure 1 As shown, the accumulation level of compound 1, which exhibits superior activity, in tumor cells was significantly higher than that of CDDP, CDDP+6, and the CDDP+VPA+CTP mixture (P<0.001). This phenomenon may be one of the main reasons for its superior antitumor activity. These results indicate that the physical mixture of CTP-VPA platinum(IV) compounds and their components may have distinctly different mechanisms of action. They also confirm that chemically bonding functional ligands to the platinum(IV) backbone to form the target CTP-VPA platinum(IV) compound is a necessary condition for enhancing antitumor activity.

[0103] Drug resistance is a core obstacle limiting the efficacy of platinum-based drugs in cancer treatment. Therefore, we assessed the resistance-overcoming ability of compound 1 by calculating the resistance factor (RF). The results showed that compound 1 possessed potent anti-resistance activity, with an RF value of 0.88, significantly lower than that of CDDP (RF = 5.14). In contrast, the RF values ​​of the physical mixtures CDDP+6 and CDDP+VPA+CTP were 3.88 and 5.49, respectively, indicating that they could not effectively reverse drug resistance. Furthermore, the in vitro toxicity of the CTP-VPA platinum(IV) compound was assessed by calculating the selectivity index (SI). Compared to CDDP (SI = 1.92), CDDP+6 (SI = 1.87), and CDDP+VPA+CTP (SI = 0.96), compound 1 had a high SI value of 5.80, suggesting that the CTP-VPA platinum(IV) system has the potential to reduce the toxic side effects of traditional platinum-based drugs.

[0104] In summary, CTP-VPA platinum (IV) compounds, especially compound 1, exhibit excellent antiproliferative activity in vitro and can effectively overcome CDDP resistance and reduce toxicity. Therefore, compound 1 has been selected as a candidate drug for further research.

[0105] 2. In vivo antitumor activity.

[0106] To evaluate in vivo antitumor activity, a 4T1 allogeneic tumor model was established (female BALB / c mice, 18-20g, purchased from Shandong Pengyue Laboratory Animal Company, and fed according to NIH guidelines). After in vitro expansion, 4T1 cells were cultured at 5 × 10⁻⁶ cells / mL. 5 One mouse was injected into the right back of each mouse. On day 3, the mice were randomly divided into 4 groups (n=6): Blank group, compound 1 group, CDDP group, and CDDP+6 group. The mice were injected intravenously with 2 mg Pt / kg three times (on days 3, 6, and 9), and tumor volume and body weight changes were monitored. On day 12, the mice were sacrificed, and serum, tumors, and organ tissues were collected for analysis.

[0107] Figure 2 In vivo experimental results showed that compound 1 had significantly better antitumor activity than CDDP: its tumor volume (139 mm) was significantly lower than that of CDDP. 3 ) much smaller than CDDP group (365mm) 3 (P<0.001) and CDDP+6 group (388mm) 3 The TGI (tumor platinum uptake) of compound 1 was 80.4% (P<0.001), significantly higher than that of the CDDP group (49.4%) and the CDDP+6 group (54.0%). Atomic absorption spectrometry confirmed that the tumor platinum uptake of compound 1 was more than 2.5 times higher than that of the CDDP group and the CDDP+6 group (P<0.001). Figure 2g), high tumor accumulation (possibly related to lipophilicity) is the key to its excellent efficacy; H&E staining showed that compound 1 can induce significant apoptosis in tumor cells.

[0108] In terms of toxicity evaluation, mice in compound 1 group showed significantly lower body weight loss than those in the CDDP group (P<0.001), and serum ALT, AST (liver-related), BUN, and CRE (kidney-related) levels were significantly lower in the compound 1 group than in the CDDP group. Figure 2 H&E staining of organs showed no obvious abnormalities (see hk), Figure 3 The results indicate that its systemic toxicity is significantly reduced, and the attenuation effect may be related to the CTP-VPA ligand.

[0109] In summary, CTP-VPA platinum(IV) compound 1 exhibits both potent antitumor activity and low systemic toxicity in vivo, and has broad application prospects.

[0110] 3. In vitro and in vivo metastasis inhibition experiments.

[0111] Platinum (II) class drugs have limited efficacy against metastatic cancer. To evaluate the anti-metastatic potential of CTP-VPA platinum (IV) compound 1, we tested its in vitro anti-metastatic activity using Transwell assays and wound healing assays. Given that lung metastasis is the main in vivo manifestation of hematogenous tumor spread, we further validated its in vivo anti-metastatic effect using a lung metastasis model.

[0112] Transwell experiments: 8μm pore size Transwell chambers were used, with 5×10⁶ seeds implanted in the upper chamber. 4 4T1 cells were resuspended in 0.2 mL of serum-free RPMI 1640. The lower chamber was filled with 10% FBS-RPMI 1640 medium containing different compounds (5 μM) and incubated at 37°C and 5% CO2 for 24 hours. After fixation with 4% paraformaldehyde and staining with 0.1% crystal violet, non-migrating cells in the upper chamber were scraped off. Migrating cells were counted in five random fields under an inverted microscope to assess anti-migrating activity.

[0113] 4. Scratch test.

[0114] 4T1 cells at 8×10 5 Inoculate 1 cell / well into a six-well plate, incubate for 12 hours until 90% confluence, then perform a scratch treatment, add 1% FBS-RPMI 1640 medium containing the compound (5 μM), and take images at 0, 12, and 24 hours to record the scratch healing degree to evaluate the anti-migration effect.

[0115] 5. In vivo anti-metastasis experiment.

[0116] Female BALB / c mice (18-20g) were injected via the tail vein with 2×10 5A lung metastasis model was constructed using 4T1 cells and randomly divided into 4 groups (n=5): Blank group, compound 1 group, CDDP group, and CDDP+6 group. Mice were administered the drug via tail vein injection at a dose of 2 mg Pt / kg three times (days 3, 6, and 9). Mice were sacrificed on day 12, and lung tissue was dissected, fixed in 4% formaldehyde, and metastatic nodules were counted and observed using H&E staining.

[0117] Transwell and wound healing assays showed that CTP-VPA platinum(IV) compound 1 exhibited significant anti-metastasis activity in vitro. This compound effectively inhibited the migration of 4T1 cells, as demonstrated in the Transwell assay. Figure 4 In the ab group, the migration rate was only 11.6% of the control group (P<0.001), significantly better than the cisplatin (CDDP) group (29.8%, P<0.001) and the physical mixture of CDDP+6 group (18.1%, P<0.001). Wound healing experiment results ( Figure 5 Further investigation confirmed that the wound healing rate of the compound 1 treatment group was significantly lower than that of the CDDP group and the CDDP+6 group (P<0.001), which verified its in vitro anti-migration efficacy from another dimension.

[0118] To evaluate the in vivo anti-metastatic effect of compound 1, we successfully constructed a lung metastasis model in female BALB / c mice. For example... Figure 4 As shown in the ce diagram, the number of metastatic nodules on the surface of the lung tissue of mice treated with compound 1 was only 16.8% of that in the blank control group (P<0.001), significantly lower than that in the CDDP group (60.8%, P<0.001) and the CDDP+6 group (48.6%, P<0.001). H&E staining results of lung tissue ( Figure 4 f) Further, it was shown that compared with the blank group, CDDP group, and CDDP+6 group, the compound 1 treatment group had fewer and smaller metastatic nodules formed in the lung tissue. In summary, CTP-VPA platinum (IV) compound 1, as a novel and highly effective anti-metastatic drug, shows great development potential and is worthy of further in-depth research.

[0119] 6. It is reduced in the tumor microenvironment and causes DNA damage.

[0120] Biostability and Reduction-Release Properties of Compound 1: Platinum (IV) compounds are generally stable during transport in biological media, but readily undergo reduction under the reducing conditions of the tumor microenvironment (TME). To verify this property, the biostability of compound 1 was evaluated by HPLC at 37°C. The results showed that it remained stable in RPMI 1640 medium for at least 48 hours. Figure 6As a three-component molecule, compound 1 is converted to its platinum(II) form and releases the functional fragment in a reducing environment. We constructed a reducing medium with 1 mM ascorbic acid (AsA, simulating the TME reducing concentration) and monitored the reduction process by HPLC. Figure 7 As incubation proceeded, the characteristic peak of compound 1 gradually decreased, while the characteristic peak of compound 6 increased accordingly, confirming that compound 1 was reduced to divalent platinum.

[0121] DNA-binding properties of compound 1: DNA damage is a core mechanism of action for platinum-based drugs. To assess the DNA-binding ability of compound 1 after reduction, guanosine-5′-monophosphate (5′-GMP) was used as a DNA base model. A solution of compound 1 was prepared in RPMI 1640 medium containing 1 mM AsA and 3 mM 5′-GMP, and incubated for 48 hours before LC-MS monitoring. Figure 8 The presence of a Platinum(II) complex peak in the solution confirmed that the platinum(II) derivative released by the reduction of compound 1 can effectively bind to DNA; simultaneously, signal peaks of VPA and CTP were detected, indicating that the functional group 6 released by the reduction of compound 1 will be further decomposed into VPA and CTP fragments.

[0122] In summary, compound 1 remains stable in biological media, can be efficiently reduced in the reduced TME, and simultaneously releases VPA, CTP and platinum(II) fragments, and the released platinum(II) can specifically bind to DNA.

[0123] Detection of Platinum Accumulation Levels in DNA: The amount of platinum accumulated in DNA directly affects its DNA damage effect. Using CDDP and CDDP+6 as references, the accumulation level of compound 1 in 4T1 cell DNA was detected by atomic absorption spectrometry (AAS). The results showed ( Figure 9 Compound 1 accumulated significantly more DNA than the CDDP group and the CDDP+6 group (P<0.001), which is consistent with the total cellular uptake trend described above.

[0124] Western Blot Detection of DNA Damage Effects: To clarify the DNA-damaging ability of compound 1, the expression levels of DNA damage marker proteins γ-H2AX and p53 were detected by Western Blot. Western Blot results confirmed ( Figure 10 Compound 1 significantly upregulated the expression levels of γ-H2AX and P53 proteins, indicating that it can induce severe DNA damage in tumor cells.

[0125] 7. Suppress HDAC to overcome MDR.

[0126] MTT assay results confirmed that CTP-VPA platinum (IV) compound 1 can significantly overcome MDR. Abnormal upregulation of HDAC can promote DNA repair by regulating key proteins such as P-gp, PARP, and PTEN, thereby inducing MDR in tumor cells. VPA is known to be a highly effective HDAC inhibitor; therefore, this study explored the core mechanism by which compound 1 overcomes MDR by evaluating its regulatory effects on HDAC3 and key DNA repair-related proteins.

[0127] Western Blot results ( Figure 11 ab) showed that CTP-VPA platinum (IV) compound 1 effectively inhibited HDAC3 activity in tumor cells both in vitro and in vivo. In in vitro experiments, compound 1 significantly inhibited HDAC3 expression levels to 40.4% of the blank control group (P<0.001); while the physical mixture CDDP+6 showed stronger HDAC3 inhibitory activity (inhibited to 20.5% of the blank group, P<0.001), suggesting that the inhibitory effect of HDAC3 may mainly originate from the VPA fragment in the functional ligand. In vivo immunostaining results showed ( Figure 11 The expression level of HDAC3 in tumor tissue treated with compound 1 was significantly downregulated compared to the control group (P<0.001). Notably, the inhibitory effect of CDDP+6 on HDAC3 in in vivo tumor tissue was lower than that of compound 1, a trend significantly different from in vitro experiments. This is speculated to be related to the different modes of action of CTP-VPA platinum (IV) compound 1 and physical mixtures in vivo. Further studies revealed that compound 1 can significantly regulate the expression of key proteins related to DNA repair (CDP+6). Figure 11 ab): Compared with the control group, the expression levels of P-gp and PARP were significantly reduced in the compound 1 treatment group (P<0.001), while the expression level of the tumor suppressor protein PTEN was significantly upregulated (P<0.001). In summary, candidate compound 1 inhibits the activity of HDAC3 in tumor cells, thereby regulating the expression of key proteins such as P-gp, PARP, and PTEN, interfering with the DNA repair process, and ultimately effectively overcoming MDR. This characteristic is of great significance for improving its anti-tumor efficacy.

[0128] 8. Induces mitochondrial-mediated apoptosis.

[0129] Apoptosis is a major antitumor mechanism of platinum-based drugs, and mitochondria are the core regulator of apoptosis. This study evaluated the apoptosis-inducing properties and mitochondrial effects of compound 1 using Annexin V-FITC / PI double staining, JC-1 staining, and Western blotting (detecting Bcl-2, Bax, caspase 3, and c-caspase 3).

[0130] Experimental procedure: 4T1 cells (1×10⁻⁶) 6 Cells were seeded in 6-well plates (cells / well) and cultured for 12 hours. Then, the cells were treated with 5 μM of the drug for 24 hours. After digestion, the cells were collected and divided into two groups for Annexin V-FITC / PI staining (apoptosis rate was detected by flow cytometry within 1 hour) and JC-1 staining (mitochondrial membrane potential was detected by flow cytometry).

[0131] The results showed that compound 1 induced apoptosis at a rate of 29.7%, significantly higher than that in the CDDP group (14.3%) and the CDDP+6 group (14.0%). Figure 12 a), and causes severe mitochondrial damage and membrane potential collapse ( Figure 13 Western blotting confirmed ( Figure 12 Compound 1 significantly downregulated the anti-apoptotic protein Bcl-2 (P<0.001), upregulated the pro-apoptotic protein Bax (P<0.001), and increased the c-caspase3 / caspase3 ratio (P<0.001).

[0132] In summary, CTP-VPA platinum (IV) compound 1 can induce mitochondrial-mediated apoptosis by damaging mitochondria and activating the Bcl-2 / Bax / caspase3 pathway.

[0133] 9. Reduces tumor fibrosis and inhibits tumor metastasis.

[0134] Inhibitory effect of compound 1 on tumor collagen fibrosis: Tumor fibrosis is closely related to tumor cell adhesion, invasion, migration, and metastasis, and captopril (CTP) has been shown to have inhibitory activity against tumor fibrosis. This study systematically evaluated the effect of CTP-VPA platinum (IV) compound 1 on tumor fibrosis using Picrosirius Red and Masson staining combined with Western blotting to detect fibrosis-related proteins. Figure 14 Staining results showed that, compared with the blank control group, the level of collagen fibrosis in tumor sections treated with compound 1 was significantly reduced (P<0.001), and the inhibitory effect was superior to that of the platinum(II) reference drug CDDP. Western blotting results further confirmed that compound 1 could significantly inhibit the expression of key regulatory proteins of collagen fibrosis, α-SMA and FSP1 (P<0.001), indicating its good potential in alleviating tumor fibrosis.

[0135] The regulatory effect of compound 1 on the TGF-β / Smad pathway and MMPs: The TGF-β / Smad signaling pathway is a core mechanism promoting extracellular matrix (ECM) remodeling and accelerating the formation of the fibrotic tumor microenvironment (TME). This pathway can enhance the fibrotic process by regulating matrix metalloproteinases (MMPs). CTP can effectively downregulate the activity of the TGF-β / Smad pathway, while VPA, as an HDAC inhibitor, also has the potential to interfere with the TGF-β cascade reaction. In this study, the expression of TGF-β / Smad pathway and MMP-related proteins (TGF-β1, Smad2, p-Smad2, MMP2, MMP9) was detected by Western blot. The results showed ( Figure 15 Compared with the Blank group, compound 1 significantly inhibited the expression of TGF-β1, Smad2, and p-Smad2 (P<0.001), while effectively downregulating the protein levels of MMP2 and MMP9 (P<0.001). Immunohistochemical staining results further confirmed this trend: the expression levels of TGF-β1 and MMP9 in tumor tissues treated with compound 1 were significantly reduced (P<0.001), and significantly lower than those in the CDDP group and the CDDP+6 physical mixture group (P<0.001). These results indicate that compound 1 may inhibit tumor fibrosis by suppressing the TGF-β1 / Smad2 signaling pathway and reducing MMP secretion.

[0136] The regulatory effect of compound 1 on the tumor inflammatory microenvironment: The formation of the fibrotic tumor microenvironment (TME) is closely related to a chronic inflammatory environment, characterized by the high expression of COX-2 and various inflammatory factors. Literature confirms that inhibiting HDAC activity in tumor cells may disrupt tumor fibrosis by improving the inflammatory environment. This study examined the expression levels of COX-2 and the inflammatory factors TNF-α and IL-6. Figure 16 The results showed that compound 1 significantly inhibited COX-2 expression (P<0.001) and significantly downregulated TNF-α and IL-6 levels (P<0.001), with significantly better inhibitory effects than the CDDP group and the CDDP+6 group. This indicates that compound 1 can inhibit chronic inflammatory responses in tumors and thus inhibit tumor fibrosis by downregulating key inflammation-related molecules COX-2, TNF-α, and IL-6.

[0137] In summary, CTP-VPA platinum (IV) compound 1 can inhibit tumor fibrosis through a dual mechanism: on the one hand, it inhibits the TGF-β1 / Smad2 signaling pathway and MMP cascade reaction, reducing ECM remodeling; on the other hand, it downregulates the expression of inflammation-related molecules, reverses inflammatory TME, and ultimately achieves effective inhibition of tumor fibrosis.

[0138] 10. Reverse EMT to inhibit metastasis.

[0139] EMT is a core mechanism of tumor metastasis, with fibrosis and inflammatory TME being important driving factors. Based on the properties of compound 1 to reverse fibrosis and inflammatory TME, this study used Western blotting and immunohistochemistry to detect the expression of key EMT proteins E-cadherin, N-cadherin, Snail1, and Vimentin, and to assess their impact on EMT.

[0140] The results showed that compound 1 could effectively reverse EMT and significantly upregulated E-cadherin expression in 4T1 cells in vitro (P<0.001), while downregulating the expression of N-cadherin, Snail1, and Vimentin (P<0.001). Figure 17 ab). Immunohistochemical staining results further confirmed the above trend ( Figure 17 In in vivo experiments, compound 1 treatment significantly upregulated E-cadherin expression and significantly downregulated N-cadherin expression in tumor tissues (P<0.001). This effect was not only superior to the blank control group, but also significantly superior to the CDDP group and the CDDP+6 physical mixture group (P<0.001).

[0141] In summary, compound 1 effectively inhibited the EMT process of tumor cells by regulating fibrosis and inflammatory TME, thereby significantly inhibiting tumor metastasis.

[0142] 11. Activate immunosuppressive metastasis.

[0143] Immunosuppressive tumor metastasis (EMT) is a core characteristic of malignant tumors, playing a crucial role in accelerating tumor proliferation and metastasis. Tumor fibrosis forms a physical barrier that hinders T cell attack, while chronic inflammation and EMT processes promote the expression of the immune checkpoint PD-L1 and induce tumor-infiltrating lymphocytes (TILs, such as CD3+). + and CD8 + T cell depletion enhances tumor immune escape capabilities. Given that CTP-VPA platinum (IV) compound 1 has shown the potential to inhibit tumor fibrosis, reduce inflammation, and reverse EMT, this study further evaluated its regulatory effects on PD-L1 expression and T cell immune function.

[0144] Inhibitory effect of compound 1 on PD-L1 expression: Experimental results ( Figure 18The results showed that, compared with the blank control group, compound 1 significantly inhibited PD-L1 expression both in vitro and in vivo (P<0.001); while the free platinum (II) drug cisplatin (CDDP) had a weaker effect on PD-L1 expression. In addition, the physical mixture CDDP+6 also showed some effect in inhibiting PD-L1 expression, but it was significantly weaker than CTP-VPA platinum (IV) compound 1.

[0145] Activation effect of compound 1 on T cell immunity: The results of T cell immunity activation showed that in the tumor tissue of the compound 1 treatment group, CD3... + and CD8 + The infiltration level of T cells was significantly increased, increasing by 3.3 times and 6.9 times compared with the control group, respectively (P<0.001), which was significantly better than the CDDP group (P<0.001). Meanwhile, the CDDP+6 mixture also enhanced T cell immunity to some extent. This indicates that the antitumor immune activation effect of CTP-VPA platinum (IV) compound 1 is mainly attributed to the CTP-VPA functional ligand, rather than the platinum nucleus itself.

[0146] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound having a captopril-valproic acid tetravalent platinum structure, the general structural formula of which is shown in (Ⅰ): in, Selected from cisplatin or oxaliplatin; L is a hydroxyl group or 2. Compound I having a captopril-valproic acid tetravalent platinum structure as described in claim 1, characterized in that, The compounds with the tetravalent platinum structure of captopril-valproic acid are monosubstituted tetravalent platinum derivatives of captopril-valproic acid Ia and disubstituted tetravalent platinum derivatives of captopril-valproic acid Ib:

3. Compound I having a captopril-valproic acid tetravalent platinum structure as described in claim 1, characterized in that, The compound with the tetravalent platinum structure of captopril-valproic acid is selected from...

4. The method for preparing compound I having a captopril-valproic acid tetravalent platinum structure as described in any one of claims 1-2, characterized in that, The first synthetic route is as follows: Compound II and compound 6 undergo a coupling reaction to give monosubstituted captopril-valproic acid tetravalent platinum compound Ia; wherein the molar ratio of compound II to compound 6 is 1:1.0 to 1.

5. The second synthetic route is as follows: Compound II and compound 6 undergo a coupling reaction to give a symmetrically disubstituted captopril-valproic acid tetravalent platinum compound Ib; wherein the molar ratio of compound II to compound 6 is 1:2.0 to 5.

0. In the synthetic route, the preparation steps of compound 6 are as follows: Preparation of compound 6: 1H-benzotriazole (H-Bt, 28 g, 240 mmol) was dissolved in 200 mL of dichloromethane, and thionyl chloride (4 mL, 60 mmol) was added. The mixture was stirred at room temperature for 15 min, and then valproic acid (8.6 g, 60 mmol) was added, and the reaction was continued at room temperature for 2.5 h. After filtration, the organic phase was washed with saturated Na₂CO₃ solution, and 200 mL of hexane was added to precipitate a white solid. The solid was filtered to obtain compound 5 (6 g, 43%). Compound 5 (0.65 g, 2.3 mmol) was dissolved in 20 mL of acetonitrile, and 10 mL of an aqueous solution of CTP (0.5 g, 2.3 mmol) and triethylamine (0.23 g, 2.3 mmol) were added. The mixture was stirred for 20 h. After the reaction was complete, the acetonitrile was removed by vacuum distillation. The residue was extracted with ethyl acetate, evaporated, and concentrated to obtain a white solid, compound 6 (0.31 g, 39.3%).

5. The method for preparing compound I having a captopril-valproic acid tetravalent platinum structure as described in claim 4, characterized in that, In an inert gas atmosphere, compound 6, condensing agent, and organic base are dissolved in an anhydrous organic solvent and reacted. Compound II is added, and after reacting in the dark, the product is separated by post-treatment to obtain monosubstituted captopril-valproic acid tetravalent platinum derivative Ia or disubstituted captopril-valproic acid tetravalent platinum derivative Ib. The molar ratio of compound II, compound 6, condensing agent, and organic base is 1:1.0-1.5:1.0-1.5:1.0-1.5; the feeding relationship between compound II and organic solvent is that 1g of compound II corresponds to 10-100ml of organic solvent, to obtain Ia; The molar ratio of compound II, compound 6, 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 1g of compound II corresponds to 10~100ml of organic solvent, to obtain Ib.

6. The method for preparing compound I having a captopril-valproic acid tetravalent platinum structure as described in claim 5, characterized in that, 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.

7. The use of compound I having a tetravalent platinum captopril-valproic acid structure as described in any one of claims 1-3 in the preparation of antitumor drugs, for use in antitumor proliferation or antitumor metastasis drugs.

8. The use of compound I having a tetravalent platinum captopril-valproic acid structure as described in claim 7 in the preparation of antitumor drugs, characterized in that, The anti-tumor activity is anti-lung cancer, anti-drug-resistant lung cancer, anti-liver cancer, or anti-breast cancer; wherein, the anti-tumor proliferation activity is anti-human lung adenocarcinoma, anti-cisplatin-resistant human lung adenocarcinoma, anti-human liver cancer, or anti-mouse breast cancer; and the anti-tumor metastasis activity is anti-mouse breast cancer.

9. A combination formulation comprising a compound or pharmaceutical composition as shown in Formula I, and a combination of a paclitaxel, fluorouracil, gemcitabine, vincristine, or antibody-based antitumor drug.

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