A derivative having a tetrahydroberberine tetravalent platinum structure, and a preparation method and use thereof

Abstract

The application provides a tetrahydroberberine platinum (IV) compound with strong anti-tumor proliferation and anti-tumor metastasis properties, a preparation method and uses thereof, and the compound with tetrahydroberberine tetravalent platinum is shown as a general formula; wherein cisplatin or oxaliplatin is selected; wherein R 3 is selected from propyl, butyl or pentyl. The tetrahydroberberine platinum (IV) complex disclosed by the application plays the effects of chemotherapy and immunotherapy by inducing DNA damage, inhibiting epithelial-mesenchymal transition (EMT) and activating immunity, and further plays the activities of anti-tumor proliferation and anti-tumor metastasis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a derivative having a tetrahydroberberine tetravalent platinum structure, its preparation method and its uses. Background Technology

[0002] Cancer remains a leading cause of unnatural death in humans. Chemotherapy is a primary treatment for malignant tumors, providing long-term clinical benefits for cancer patients. However, mounting evidence suggests that the toxic side effects of conventional chemotherapy are a contributing factor to the formation of a metastatic tumor microenvironment (TME) and promote tumor metastasis. Chemotherapy-induced metastasis has become a leading cause of death in cancer patients, accounting for approximately 90% of deaths. Therefore, preventing or reversing metastatic drivers during chemotherapy is a crucial strategy for designing novel anti-tumor proliferation and anti-metastatic drugs.

[0003] Epithelial-mesenchymal transition (EMT) and immunosuppression are key drivers of tumor metastasis. EMT is a crucial process in cancer progression, causing tumor cells to lose inherent epithelial cell characteristics such as polarity and tight junctions, and is a core marker of tumor migration. Simultaneously, immunosuppression in the tumor microenvironment is also a major characteristic of cancer, promoting cancer cell evasion of immune surveillance and accelerating chemotherapy-driven metastasis. The feedback loop between EMT and immunosuppression has been validated in tumors: EMT promotes the expression of immunosuppressive checkpoint molecules, while immunosuppressive factors further accelerate tumor cell EMT. Subsequently, this feedback loop stimulates angiogenesis by promoting hypoxia and increasing the secretion of vascular endothelial growth factor, thereby leading to tumor cell metastasis. Therefore, simultaneously inhibiting EMT and activating the immune response of tumor cells offers new hope for chemotherapy in metastatic tumors.

[0004] Platinum(II) drugs are the cornerstone of cancer chemotherapy, exerting their anti-tumor effects by damaging DNA. However, their efficacy against metastatic cancer is quite limited in clinical practice. Increasing evidence suggests that long-term use of platinum(II) drugs can induce EMT transformation in tumor cells, cause inflammatory TME, thereby suppressing immunity and increasing the risk of tumor metastasis. Developing novel platinum-based drugs with EMT-inhibiting and immunomodulatory properties may be a feasible approach to overcome the inherent drawbacks of traditional platinum(II) drugs and improve their anti-metastatic capabilities. Platinum(IV) compounds, as prodrugs for platinum(II) drugs, contain two functional ligands in their structure, making them easy to modify and providing an effective strategy for the development of the aforementioned multifunctional platinum(IV) compounds.

[0005] Natural products are an important chemical resource for drug discovery. Among them, isoquinoline alkaloids possess various pharmacological properties, including anticancer and anti-inflammatory effects. Tetrahydroberberine and its derivatives have been shown to have significant potential for anti-proliferation and anti-metastasis, and are unique isoquinoline alkaloids. Studies have found that tetrahydroberberine exerts its antitumor activity through multiple mechanisms, inhibiting the EMT process and further significantly suppressing tumor metastasis. In addition, tetrahydroberberine derivatives can also regulate immunity by blocking the immune checkpoint PD-1 / PD-L1 axis and CD47, thereby inducing macrophage polarization and T cell activation in the TME. Therefore, introducing tetrahydroberberine into the platinum (IV) system holds promise for obtaining novel platinum (IV) drugs with anti-tumor proliferation and anti-metastasis properties. Summary of the Invention

[0006] In view of this, this invention proposes a compound with a tetrahydroberberine tetravalent platinum structure. By introducing the tetrahydroberberine molecule into the tetravalent platinum core, a series of novel tetravalent platinum compounds modified with tetrahydroberberine derivatives were synthesized. These derivatives exhibit good therapeutic effects against tumor proliferation and metastasis. Mechanistic studies have revealed that these compounds exert dual effects of chemotherapy and immunotherapy by inducing DNA damage, inhibiting epithelial-mesenchymal transition (EMT), and activating immunity, thereby exerting anti-tumor proliferation and anti-tumor metastasis activities. This provides a new direction for the development of novel platinum-based antitumor drugs, especially drugs for metastatic malignant tumors. This invention also provides a method for preparing compounds with a tetrahydroberberine tetravalent platinum structure and their use in antitumor drugs.

[0007] To achieve the above objectives, the present invention provides a compound having a tetrahydroberberine tetravalent platinum structure, the technical solution of which is as follows:

[0008] A compound as shown in general formula (Ⅰ):

[0009]

[0010] in, Selected from cisplatin or oxaliplatin;

[0011] Among them, R 3 Selected from propyltrimethyl, butyltrimethyl, or pentyltrimethylmethyl.

[0012] In another aspect, the present invention provides a method for preparing a compound as shown in general formula (I), wherein the synthetic route of the compound is as follows:

[0013]

[0014] Compound 3 and compound 4 undergo a coupling reaction to obtain tetrahydroberberine-modified tetravalent platinum compound 1; wherein the molar ratio of compound 3 to compound 4 is 1:2.0 to 4.0.

[0015] Furthermore, in the synthetic route, the preparation steps are as follows:

[0016] In an inert gas atmosphere, compound 4, condensing agent, and organic base were dissolved in an anhydrous organic solvent and reacted. Compound 3 was added, and after reacting in the dark, compound 1 was obtained by post-treatment.

[0017] The molar ratio of compound 3, compound 4, condensing agent, and organic base is 1:2.0~4.0:2.0~4.0:2.0~4.0; the feeding relationship between compound 3 and organic solvent is that 30~80ml of organic solvent is added for every 1g of compound 3.

[0018] The inert gas in this invention 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.

[0019] The specific preparation process is as follows: TBTU and compound 4 (tetrahydroberberine derivative) are added to the reaction vessel, the air in the system is replaced with nitrogen, anhydrous DMF is added, the reaction is stirred at room temperature, anhydrous triethylamine is added to the reaction system, the reaction is stirred at room temperature, tetravalent platinum compound 3 is added to the reaction system, the air in the system is replaced with nitrogen again, and the reaction system is placed at 25-120℃ in the dark for 12-72 hours. After the reaction is completed, the solvent is removed under reduced pressure, and the tetrahydroberberine-modified tetravalent platinum compound 1 is obtained by column chromatography.

[0020] Furthermore, compound 3 is composed of It is prepared by oxidation with hydrogen peroxide.

[0021] The preparation process can be specifically as follows:

[0022]

[0023] Divalent platinum compounds Dihydroxytetravalent platinum compound 3 was prepared by oxidation with hydrogen peroxide at 60–70 °C for 1–8 hours.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising an effective therapeutic amount of a compound as shown in general formula (I), and pharmaceutically acceptable excipients thereof.

[0025] Pharmaceutically acceptable excipients in this invention include one or more of the following: carriers, excipients, and diluents; such as binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorants, flavoring agents, preservatives, solvents, and matrices. Pharmaceutically acceptable excipients can be aqueous or non-aqueous. Conventional excipients include gums, such as gelatin; starches, such as corn starch and potato starch; sugars, such as lactose, glucose, and sucrose; and cellulose materials and mixtures thereof, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate. Pharmaceutically acceptable excipients include, but are not limited to, tragacanth gum 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, glycerin, 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), alginate, pyrogen-free water, isotonic saline, and Ringer's solution.

[0026] The tetrahydroberberine tetravalent platinum compound or pharmaceutical composition described in this invention is administered in dosage forms such as tablets, capsules, aerosols, dispersible tablets, oral liquids, suppositories, drop pills, large-volume infusions, small injections, lyophilized powder injections, ointments, or liniments, including various sustained-release, controlled-release, or nano-formulations prepared using currently accepted pharmaceutical knowledge.

[0027] The tetrahydroberberine 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.

[0028] The tetrahydroberberine tetravalent platinum compound of the present invention can be administered intravenously. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, and acupoint injection.

[0029] 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.

[0030] The present invention also provides the use of compounds or pharmaceutical compositions of general formula (I) in the preparation of drugs for antitumor proliferation and antitumor metastasis.

[0031] The tetrahydroberberine tetravalent platinum derivative described in this invention can effectively inhibit EMT, activate tumor immune response, and has excellent anti-tumor proliferation and anti-metastasis activity, and has a good therapeutic effect on malignant tumors.

[0032] Furthermore, the anti-tumor proliferation effect is against human lung adenocarcinoma, against cisplatin-resistant human lung adenocarcinoma, against human liver cancer, and against mouse breast cancer. The anti-tumor metastasis effect is against mouse breast cancer cells.

[0033] Furthermore, the invention provides a combination formulation comprising a compound or pharmaceutical composition as shown in general formula (I) and other types of antitumor drugs.

[0034] The tetrahydroberberine derivative tetravalent platinum compounds described in this invention are expected to be used alone or in combination with commercially available platinum compounds, 5-fluorouracil compounds, paclitaxel compounds, etc., 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.

[0035] Compared with existing technologies, the compound with a tetrahydroberberine tetravalent platinum structure, its preparation method, and its uses described in this invention have the following advantages:

[0036] (1) In this invention, tetrahydroberberine molecules are introduced into the tetravalent platinum core to synthesize a series of novel tetrahydroberberine tetravalent platinum structure derivatives. Antitumor activity tests have shown that they have good antitumor and anticancer abilities. These tetrahydroberberine tetravalent platinum derivatives have good therapeutic effects on malignant tumors and have excellent antitumor proliferation and anti-metastasis activities.

[0037] (2) Tetrahydroberberine tetravalent platinum structure derivatives can induce severe DNA damage in tumor cells and induce mitochondrial-mediated apoptosis, thereby exerting anti-tumor effects.

[0038] (3) The antitumor proliferation and antitumor metastasis activities of tetrahydroberberine tetravalent platinum derivatives are related to the inhibition of epithelial-mesenchymal transition (EMT) in tumor cells. These tetrahydroberberine tetravalent platinum derivatives can significantly inhibit the Wnt / β-Catenin pathway, reverse the inflammatory environment, and inhibit the HIF-1α pathway, thereby inhibiting the EMT process and inhibiting tumor angiogenesis, thus exerting significant antitumor proliferation and anti-metastasis activities;

[0039] (4) The antitumor activity of tetrahydroberberine tetravalent platinum derivatives is related to the activation of immune responses within tumor cells. These tetrahydroberberine tetravalent platinum derivatives can activate tumor immunity by inhibiting immune checkpoints PD-L1 and CD47. By inhibiting PD-L1, CD37 is activated. + and CD8 +T-cell immunity; by inhibiting CD47, it induces the transformation of macrophages from M2 to M1, thereby activating immunity;

[0040] (5) The innovative compound structure of Formula I described in this invention is expected to yield a variety of lead molecules effective against tumors, providing new candidate drug molecules to address the shortcomings of traditional divalent platinum drugs, and opening up new avenues for the modification of tetravalent platinum compounds. This invention represents innovative drug research and is of great significance to human health and socio-economic development. Attached Figure Description

[0041] Figure 1 In vivo antitumor activity of compounds 1-3, CDDP, and OXP against 4T1 tumors in female BALB / c mice (n=6); *p<0.05, ***p<0.001; (a) Schematic diagram of experimental design; (b) Functional relationship of tumor growth over time; (c) Tumor weight of each group at the end of the experiment; TGI of the test drugs compared with the control group is marked in red [TGI = (1 - tumor weight of the drug treatment group / tumor weight of the control group) × 100%]; (d) Tumor images after mouse sacrifice; (e) Relative body weight of mice during treatment; (f) H&E staining of tumor tissue.

[0042] Figure 2 Organ indices (heart, liver, spleen, lung, kidney) in BALB / c mice treated with compounds 1-3, CDDP, and OXP, and in the blank control group (n=5); Organ index = organ weight / body weight × 100%; ***P<0.001.

[0043] Figure 3 H&E staining of liver, spleen and kidney tissues of mice in blank, CDDP, OXP and complex 1-3 treatment groups.

[0044] Figure 4 Transwell assays were used to evaluate the inhibitory properties of complexes 1–3, CDDP, and OXP (4 μM) on 4T1 cell migration after tumor cells were treated with the drugs for 24 hours; (a) representative images; (b) relative migration analysis; ***P<0.001.

[0045] Figure 5 The scratch healing assay was used to detect the inhibitory effects of complexes 1-3, CDDP, and OXP (4 μM) on the migration of 4T1 cells in vitro. (a) The degree of scratch healing was observed at 0, 12, and 24 hours. (b) Scratch closure analysis; *P<0.05, **P<0.01, ***P<0.001, ns: no significant difference compared with the control group.

[0046] Figure 6Inhibitory effects of CDDP, OXP, and compounds 1-3 on lung metastases of 4T1 tumors in vivo (n=5). ***P<0.001. (a) Schematic diagram of experimental design; (b) Representative photographs of the front and back of the lungs in each group at the end of the experiment; (c) Statistical count of lung nodules in each group. The inhibition rate compared with the control group is listed above; (d) H&E staining of lung metastatic nodules; nodules are indicated by arrows.

[0047] Figure 7 Platinum accumulation in 4T1 cells in vitro and in vivo in 4T1 tumor tissues. (a) Platinum content in whole cells of tumor cells treated with complex 1-1-1-4, CDDP, and OXP; (b) Platinum content in tumor tissues treated with complex 1-3, CDDP, and OXP in vivo; (c) Subcellular distribution of platinum in cell membrane, cytoplasm, and DNA of tumor cells treated with complex 1-1-1-4, CDDP, and OXP; **p<0.01, ***p<0.001.

[0048] Figure 8 Quantitative analysis of 4T1 cell apoptosis was performed by flow cytometry using Annexin V-FITC / PI staining. Tumor cells were cultured with platinum complex at 37°C for 24 hours. (a) Blank; (b) CDDP (10 μM); (c) OXP (10 μM); (d) Compounds 1-3 (10 μM); (e) Stacking column.

[0049] Figure 9 Mitochondrial membrane potential (ΔΨm) was analyzed by flow cytometry. 4T1 cells were treated with a platinum complex at 37°C for 24 hours and stained with JC-1. (a) Blank; (b) CDDP (10 μM); (c) OXP (10 μM); (d) 1–3 (10 μM).

[0050] Figure 10 ROS in 4T1 cells were stained with DCFH-DA. Tumor cells were treated with CDDP (10 μM), OXP (10 μM), and complex 1-3 (10 μM) at 37 °C for 24 h. (a) Representative images; (b) Statistical analysis of fluorescence intensity by flow cytometry; ***P<0.001.

[0051] Figure 11 The expression of Bcl-2, Bax, caspase 3, and c-caspase 3 in tumor cells was determined by Western blotting. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative grayscale intensity analysis; ***P<0.001.

[0052] Figure 12 HPLC was used to detect the ability of compounds 1-3 to be reduced and induce DNA damage. (a) Stability of complex 1-3 in biological media: A solution of complex 1-3 (0.5 mM) in RPMI 1640 was prepared and stored at 37 °C for 48 hours; (b) Reduction ability of complex 1-3: An RPMI 1640 solution containing complex 1-3 (0.5 mmol) and ascorbic acid (AsA, 1 mmol, similar to TME) was prepared and incubated at 37 °C for 48 hours; (c) DNA binding ability of complex 1-3: An RPMI 1640 solution containing ascorbic acid (AsA, 1 mM), 5'-GMP (3 mM), and complex 1-3 (0.25 mM) was prepared and incubated at 37 °C for 48 hours. Spectra were recorded at 0 hours and 48 hours. The adduct of cisplatin and 5'-GMP, Platinate GMP, was verified by ESI-MS.

[0053] Figure 13 The expression of γ-H2AX and P53 in tumor cells was determined by Western blot. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative gray intensity analysis; ***P<0.001.

[0054] Figure 14 The expression of COX-2 and MMP-9 in tumor cells was determined by Western blotting. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative grayscale intensity analysis; ***P<0.001.

[0055] Figure 15 Immunohistochemical staining of COX-2 in tumor tissues in vivo. (a) Representative photographs; (b) Quantitative data; ***P<0.001.

[0056] Figure 16 The expression of E-cadherin, N-cadherin, Vimentin, and Snail1 in tumor cells was determined by Western blot. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative grayscale intensity analysis; ***P<0.001.

[0057] Figure 17Immunohistochemical staining of E-cadherin and N-cadherin in tumor tissues in vivo. (a) Representative micrographs; (b) Quantitative data; ***P<0.001.

[0058] Figure 18 The expression of β-catenin, cylcin D1, and c-Myc in tumor cells was determined by Western blot. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative gray intensity analysis; ***P<0.001.

[0059] Figure 19 The expression of HIF-1α and VEGFA in tumor cells was determined by Western blot. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative gray intensity analysis; ***P<0.001.

[0060] Figure 20 Immunohistochemical staining of CD34 in tumor tissue in vivo. (a) Representative micrographs; (b) Quantitative data; ***P<0.001.

[0061] Figure 21 In vivo antitumor immunity of compounds 1-3, CDDP, and OXP in BALB / c mice (n=5) with bilateral 4T1 tumors; *P<0.05, **P<0.01, ***P<0.001. (a) Schematic diagram of experimental design. (b) Image of mice at the end of the experiment; (c) Relationship between the volume of primary and distant tumors and time; (d) Weight of primary and distant tumors at the end of the experiment; (e) Image of tumors at the end of the experiment.

[0062] Figure 22 The expression of PD-L1 and CD47 in tumor cells was determined by Western blot. 4T1 cells were treated with compounds 1-3 (10 μM), CDDP (10 μM), and OXP (10 μM) for 24 h. (a) Blot images; (b) Relative gray intensity analysis; ***P<0.001.

[0063] Figure 23 Immunohistochemical staining of PD-L1 expression, T cells, and M1 / M2 macrophages in tumor tissue. (a) Representative photograph; (b) PD-L1, CD3 + and CD8 + TILs, CD86 + M1 macrophages and CD206 +Quantitative data of M2 macrophages; **P<0.01, ***P<0.001.

[0064] Figure 24 Schematic diagram of the antitumor activity mechanism of the drug of this invention. Detailed Implementation

[0065] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods. The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0066] I. Preparation of tetravalent platinum shown in compound 3

[0067] 1. Synthesis of dihydroxycisplatin(IV)3-1

[0068]

[0069] Add 1.0 g of cisplatin and 30 mL of distilled water to a 250 mL round-bottom flask, stir to disperse, and slowly add 50 mL of 30% hydrogen peroxide dropwise to the reaction system. Raise the temperature to 60 °C and stir for 4 h. Stop the reaction, distill under reduced pressure to obtain a yellow solid, then add an appropriate amount of distilled water, heat to 80 °C to dissolve, and finally let it stand at 4 °C for 12 hours to crystallize. Filter to separate and obtain 3-1 yellow needle-like solid (0.82 g, 74%).

[0070] 2. Synthesis of dihydroxyoxaliplatin (IV) 3-2

[0071]

[0072] Add 1.0 g of oxaliplatin and 30 mL of distilled water to a 250 mL round-bottom flask, stir to disperse, and slowly add 50 mL of 30% hydrogen peroxide dropwise to the reaction system. Raise the temperature to 60 °C and stir for 4 h. Stop the reaction, let it crystallize at 4 °C for 12 h, filter to obtain a yellow solid, add an appropriate amount of distilled water, heat to 80 °C to dissolve, let it crystallize at 4 °C for 12 h, filter to obtain compound 3-2 white crystals (0.85 g, 78%).

[0073] II. Preparation of the tetrahydroberberine derivative shown in compound 4

[0074] 1. Preparation of tetrahydroberberine derivative 4-1

[0075]

[0076] Compound 3a (1 g, 3.07 mmol) and methyl 4-bromobutyrate (667.2 mg, 3.69 mmol) were dissolved in 10 mL of DMF. Anhydrous potassium carbonate (847.3 mg, 6.14 mmol) was then added, and the mixture was stirred for 24 h. The reaction was stopped, the solvent was removed under reduced pressure, and the solution was obtained by column chromatography as a yellow oily liquid (750 mg, 57.4%).

[0077]

[0078] Compound 3a-1 (265 mg, 0.62 mol) was dissolved in 10 mL of ethanol. Then, 5% NaOH / H2O (6.2 mL) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the resulting solution was acidified to pH 3-4 and extracted with dichloromethane. The organic phase was collected and concentrated under reduced pressure to give a pale yellow solid 4-1 (117 mg, 45.8%).

[0079] 2. Preparation of tetrahydroberberine derivative 4-2

[0080]

[0081] Compound 3a (1 g, 3.07 mmol) and methyl 5-bromopentanoate (720 mg, 3.69 mmol) were dissolved in 10 mL of DMF. Anhydrous potassium carbonate (847.3 mg, 6.14 mmol) was then added, and the mixture was stirred for 24 h. The reaction was stopped, the solvent was removed under reduced pressure, and the solution was obtained by column chromatography as a yellow oily liquid (800 mg, 59.3%).

[0082]

[0083] Compound 3a-2 (800 mg, 1.9 mmol) was dissolved in 10 mL of ethanol. Then, 5% NaOH / H2O (19 mL) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the resulting solution was acidified to pH 3-4 and extracted with dichloromethane. The organic phase was collected and concentrated under reduced pressure to give a pale yellow solid 4-2 (443 mg, 54.8%).

[0084] 3. Preparation of tetrahydroberberine derivative 4-3

[0085]

[0086] Compound 3a (1 g, 3.07 mmol) and methyl 6-bromohexanoate (772 mg, 3.69 mmol) were dissolved in 10 mL of LDM. Anhydrous potassium carbonate (847.3 mg, 6.14 mmol) was then added, and the mixture was stirred for 24 h. The reaction was stopped, the solvent was removed under reduced pressure, and the solution was obtained by column chromatography as a yellow oily liquid (905 mg, 54.3%).

[0087]

[0088] Compound 3a-3 (745.2 mg, 1.66 mol) was dissolved in 10 mL of ethanol. Then, 5% NaOH / H2O (16.6 mL) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the resulting solution was acidified to pH 3-4 and extracted with dichloromethane. The organic phase was collected and concentrated under reduced pressure to give a pale yellow solid 4-3 (383 mg, 52.5%).

[0089] III. Synthesis of the Representative Compound of this Invention

[0090] 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.

[0091] Example 1

[0092] Synthesis of Symmetrical Bistetrahydroberberine Oxaliplatin Tetravalent Platinum Compound 1-1

[0093]

[0094] Acid 4-2 (298 mg, 0.70 mmol) and TBTU (224 mg, 0.7 mmol) were dissolved in 5 mL of DMF, and the mixture was stirred at room temperature for 15 min. Then, triethylamine (97 μL, 0.7 mmol) was added, and the mixture was stirred for another 15 min. Subsequently, platinum oxide 3-2 (100 mg, 0.23 mmol) was added. The mixture was stirred vigorously at 55 °C in the dark for 72 hours. After the reaction was complete, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to give a yellow solid compound 1-1 (80.0 mg, 27.9%). The purity was determined by HPLC to be 96.3%.

[0095] Compound 1-1: 1H NMR(500MHz,Methanol-d4)δ7.03(s,4H),6.93(s,2H),6.73(s,2H),5.99–5.95(m,4H), 4.72–4.66(m,2H),4.57–4.49(m,2H),4.36–4.30(m,2H),4.13–4.06(m,4H),3.85(s,6H) ,3.79(ddd,J=11.9,5.5,2.7Hz,2H),3.68(dd,J=17.0,4.6Hz,2H),3.34(s,8H),3.26–3 .18(m,2H),3.09–2.79(m,6H),2.44–2.36(m,4H),1.87–1.73(m,8H),1.32–1.26(m,2H). 13 C NMR(126MHz,DMSO-d6,Methanol-d4)δ175.0,170.8,164.2,150.7,146.8,144.0,126.7,126.6,126.5,126.0,124.2 ,112.6,108.5,106.0,101.4,72.1,61.1,60.2,59.0,56.1,33.7,30.9,29.6,24.1,21.5,21.0,14.4.MS-ESI:calcd for[M+H] + :1246(M=C 56 H 66 N4O 16 Pt),found:1246.MS-ESI:calcd for[M] + :1246.4200,(M=C 56 H 66 N4O 16 Pt), found: 1246.4243.

[0096] Example 2

[0097] Synthesis of tetrahydroberberine derivative cisplatin tetravalent platinum compounds 1-2

[0098]

[0099] 4-1 (370 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL of DMF, and the mixture was stirred at room temperature for 15 min. Then, triethylamine (125 μL, 0.90 mmol) was added, and the mixture was stirred for another 15 min. Subsequently, platinum oxide 3-1 (100 mg, 0.30 mmol) was added. The mixture was stirred vigorously at 55 °C in the dark for 72 hours. After the reaction was complete, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to give a yellow solid compound 1-2 (70.0 mg, 20.8%). The purity was determined by HPLC to be 96.2%.

[0100] Compounds 1-2: 1 HNMR(500MHz,Methanol-d4)δ6.99(s,4H),6.89(d,J=2.7Hz,2H),6.68(s,2H),5.94(s,4H),4.57–4.51(m,2H),4.08–4.02(m,4H) ,3.84(s,6H),3.59–3.52(m,4H),3.19–3.10(m,4H),2.95–2.85(m,4H),2.41–2.35(m,4H),1.82–1.79(m,4H),1.69–1.56(m,4H). 13 C NMR (126MHz, Methanol-d4) δ176.4,150.7,147.3,147.1,144.1,127.1,125.7,125.1,124.0,123. 8,112.2,107.9,105.1,101.2,71.9,59.7,55.0,52.6,50.6,33.7,29.4,26.9,21.5.MS-ESI:calcd for[M] + :1121(M=C 46 H 54 Cl2N4O 12 Pt),found:1121.MS-ESI:calcdfor[M+H] + :1120.2841(M=C 46 H 54 Cl2N4O 12 Pt), found: 1120.2851.

[0101] Example 3

[0102] Synthesis of tetrahydroberberine derivatives cisplatin tetravalent platinum compounds 1-3

[0103]

[0104] 4-2 (286 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL of DMF, and the mixture was stirred at room temperature for 15 min. Then, triethylamine (125 μL, 0.90 mmol) was added, and the mixture was stirred for another 15 min. Subsequently, platinum oxide 3-1 (100 mg, 0.3 mmol) was added. The mixture was stirred vigorously at 55 °C in the dark for 72 hours. After the reaction was complete, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to give a yellow solid compound 1-3 (90.0 mg, 26.1%). The purity was determined by HPLC to be 98.7%.

[0105] Compounds 1-3: 1 H NMR(500MHz, Methanol-d4)δ7.12(d,J=8.3Hz,4H),7.07(d,J=8.5Hz,2H),6.88(s,2H),6.17–6.06(m,4H),4.18–4.12(m,2H),4.11–4.03(m,4H),3 .92(s,6H),3.48–3.42(m,2H),3.19–3.10(m,2H),2.68–2.62(m,8H),2.4 7–2.41(m,4H),2.14–2.05(m,2H),1.88–1.80(m,8H),1.70–1.74(m,2H). 13 C NMR(126MHz,Methanol-d4)δ176.2,150.9,147.7,147.5,144.1,125.5,124.9,124.1,123.8,122.3,1 12.7,107.9,105.1,101.4,72.0,59.8,55.0,52.1,50.3,33.2,32.9,29.4,26.1,21.3.MS-ESI:calcd for[M] + :1149(M=C 48 H 58 Cl2N4O 12 Pt),found:1149.HRMS:calcd for[M+H] + :1148.3154(M=C 48 H 58 Cl2N4O 12 Pt), found: 1148.3151.

[0106] Example 4

[0107] Synthesis of tetrahydroberberine derivatives cisplatin tetravalent platinum compounds 1-4

[0108]

[0109] 4-3 (295 mg, 0.90 mmol) and TBTU (289 mg, 0.90 mmol) were dissolved in 5 mL of DMF, and the mixture was stirred at room temperature for 15 min. Then, triethylamine (125 μL, 0.90 mmol) was added, and the mixture was stirred for another 15 min. Subsequently, platinum oxide 3-1 (100 mg, 0.3 mmol) was added. The mixture was stirred vigorously at 55 °C in the dark for 72 hours. After the reaction was complete, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to give a yellow solid compound 1-4 (81.2 mg, 23.0%). The purity was determined by HPLC to be 97.7%.

[0110] Compounds 1-4: 1 HNMR(500MHz,Methanol-d4)δ6.95(d,J=1.5Hz,4H),6.86(s,2H),6.65(s,2H),5.92(s,4H),4.44–4.36(m,2H),4.07–4.00(m,4H),3.88–3.78 (m,6H),3.51–3.44(m,4H),2.99–2.93(m,2H),2.87–2.79(m,4H),2.34 –2.22(m,6H),1.80–1.74(m,4H),1.71–1.65(m,4H),1.63–1.50(m,8H). 13 C NMR (126MHz, DMSO) δ175.0,173.8,150.3,146.2,145.9,144.0,131.4,128.8,128.0,127.9,124.0,111.6,108.5,106. 2,101.0,72.1,59.5,56.2,54.0,51.7,51.2,45.7,36.2,34.3,33.8,30.1,29.5,25.6,24.8,9.6.MS-ESI:calcdfor[M] + :1177(M=C 50 H 62 Cl2N4O 12 Pt),found:1177.HRMS:calcd for[M+H] + :1176.3467(M=C 50 H 62 Cl2N4O 12 Pt), found: 1176.3455.

[0111] IV. Experimental Testing

[0112] 1. In vitro antitumor activity experiment

[0113] Experimental Methods: Cell viability was determined using the MTT assay. The half-maximal inhibitory concentration (IC50) of each test sample on cell growth was used as the baseline. 50 The in vitro anticancer activity of the complex is measured by the α value.

[0114] 100 μL of tumor cells in logarithmic growth phase were seeded into 96-well plates at a density of 3000-5000 cells / well, with the last column reserved as a zeroing well. 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 in the dark for 20 min. The absorbance (OD) of each well was measured at 570 nm using an ELISA reader, and the IC50 was calculated. 50 Value. Each experiment should be repeated at least three times.

[0115] 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 LO2.

[0116] Discussion of antitumor activity:

[0117] Table 1 shows the in vitro antitumor proliferative activity of the compounds.

[0118]

[0119] 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-4-2: A mixture of CDDP and 4-2 in a molar ratio of 1:2.

[0120] The experimental results are shown in Table 1. The antiproliferative activity of CND-platinum(IV) complex 1-1-1-4 against four tumor cell lines—human lung cancer (A549), CDDP-resistant human lung cancer (A549R), mouse breast cancer (4T1), and human liver cancer (HepG2)—as well as a normal human liver cell line (LO2) was evaluated using the MTT assay. Platinum(II) drugs cisplatin (CDDP) and oxaliplatin (OXP), and platinum(IV) drug Satraplatin (STP, JM216) were used as reference drugs. CND, CND acid 4-2, and a mixture of CDDP and 4-2 (CDDP-4-2) were then evaluated. The results calculated from three parallel experiments after 48 hours of treatment are shown in Table 1.

[0121] All CND platinum(IV) complexes exhibited moderate to potent antitumor activity against tumor cell lines, significantly stronger than oxaloplatin 3-1, CND, and acid 4-2. These facts suggest that introducing CND into the platinum(IV) system to prepare CND platinum(IV) compounds is key to improving antitumor efficacy.

[0122] The linking group between CND and the platinum core significantly affects antitumor activity; the complex 1-3 with the butyl group exhibits superior antitumor activity compared to the complexes 1-2 and 1-4 derived from the propyl and pentyl groups. In enhancing the antitumor potency of CND-platinum(IV) complexes, the CDDP core is more promising than the OXP core; the antitumor activity of the OXP core complex 1-1 is decreased compared to the corresponding CDDP-derived complex 1-3. In summary, compound 1-3 shows the most promising antitumor activity, with an IC50 of [missing value] against all tested tumor cell lines. 50 The values ​​were all below 1.71 μM, making them more effective than the reference drugs CDDP and OXP, STP, and the mixture CDDP-4-2.

[0123] In cancer treatment, resistance of tumor cells to platinum-based drugs is a major obstacle affecting clinical efficacy. To investigate the potential of CND platinum (IV) mixtures in overcoming resistance, the IC50 resistance factor (RF = A549R) was calculated. 50 IC value / A549 50 The RF of complex 1-3 was 1.02, which was 6.44 times lower than that of CDDP (RF = 6.57). However, the mixture CDDP-4-2 had a weaker effect on resistance (RF = 4.03). These results indicate that introducing CND as a functional ligand into the platinum (IV) system to construct CND-platinum (IV) mixtures has a significant positive impact on eliminating CDDP resistance. Subsequently, we evaluated its toxicity by testing the normal hepatocyte cell line LO2 and calculated the selectivity index (SI = IC50 of LO2). 50 IC value of HepG2 50The SI value of complex 1-3 was 2.08, which was higher than that of CDDP and OXP (SI = 0.46 and 0.18, respectively), indicating its potential to reduce in vitro toxicity.

[0124] Therefore, CND platinum (IV) complexes are a promising scaffold for anticancer drug research. In particular, complexes 1-3 demonstrated favorable antitumor properties against all tested tumor cell lines, outperforming reference drugs CDDP, OXP, STP, and CDDP-4-2. Furthermore, they show great potential in overcoming drug resistance and reducing toxicity. Therefore, complexes 1-3 were selected as candidates for further evaluation.

[0125] 2. In vivo antitumor activity experiment

[0126] To evaluate the in vivo antitumor efficacy of tetrahydroberberine platinum (IV) complexes, we assessed the antitumor activity of tetravalent platinum complexes 1–3 with tetrahydroberberine ligands in female BALB / c mice bearing 4T1 tumors. Cisplatin (CDDP) and oxaliplatin (OXP) were used as positive controls, and the blank solvent treatment group served as a blank.

[0127] Experimental methods: Female BALB / c mice (18-20g) were purchased from Shandong Pengyue Laboratory Animal Breeding Co., Ltd. All animals were fed according to the National Institutes of Health (NIH) guidelines for laboratory animal care and use. The experiment was approved by the Animal Ethics Committee of Liaocheng University.

[0128] After in vitro expansion of 4T-1 cells, the cells were digested and collected, and washed three times with physiological saline. The cells were then resuspended in physiological saline solution. Tumor cells were inoculated into the right dorsal side of female BALB / c mice at a density of 5 × 10⁻⁶ cells / mL. 5 On day 4 post-inoculation, the tumors were palpable. Mice were randomly divided into four groups of six each: a blank control group, compound 1-3 groups, a CDDP group, and an OXP group, with a dosage of 2 mg Pt / kg. The drugs were administered on days 3, 6, and 9, for a total of three times, via tail vein injection. Tumor volume changes were recorded during the experiment to assess tumor growth rate; mouse weight changes were recorded to evaluate drug toxicity. Mice were sacrificed on day 10, and serum, tumor tissue, and organ tissues (heart, lung, liver, spleen, and kidney) were collected. Tumor tissues were weighed. Tissue samples were fixed in formalin and evaluated by hematoxylin and eosin (HE) staining and immunohistochemical analysis.

[0129] Figure 1 The results showed that complex 1-3 had a strong anti-tumor effect on 4T1 tumors in vivo, significantly inhibiting tumor volume to 11.8 mm on day 9. 3The tumor growth inhibition rate (TGI = 87.2%) was significantly higher than that of the reference drug CDDP (266.2 mm). 3 (TGI = 53.2%) and OXT (405.9 mm) 3 (TGI = 18.0%). Figure 1 Images stained with hematoxylin and eosin (H&E) as shown in f indicate that compounds 1-3 induced severe apoptosis in tumor cells, with severe degeneration, necrosis, and dispersal of the cell nuclei, similar to the effects of CDDP and OXP.

[0130] Then, systemic toxicity was measured by monitoring weight loss during the experiment. Figure 1 e). The results showed that complexes 1-3, which exhibited superior antitumor activity compared to CDDP, had lower toxicity than CDDP (P<0.05). Then, the organ index, the ratio of organ weight to body weight, was calculated to reflect the drug's toxicity to organs in vivo. Figure 2 Compared with the control group, the spleen, one of the most important immune organs, was significantly inhibited by platinum(II) drugs CDDP and OXP (P<0.001), while the inhibition of the spleen by platinum(IV) complex 1-3 was negligible. This may be because the drug accumulation of complex 1-3 in the spleen was reduced compared with CDDP and OXP. Figure 2 This effect on the spleen can further influence the body's immune response. Figure 3 The H&E staining results also confirmed the low toxicity of compounds 1-3. Compared with the blank group, no significant histological differences were observed in the liver, spleen and kidney tissues of the compound 1-3 treatment group.

[0131] In vivo experiments showed that the tetrahydroberberine platinum (IV) complex 1-3 exhibited potent antitumor activity against 4T1 tumors, superior to CDDP and OXP. Furthermore, it showed reduced toxicity in mice compared to the platinum (II) reference drug, possibly related to weakened splenic inhibition. Subsequently, researchers further evaluated its effects on tumor metastasis and immunity in vitro and in vivo. These findings further validate the potential of the tetrahydroberberine platinum (IV) conjugate 1-3 as an antitumor drug.

[0132] 3. In vitro and in vivo metastasis inhibition experiments

[0133] To evaluate the potential of the tetrahydroberberine platinum (IV) complex as an anti-metastasis agent, researchers examined its metastasis-inhibiting activity. Using CDDP and OXP as reference drugs, in vitro Transwell and scratch healing assays were performed, followed by further testing of its anti-metastasis activity in an in vivo lung metastasis model.

[0134] Experimental Methods: Transwell Assay: The experiment was conducted in a Transwell chamber containing micropores (8 μm pores). First, 4 T1 cells (5 × 10⁶ cells / mL) were placed in the chamber. 4 Cells were resuspended in RPMI 1640 medium and seeded in the upper chamber. 10% FBS-RPMI 1640 medium containing different compounds (1-3, CDDP, OXP: 4 μM) was added to the lower chamber. The cells were then incubated at 37°C for 24 hours in a 5% CO2 incubator. Next, the cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet for 20 minutes. Non-migrating cells in the upper chamber were gently scraped off with a cotton swab. Migrating cells on the lower surface of the chamber were photographed in five random fields using an inverted microscope to assess the anti-migrating effect of the drugs.

[0135] Scratch assay: 4T1 cells (8×10⁻⁶) were used to scratch the skin of the skin. 5 Cells were seeded in six-well cell culture plates and incubated at 37°C with 5% CO2 for 12 hours. Once the cell density reached 90%, scratch treatment was performed in each well. Cells were then treated with 1% FBS-RPMI 1640 medium (1-3, CDDP, OXP: 4 μM) containing the compounds, and the healing progress of the scratches was recorded by photographing at 0, 12, and 24 hours.

[0136] In vivo anti-metastasis experiment: BALB / c female mice (18-20g) were used as experimental subjects, and 4T1 cells (1×10⁻⁶) were injected via tail vein. 5 A lung metastasis model was established. Mice were randomly divided into four groups (n=5): 1-3, CDDP, OXP, and a blank control group. The mice were administered the drug via tail vein three times on days 4, 7, and 10 after cell inoculation, at a dose of 2 mg Pt / kg. Mice were sacrificed on day 11, and lung tissue was dissected. After fixation with 4% formaldehyde solution for 24 h, lung metastasis nodules in each mouse were counted and analyzed, and lung tissue was stained with hematoxylin and eosin (HE) for observation.

[0137] Figure 4 Transwell assay results showed that complex 1-3 significantly inhibited tumor cell migration, reducing it to 14.3% in the control group (P < 0.001), which was significantly better than that of CDDP (54.7%, P < 0.001) and OXP (79.5%, P < 0.001) under the same conditions. Furthermore, the scratch healing results ( Figure 5 Further verification of the potent anti-transfer activity of complex 1-3 showed that the scratch healing rate of complex 1-3 was lower than that of CDDP and OXP (P<0.001), which is basically consistent with the trend shown by transwell results.

[0138] Results of in vivo anti-tumor metastasis activity: Figure 6 As shown, complex 1-3 effectively inhibited metastasis, reducing pulmonary nodules to 4.9% in the control group (P<0.001), which was more than 8 times lower than that of CDDP (41.3%, P<0.001) and OXP (49.0%, P<0.001). Furthermore, Figure 6 H&E staining images of lung tissue in d showed that, compared with the blank group and the CDDP and OXP treatment groups, fewer and smaller lung metastatic nodules were observed in the compound 1-3 treatment group, which further validated the anti-metastatic properties of compound 1-3 in vivo.

[0139] Conclusion on anti-tumor metastasis:

[0140] Tetrahydroberberine-platinum (IV) hybrids 1-3 not only exhibit good anti-proliferative activity against tumor cells, but also demonstrate attractive anti-metastatic efficacy both in vitro and in vivo, with significantly higher efficacy than platinum (II) drugs CDDP and OXP. These facts reflect its development potential as an anti-metastatic drug and broaden the application of platinum-based drugs in the field of anti-metastatic therapy.

[0141] 4. In vitro and in vivo tumor uptake experiments

[0142] The uptake of chemotherapy drugs in tumor cells directly affects their antitumor efficacy. Platinum-based drugs enter the cell nucleus and exert their antitumor activity by causing severe DNA damage. Therefore, atomic absorption spectrometry (AAS) was used to assess the cellular uptake and subcellular distribution of tetrahydroberberine platinum (IV) conjugate in 4T1 cells, and its uptake level in tumor tissue was also detected.

[0143] Experimental method: Logarithmic growth phase 4T1 cells were placed in 6-well plates (10... 6 Cells were cultured in 37°C, 5% CO2 incubator for approximately 12 hours, then treated with the drug and cultured for another 24 hours. Cells were digested with EDTA-free trypsin and collected. Cells were washed twice with PBS, digested with concentrated nitric acid, and the platinum content in the cells was determined by atomic absorption spectrometry (AAS) to determine the drug uptake in tumor cells. Cell membranes, cytoplasm, and nuclei were separated using a kit for AAS testing. Tumor tissues from different experimental groups were weighed, digested, and then the platinum content was quantified by AAS to calculate the drug uptake.

[0144] The uptake of chemotherapy drugs in tumor cells directly affects the anti-tumor effect. Platinum-based drugs enter the cell nucleus, causing severe DNA damage and further enhancing their anti-tumor capabilities. Therefore, we used atomic absorption spectrophotometry (AAS) to assess the cellular uptake and subcellular distribution of CND platinum(IV) conjugates in 4T1 cells. In addition, the drug uptake level in in vivo in tumor tissues was also measured. The lipophilicity of the CND platinum(IV) conjugate was a key factor affecting the uptake level. With the elongation of the 1-2-1-4 carbon chain of the complex, its uptake in 4T1 cells gradually increased. Figure 7 a) significantly higher than CDDP and OXP (P<0.001). Furthermore, complex 1-3 accumulated at high levels in tumor tissues in vivo ( Figure 7 (b) The concentrations of compounds 1-3 in tumor cell DNA were more than 3.9 times that of CDDP and OXP (P<0.001). Subcellular distribution results showed that compounds 1-3 accumulated in tumor cell DNA at levels 8.2 times and 24.1 times that of CDDP and OXP, respectively (P<0.001). In conclusion, CND platinum (IV) compounds 1-3 can accumulate at high levels in tumor cells both in vitro and in vivo. After entering tumor cells, they easily reach the cell nucleus, further inducing severe DNA damage and promoting tumor cell apoptosis.

[0145] 5. Apoptosis experiment

[0146] To determine the antitumor mechanism of the tetrahydroberberine platinum (IV) complex, using CDDP and OXP as reference drugs, the apoptosis-inducing properties of complexes 1-3 on 4T1 cells were assessed using Annexin V-FITC / propidium iodide (PI) double staining. Tumor cell apoptosis is closely related to mitochondrial damage. The mitochondrial-mediated apoptosis pathway is closely related to the decrease in mitochondrial membrane potential (ΔΨm) and ROS generation. Subsequently, we used JC-1 and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining methods to measure the loss of ΔΨm and the generation of ROS in tumor cells treated with complexes 1-3. The Bcl-2 signaling pathway controls the mitochondrial-mediated apoptosis pathway. Western blot was used to detect key proteins related to the Bcl-2 pathway, including Bcl-2, Bax, caspase3, and c-caspase3.

[0147] Experimental method: Healthy 4T1 cells in logarithmic growth phase were placed in 6-well plates (10... 6Cells were cultured in 37°C, 5% CO2 incubator for approximately 12 hours, then treated with drugs and cultured for another 24 hours. Cells were digested with EDTA-free trypsin, collected, and divided into three aliquots. For the first aliquot, cells were washed twice with PBS, and 5 μL each of Annexin V-FITC staining agent and PI staining agent were added, mixed, and incubated at room temperature in the dark for 5-15 minutes. The samples were then analyzed by flow cytometry within 1 hour. For the second aliquot, DCFH-DA staining was used to detect intracellular ROS production, and the results were analyzed by flow cytometry. For the third aliquot, JC-1 staining was used, and the results were analyzed by flow cytometry to examine changes in mitochondrial membrane potential.

[0148] Cells were collected using the above method, proteins were extracted, and proteins were separated by SDS-Page electrophoresis. The proteins were then transferred to a PVDF membrane, blocked in 5% skim milk for 1 hour, incubated with primary antibody at 4°C overnight, incubated with secondary antibody for 1 hour, and then developed using ECL chemiluminescence staining solution. The membrane was then imaged using a Tanon 46000SF scanning system.

[0149] Figure 8 The results showed that complex 1-3 could effectively induce tumor cell apoptosis, with an apoptosis rate as high as 30.5%, which was similar to that of CDDP (28.28%) and OXP (25.67%). Figure 9 The results showed that complexes 1-3 effectively disrupted mitochondrial membrane potential and significantly improved ROS generation in tumor cells, with effects similar to those of platinum(II) reference drugs CDDP and OXP. Figure 10 ). Figure 11 The results showed that, compared with the control group, the anti-apoptotic protein Bcl-2 in tumor cells was significantly reduced after treatment with complex 1-3 (P<0.001), while the pro-apoptotic protein Bax showed no significant change (P<0.001). Subsequently, the apoptosis-executing proteins caspase3 and c-caspase3 were also increased under the action of complex 1-3. This indicates that tetrahydroberberine platinum (IV) complex 1-3 can effectively induce mitochondrial membrane potential collapse and ROS generation through the Bcl-2 / Bax / caspase3 pathway, thereby leading to severe mitochondrial-mediated apoptosis.

[0150] 6. DNA damage experiment

[0151] The DNA-binding properties of platinum-based drugs are crucial to their antitumor efficacy. Platinum(IV) complexes, the parent compounds of platinum(II) drugs, must be reduced to their divalent state before interacting with DNA. Here, we used high-performance liquid chromatography (HPLC) to verify the characteristic that tetrahydroberberine-platinum(IV) complexes 1–3 are reduced and induce DNA damage. To further verify its DNA damage mechanism, we used Western blot to detect the expression of phosphohistones γ-H2AX and p53, which are associated with DNA damage.

[0152] Experimental methods: The ability of platinum (IV) compounds 1-3 to be reduced in ascorbic acid (ASA) was determined by HPLC, and their reduction ability was examined. The binding ability of the divalent platinum fragments released after reduction of 1-3 in ASA to 5'-GMP was tested, and their DNA binding characteristics were examined. The expression of DNA damage-related proteins γ-H2AX and P53 was detected by Western blotting, and the DNA damage characteristics were examined.

[0153] Figure 12 The results showed that complexes 1-3 remained stable in RPMI 1640 for at least 48 hours. Then, in the presence of the reducing agent ascorbic acid (AsA, 1 mM, concentration similar to that in the TME), complexes 1-3 were readily reduced. The released platinum(II) complex could bind to guanosine-5'-monophosphate (5'-GMP), added as a DNA base model, and further lead to the appearance of a platinum(II) GMP peak. These facts demonstrate that tetrahydroberberine-platinum(IV) complexes 1-3 are reduced during the reduction of the TME and further induce DNA damage. Figure 13 The results showed that complex 1-3 simultaneously increased the expression of γ-H2AX and p53 in tumor cells, similar to the platinum(II) drugs CDDP and OXP. Therefore, tetrahydroberberine-platinum(IV) complex 1-3 can effectively induce DNA damage both in vitro and in vivo, and further lead to severe apoptosis in tumor cells.

[0154] 7. Inhibits tumor inflammation

[0155] Cancer-associated inflammation (TAI) is a hallmark of cancer and has become an important target for cancer therapy. Chronic inflammatory responses significantly promote immunosuppression and endometrial metastasis (EMT), further leading to tumor metastasis. Cyclooxygenase-2 (COX-2), a key enzyme in the inflammatory response, plays a crucial role in maintaining the inflammatory TME. Furthermore, overexpression of COX-2 promotes the expression of matrix metalloproteinases (MMPs), which are responsible for basement membrane degradation. High expression of MMPs and COXs synergistically promotes TME and tumor invasion and metastasis within the TME. Tetrahydroberberine derivatives, as isoquinoline alkaloids, show great potential in suppressing inflammation. This study evaluated COX-2 and MMP-9 proteins in tumor cells to investigate whether the antitumor properties of tetrahydroberberine platinum (IV) compounds are related to inflammation within the TME.

[0156] Experimental methods: The expression of COX-2 and MMP-9 proteins was monitored by Western blotting of tumor cells in vitro. COX-2 expression was detected by immunohistochemical staining of tumor tissues in vivo.

[0157] Figure 14 The results showed that, compared with the blank group, complexes 1-3 significantly inhibited COX-2 and MMP-9 (P<0.001). Furthermore, Figure 15 Immunohistochemical results showed that complexes 1-3 also significantly inhibited COX-2 expression in tumor tissue, outperforming CDDP and OXP. This indicates that the tetrahydroberberine platinum (IV) complex can effectively inhibit tumor inflammatory responses by downregulating key proteins COX-2 and MMP-9, thereby further influencing tumor immune responses, EMT processes, and metastasis.

[0158] 8. EMT Suppression Experiment

[0159] Inhibition of EMT to suppress metastasis: EMT is a key event in promoting tumor metastasis and suppressing the immune response in TME. It includes the loss of the epithelial marker E-cadherin and the upregulation of stromal markers such as N-cadherin, Snail, and Vimentin. Given the good anti-metastatic activity of platinum (IV) complexes 1-3 and the potential of tetrahydroberberine to inhibit EMT, we evaluated key proteins E-adherin, N-adherin, Snail, and Vimentin in in vitro tumor cells and in vivo tumor tissues using Western blot to determine whether the antitumor properties of the tetrahydroberberine-platinum (IV) mixture are related to the EMT process. The Wnt / β-catenin signaling pathway is a highly conserved pathway and a core pathway in EMT and metastasis. This patent used Western blot to determine the expression of β-catenin, c-Myc, and cyclin D1, which are closely related to the Wnt / β-catenin pathway, in in vitro tumor cells.

[0160] Experimental methods: The expression of E-cadherin, N-cadherin, Snail, and Vimentin proteins was monitored by Western blotting of tumor cells in vitro. The expression of β-catenin, Cyclin D1, and c-Myc proteins was also monitored by Western blotting of tumor cells in vitro.

[0161] Figure 16 This indicates that complexes 1-3 effectively reversed the EMT phenotype. Compared with the control group, E-cadherin was significantly upregulated (P<0.001), while the expression of N-cadherin, Snail, and Vimentin was also significantly inhibited (P<0.001). Subsequent immunohistochemical staining further confirmed the inhibitory effect of compounds 1-3 on tumor EMT in vivo. Figure 17 As can be seen, compounds 1-3 effectively improved E-cadherin secretion in tumor tissue (P<0.001), while E-cadherin levels decreased compared to the control group (P<0.001). Therefore, tetrahydroberberine platinum (IV) conjugates can effectively inhibit EMT, which may be related to the tetrahydroberberine ligand and will further have a positive effect on inhibiting tumor metastasis and activating immune responses. Figure 18 The results showed that, compared with the control group, complexes 1-3 simultaneously inhibited the expression of β-catenin, c-Myc, and cylcin D1 (P < 0.001). These facts indicate that tetrahydroberberine platinum (IV) complexes 1-3 significantly inhibited the Wnt / β-catenin pathway and further had a positive effect on inhibiting EMT and metastasis.

[0162] 9. Experiments on inhibiting EMT and angiogenesis by inhibiting the HIF-1α pathway

[0163] The hypoxic microenvironment is a common feature of malignant tumors, typically characterized by the overexpression of HIF-1α. It is also a key factor in promoting endogenous metastasis (EMT) through the formation of the HIF-1α / β-catenin complex. Hypoxia-induced EMT can then promote angiogenesis via the HIF-1α / VEGFA pathway, thereby facilitating metastasis. Therefore, the expression of HIF-1α and VEGFA in in vitro tumor cells treated with tetrahydroberberine platinum (IV) compounds 1–3 was detected by Western blot. Immunohistochemical staining was also used to detect CD34, a marker of angiogenesis, in in vivo tumor tissues.

[0164] Experimental methods: The expression of HIF-1α and VEGFA proteins was monitored by Western blotting of tumor cells in vitro. CD34 is a biomarker of angiogenesis, and its expression was detected by immunohistochemistry to determine the microvessel density in tumor tissue.

[0165] Figure 19 The results showed that, compared with the control group, compounds 1-3 effectively inhibited the expression of HIF-1α and VEGFA in tumor cells (P<0.001). Subsequently, Figure 20 It was found that complexes 1-3 also significantly reduced the density of CD34-labeled tumor microvessels in vivo, with effects significantly superior to CDDP and OXP (P<0.001). Therefore, it can be concluded that tetrahydroberberine (IV) compounds 1-3 can effectively inhibit the HIF-1α pathway, which is beneficial for synergistic inhibition of EMT with the inhibited Wnt / β-Catenin pathway. Subsequently, angiogenesis was significantly inhibited, which will further affect the metastatic properties of tumor cells.

[0166] 8. Immune activation experiment

[0167] Immunosuppressive tumor microenvironment (TME) is associated with cancer progression, metastasis, and poor prognosis. The EMT process and chronic inflammation are key drivers of tumor immunosuppression. Tetrahydroberberine platinum (IV) complex has the efficacy of inhibiting inflammation and suppressing EMT within the TME, and holds promise for activating anti-tumor immunity during cancer treatment.

[0168] Experimental Methods: A bilateral tumor model in immune-functional female BALB / c mice provides an effective strategy for determining in vivo antitumor immunity. On day 0, mice were injected with 1×10⁻⁶ mol / L of the left side. 6 Cells formed a primary tumor; on day 7, 1×10⁻⁶ cells were injected into the right side of the mouse. 6Cells formed distal tumors. Mice were divided into four groups: blank control group, CDDP, OXP, and groups 1-3. The drug was administered intratumorally (it) to the primary tumor at a dose of 2 mg Pt / kg on days 6, 9, and 12, while distal tumors were not injected. Tumor growth was continuously monitored, and bilateral tumors were collected at the end of the experiment for TGI calculation.

[0169] Figure 21 The results showed that the tetrahydroberberine platinum (IV) complex 1-3 had a strong inhibitory effect on both tumors. It reduced the volume of the primary tumor to 21.7 mm on day 12. 3 It is significantly smaller than the control group (840.3 mm). 3 (P < 0.001) and CDDP group (413.6 mm) 3 (P < 0.001) and OXP group (566.3 mm) 3 (P < 0.001). Simultaneously, compared with the CDDP group (TGI = 70.5%, P < 0.05), the OXP group (TGI = 50.6%, P < 0.01), and the blank group (P < 0.001), compound 1-3 also significantly increased the TGI of primary tumors to 90.8%. These facts further validate the potent antitumor activity of the tetrahydroberberine platinum (IV) mixture 1-3. More importantly, it effectively inhibited the proliferation of distant tumors, mainly due to the induction of tumor immune activation by the tetrahydroberberine platinum (IV) complex 1-3. The inhibitory effect of compound 1-3 on distant tumors was 25.1 mm. 3 (TGI = 88.5%), while CDDP (242.1 mm) 3 The inhibitory effects of tetrahydroberberine platinum (TGI = 31.7%, P < 0.01) and OXP (267.9 mm³, TGI = 46.1%, P < 0.001) were relatively low. The significant ascites effect indicates that the tetrahydroberberine platinum (IV) complex 1-3 can effectively stimulate antitumor immunity.

[0170] 9. Experiments on inducing T cell and macrophage polarization and activating anti-tumor immunity.

[0171] T cell activation and macrophage polarization are crucial to the human immune system. Programmed cell death ligand 1 (PD-L1) and CD47 are considered key immune checkpoints (ICPs) in tumors, playing a significant role in reducing immunity and promoting cancer immune evasion. Overexpression of PD-L1 leads to T cell inactivation and exhaustion under the influence of the PD-1 / PD-L1 axis. The immunostimulatory capacity of tetrahydroberberine platinum (IV) complex 1–3 was tested in vitro on 4T1 cells and in vivo on Balb / c mice carrying bilateral 4T1 tumors.

[0172] Experimental methods: Western blot was used to detect the expression of PD-L1 and CD47 in 4T1 tumor cells. Immunohistochemistry was used to detect the expression of PD-L1 and CD87 in tumor tissues. + M1 and CD206 + M2 and CD3 + and CD8 + Tumor-infiltrating lymphocytes (TILs) were detected to examine the effect of tetrahydroberberine platinum (IV) complex on tumor immunity.

[0173] T cell activation was observed in tumor cells treated with complexes 1-3. Figure 22 and 23 As shown, compared with the control group, the expression of PD-L1 was significantly inhibited in the complex 1-3 treatment group (P<0.001). Complex 1-3 also effectively inhibited the secretion of PD-L1 in tumor tissue in vivo, reducing PD-L1 expression to 10.3% of that in the control group (P<0.001). Subsequently, the number of CD3+ and CD8+ T cells increased by 2.7-fold and 2.0-fold, respectively, compared with the control group (P<0.001), which was also significantly better than the CDDP and OXP treatment groups (P<0.001). In addition, complex 1-3 also induced macrophage polarization in tumors from M2 to M1 type. Compared with the control group, CD47 protein was significantly inhibited in cells treated with complex 1-3 (P<0.001). Subsequently, the number of CD206+ M2 phenotype macrophages in tumor tissue decreased to 12% of the control group (P<0.001), while the CD86+ M1 phenotype significantly increased in mice treated with compounds 1-3 (419%, P<0.001). Therefore, the tetrahydroberberine platinum (IV) complex, by blocking key PD-L1 and CD47, further increased the density of CD3+ and CD8+ T lymphocytes, while simultaneously inducing macrophage polarization from M2 to M1, thereby effectively activating anti-tumor immunity. Angiogenesis is key to invasive tumor metastasis and growth, and is an important step in controlling tumor development. Vascular endothelial growth factor (VEGF) is a key factor associated with angiogenesis. Furthermore, MMP-9 promotes tumor metastasis by degrading extracellular matrix proteins. Simultaneously, the study confirmed that MMP-9 and VEGFA exhibit a synergistic effect in increasing vascular density and promoting tumor metastasis.

[0174] The antitumor activity mechanism of the drug of this invention is shown in the figure. Figure 24 .

[0175] 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 tetrahydroberberine tetravalent platinum structure as shown in general formula (Ⅰ): in, Selected from cisplatin or oxaliplatin; wherein, R 3 Selected from propylidene, butylidene, or pentylidene.

2. The compound according to claim 1, characterized in that: The compound is selected from:

3. A method for preparing the compound according to any one of claims 1 to 2, characterized in that: The synthesis route is as follows: The molar ratio of compounds 3 and 4 is 1:2.0 to 4.

0.

4. The method for preparing the compound according to claim 3, characterized in that: The preparation steps of the synthetic route are as follows: under an inert gas atmosphere, compound 4, condensing agent, and organic base are dissolved in an anhydrous organic solvent and reacted. Compound 3 is added, and after reacting in the dark, compound 1 is obtained by post-processing. The molar ratio of compound 3, compound 4, condensing agent, and organic base is 1:2.0~4.0:2.0~4.0:2.0~4.0; the feeding relationship between compound 3 and organic solvent is that 30~80ml of organic solvent is added for every 1g of compound 3.

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 TBTU, HATU, or EDCI; the organic base is triethylamine or N,N-diisopropylethylamine, 4-dimethylaminopyridine; and the organic solvent is DMF or DMSO.

6. A pharmaceutical composition, characterized in that: This includes compounds of general formula (Ⅰ) as described in claim 1 or 2, and pharmaceutically acceptable excipients thereof.

7. The use of the compound of claim 1 or 2 or the pharmaceutical composition of claim 6 in the preparation of an antitumor drug.

8. The application as described in claim 7, characterized in that, Application in the preparation of drugs that inhibit tumor proliferation and metastasis.

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

10. The application according to claim 8, characterized in that: The antitumor proliferation mentioned refers to the proliferation of cisplatin-resistant human lung adenocarcinoma.

11. A combination formulation comprising a compound of general formula (I) as described in claim 1 or 2, or a pharmaceutical composition as described in claim 6, and a platinum-based, 5-fluorouracil-based, or paclitaxel-based antitumor drug.

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